Note: Descriptions are shown in the official language in which they were submitted.
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MECHANISMS OF OSTEOINDUCTION BY LIM MINERALIZATION
PROTEIN-1 (LMP-1)
BACKGROUND OF THE INVENTION
Many Americans are afflicted by low back pain, degenerative spinal disease, or
bone fractures. These musculoskeletal problems are responsible for a major
portion of the
health care budget and are among the greatest causes of chronic disability and
lost
productivity in the United States. Qrthopaedic surgical treatment of these
problems
frequently requires bone grafting to promote healing. Fusion of two or more
bones with
cancellous bone graft may fail to heal in 25-45% of patients, and in even
higher
percentage of smokers and diabetic patients, co-morbidities which are more
prevalent in
the Veteran population. Use of osteoinductive proteins such as BMP-2 to induce
bone
formation in these patients is now possible. In 2002 the U.S. Food and Drug
Administration approved rhBMP-2 for use as a bone graft substitute in
interbody spine
fixsions. Despite this regulatory milestone for BMP-2, this technology is not
feasible for
many patients with bone healing needs due to an unexpectedly high dose
required in
humans which has resulted in a very high cost (Boden SD, Zdeblick TA, Sandhu
HS, and
Heim SE. Spine 2000;25:376-81; Ackerman SJ, Mafilios MS, and Polly DW, Jr.
Spine
2002;27: S94-S99).
A 15,000-fold higher concentration of BMP-2 is required to induce bone in
hunians (1.5 mg/mL) than in cell culture (100 ng/mL). Thus, without a dramatic
improvement in BMP-2 responsiveness, healthcare economics may severely limit
translation of one of the most seminal discoveries related to osteoblast
differentiation in
the last 50 years from helping large numbers of patients.
Consequently, a fiu-ther understanding of the complex regulation of BMP-2
during
osteoblast differentiation and the cellular responsiveness to such important
bone forming
proteins is critical so that their effect can be enhanced or their required
dose limited to a
more affordable quantity of protein especially in the most challenging
orthopaedic healing
environment - posterolateral lumbar spine fusion.
Several years ago a novel intracellular LIM domain protein critical to fetal
and
post-natal bone formation was identified (Boden SD, Liu Y, Hair GA et al.
Endocrinology
1998;139:5125-34).
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Termed LIM Mineralization Protein (LMP-1) it was the first LIM domain protein
to be directly associated with osteoblast differentiation. Blocking LMP-1
expression
prevents osteoblast differentiation in vitro, suggesting a critical functional
role of this
novel intracellular protein. Leukocytes expressing the LMP-1 cDNA (via plasmid
or
adenoviral transduction) that are implanted into rabbits or athymic rats
induce bone
formation in bony and ectopic locations (Boden SD, Titus L, Hair G et al.
Spine
1998;23:2486-92). The feasibility of LMP-1 delivery by ex vivo gene therapy
for spine
fusion and bone defect applications in rabbits and primates is currently being
evaluated.
LMP-1 also has considerable potential as a local, regional, or systemic
anabolic strategy
for increasing bone density in patients with osteoporosis. However, before
clinical
applications can be seriously considered it will be critical to understand the
mode of action
of this protein. The present invention addresses this problem.
Osteoblasts are thought to differentiate from pluripotent mesenchymal stem
cells,
the maturation of which results in the secretion of an extracellular matrix
which can
mineralize and form bone. The regulation of this complex process involves a
group of
signaling glycoproteins known as bone morphogenetic proteins (BMPs), members
of the
transforming growth factor-beta (TGF-13) superfamily. Some BMPs are uniquely
capable
of initiating the entire osteoblast differentiation cascade and BMP-2 is one
of the most
extensively studied.
With Applicant's discovery that LMP-1 can dramatically increase cellular
responsiveness of mesenchymal stem cells (MSCs) to BMP-2 and mechanistic
elucidation
of various aspects of the signaling pathway of LMP-1, the present invention
provides
combinatorial strategies including small molecules and peptide mimics, to
overcome the
dose-related translational barriers for BMP-2 therapeutics.
SUMMARY OF THE INVENTION
The present invention relates to combinatorial therapeutic strategies
including
small molecules and peptide mimics of LIM mineralization proteins, primarily
LMP-l, to
overcome the dose-related translational barriers for BMP-2 therapeutics.
DETAILED DESCRIPTION
The present invention derives from studies designed to elucidate the mechanism
of
LIM mineralization protein (LMP) action in modulating growth factor
responsiveness in
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cells, tissues and organisms. As a result of these studies, Applicant's have
discovered that
there is an unexpected synergistic result when an osteogenic composition
comprising at
least one LIM mineralization protein and at least one growth factor is
administered to
induce bone formation in a subject in need of bone repair, replacement or
augmentation,
for example subject suffering from compromised bone conditions.
Overview
The studies disclosed herein demonstrate that LMP-1 enhances responsiveness to
BMP-2 in MSCs. To elucidate the mechanism underlying this observation it is
further
demonstrated that LMP- 1 interacts in vitro with an 85 kDa protein, identified
as Smurfl, a
key regulator of the degradation of BMP-2 signaling molecules, Smadl and
Smad5. It is
also demonstrated here that endogenous Smurfl and LMP-1 co-immunoprecipitate
from
cells, suggesting the physiological relevance of the interaction. The
importance of the
Smurfl/LMP-1 interaction is further documented by the fact that LMP-1
overexpression
increases levels of phosphorylated Smadl (P-Smadl) in the nucleus aiid
increases
expression of BMP-2 regulated genes, expected outcomes of Smurfl/LMP-1
interaction.
LMP- 1 -induced inhibition of Smurfl WW domain antibody binding to Smurf
identified
the WW domain as the region of Smurfl that LMP-1 binds. Further, analysis of
LMP-1
sequence has identified two potential WW domain interacting motifs within an
osteoinductive region of LMP-1. It is also demonstrated that LMP-1 increases
BMPRIA
levels in support of the liypothesis that LMP interrupts the Smurfl/Smad6
mediated
degradation of the BMP receptor. LMP-1 is shown herein to interact with Jab 1,
an adaptor
protein which regulates degradation of the common Smad, Smad4 resulting in
increased
nuclear Smad4.
Furthermore, identified herein is the precise region of LMP which interacts
with
Smurfl. This discovery facilitates design of small compounds that mimic LMP's
effects.
The compounds include small proteins and peptides. In addition, the ability to
use a single
exposure dose of a recombinant TAT-LMP fusion protein is demonstrated,
confirming that
continuous LMP-1 expression is not required for an effective therapeutic
outcome and
opens the door for design of an LMP-mimic small compound.
Also discovered is a novel interaction between LMP-1 and Smurfl which
represents a powerful control mechanism over BMP signaling and responsiveness.
This
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LMP-1 interaction occurs with the Sniurf WW2 domain, is dependent on a
specific PY
motif in LMP-1, and can be mimicked by a small peptide containing only that
motif.
Further, LMP- 1 competitively binds to Smurfl, preventing ubiquitin-mediated
proteasomal degradation of Smads, contributing to an enhanced cellular
responsiveness to
BMP-2. These findings allow for the design of small molecule therapeutics that
more
efficiently control responsiveness of the BMP signaling pathway which would
make
clinical translation easier. Such small molecules would be more easily
synthesized, stored,
and delivered for clinical use to induce bone fonnation alone or with much
lower doses of
BMP-2 than are currently required in the clinical setting.
Thus, therapeutics that modulate the effects of LMP-1 have the potential to
either
replace BMP-2 as a strategy to induce bone formation or to serve as a method
to enhance
the efficacy of rhBMP-2, lowering the dose and cost of its use as an inducer
of bone
formation.
Combination therapy
The invention relates to treatment of diseases using combination therapy. In
particular, the novel LMP agents described herein may be used in conjunction
with BMP
agents. The present invention provides a method of inducing bone deposition by
co-
administration of at least one LMP agent and a therapeutically effective dose
of at least
one BMP agent. It has been found that LMP agents are capable of accelerating
bone
formation by enhancing the BMP agent's responsiveness. In the method of the
invention,
the LMP agent accomplishes this by affecting a BMP agent including but not
linzited to
endogenous BMP protein, exogenous BMP protein, exogenous BMP protein fragment,
and exogenous BMP protein variant fragment. The present invention may
therefore be
used to decrease the time required to form new bone in the presence of a BMP
agent
comprising administering at least one LMP agent.
As used herein LIM mineralization protein (LMP) "LMP" includes LMP- 1 and
biologically active fragments thereof, LMPlt and biologically active fragments
thereof,
and LMP-3 and biologically active fragments thereof. More detailed
descriptions,
including sequences, can be found in US Patent 6,300,127, pending application
USSN
10/951,236, and pending application USSN 09/959,578 filed by Boden et al., the
entire
teachings of which are incorporated herein by reference. LMP-2 is excluded as
it is non-
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osteogenic. Growth factors suitable in the invention include bone morphogenic
proteins
(BMP) including BMP-2.
As used herein the term "LMP agent" includes a functional fragment of an LMP
protein, a functional fragment of an LMP protein with a protein transduction
domailn
(PTD) attached, an LMP protein with a PTD attached, an LMP protein without a
PTD
attached, a functional fragment of an LMP protein variant, an LMP protein
variant with a
PTD attached, an LMP protein variant without a PTD attached, an
oligonucleotide
sequence encoding any of the above, and an LMP gene.
As used herein, the term "BMP agent" includes a functional fragment of a BMP
protein, a functional fragment of a BMP protein with a PTD attached, a BMP
protein, a
functional fragment of a BMP protein variant, a BMP protein variant, an
endogenous BMP
protein, exogenous BMP protein, an exogenous BMP protein fragment, an
exogenous
BMP protein variant fragment, an oligonucleotide sequence encoding any of the
above,
and a BMP gene. Particularly useful BMP is BMP-2, especially rhBMP-2.
Protein and peptide variants and derivatives
Those skilled in the art will understand that one may make many molecules
derived in sequence from the aforementioned LMP agents or BMP agents in which
amino
acids have been deleted ("deletion variants"), inserted ("addition variants"),
or substituted
("substitution variants"). Molecules having such substitutions, additions,
deletions, or any
combination thereof are termed individually or collectively "variant(s)." Such
variants
should, however, maintain at some level (including a reduced level) the
relevant activity of
the unmodified or "parent" molecule (e.g., an LMP variant possesses the
ability to
modulate BMP responsiveness or to bind Smurfl). Hereinafter, "parent molecule"
refers to
an unmodified molecule or a variant molecule lacking the particular variation
under
discussion. There are two principal variables in the construction of amino
acid sequence
variant(s): the location of the mutation site and the nature of the niutation.
In designing
variant(s), the location of each mutation site and the nature of each mutation
will depend
on the biochemical characteristic(s) to be modified. Each mutation site can be
modified
individually or in series, e.g., by (1) deleting the target amino acid
residue, (2) inserting
one or more amino acid residues adjacent to the located site or (3)
substituting first with
conservative amino acid choices and, depending upon the results achieved, then
with more
radical selections.
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An amino acid sequence addition may include insertions of an amino- and/or
carboxyl-terminal fusion ranging in length from one residue to one hundred or
more
residues, as well as internal intra-sequence insertions of single or multiple
amino acid
residues. Internal additions may range generally from about 1 to 20 amino acid
residues,
preferably from about 1 to 10 amino acid residues, more preferably from about
1 to 5
amino acid residues, and most preferably from about 1 to 3 amino acid
residues.
An example of an amino- or a carboxy-terminus addition includes chimeric
proteins
comprising the amino-terminal or carboxy-terminal fusion of the parent
molecules with all
or part of a transduction peptide or other conjugate moiety.
Amino acid sequence deletions generally range from about 1 to 30 amino acid
residues, preferably from about 1 to 20 amino acid residues, more preferably
from about 1
to 10 amino acid residues and most preferably from about 1 to 5 contiguous
residues.
Amino-terminal, carboxy-terminal and internal intrasequence deletions are
contemplated
by the present invention. As used herein a "functional fragment" of a protein
is any
fragment or portion of a protein which retains the characteristic of interest
of the parent
protein or peptide. As used herein "biologically active" means retaining that
characteristic
or property in question from the parent molecule.
In one embodiment the protein or peptide may possess multiple activities such
as
would be provided by multiple binding sites. These binding sites or domains
may be
identical or variable and may be in sequence or separated by non-binding site
amino acids.
In yet another embodiment, recombinant proteins, peptides or fusion proteins
may
be produced.
In another embodiment the LMP agents or BMP agents of the present invention
are
conjugated to other proteins or peptides. Protein transduction domains (PTDs)
and
attachment of these to proteins and peptides are contemplated. In one
embodiment of the
present invention, the PTD is the HIV-TAT protein.
In one embodiment, a variant protein or peptide will preferably be
substantially
homologous to the amino acid of the parent molecule or a portion or a domain
of the
parent molecule from which it is derived. The term "substantially homologous"
as used
herein means a degree of homology that is in excess of 80%, preferably in
excess of 90%,
more preferably in excess of 95% or most preferably even 99%. Homology is
determined
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relative to the smaller peptide or variant and is measured across that domain,
site or
fragment in the parent from which the variant or peptide is derived.
The invention also comprises chemically modified derivatives of the parent
molecule(s) in which the peptide is linked to a nonproteinaceous moiety (e.g.,
a polymer)
in order to modify its properties. These chemically modified molecules are
referred to
herein as "derivatives". Such derivatives may be prepared by one skilled in
the art given
the disclosures herein. Conjugates may be prepared using glycosylated, non-
glycosylated
or de-glycosylated parent molecule(s) and suitable chemical moieties.
Typically non-
glycosylated molecules and water-soluble polymers will be used. Other
derivatives
encompassed by the invention include post-translational modifications (e.g., N-
linlced or
O-linlced carbohydrate chains, processing of N-terminal or C-terminal ends),
attachment of
chemical moieties to the amino acid backbone, and chemical modifications of N-
linlced or
0-linked carbohydrate chains. The polypeptides may also be modified with a
detectable
label, such as an enzymatic, fluorescent, isotopic or affmity label to allow
for detection
and isolation of the protein peptide.
Water-soluble polymers are desirable because the protein or peptide to wliich
each
is attached will not precipitate in an aqueous environment, such as a
physiological
environment. Preferably, the polymer will be pharmaceutically acceptable for
the
preparation of a therapeutic product or composition. One skilled in the art
will be able to
select the desired polymer based on such considerations as whether the
polymer/protein
conjugate will be used therapeutically and, if so, the therapeutic profile of
the protein (e.g.,
duration of sustained release; resistance to proteolysis; effects, if any, on
dosage;
biological activity; ease of handling; degree or lack of antigenicity and
other known
effects of a water-soluble polymer on a therapeutic proteins).
Variants and/or derivatives may be screened to assess their physical
properties in
vitt o and can be subsequently screened in vivo in the models described
herein. It will be
appreciated that such variant(s) will demonstrate similar properties to the
unmodified
molecule, but not necessarily all of the same properties and not necessarily
to the same
degree as the corresponding parent molecule.
Oligonucleotides
Oligonucleotide sequences of the present invention include those polymeric
nucleic acid sequences which would "code for" the protein or peptide of
interest. Those of
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ordinary skill in the art will appreciate the degeneracy of the genetic code
and that variable
codons may still produce the same protein on translation. As used herein the
term
"oligonucleotide" includes nucleic acid sequences which code for the proteins
or peptides
of the invention or their parent molecules, including but not limited to the
LMP and BMP
agents and vectors encoding said agents as well as small interfering RNAs
(siRNAs)
designed to target the genes disclosed herein, especially those involved in
BMP and LMP
signalling pathways. Particular oligonucleotides of the present invention
include siRNAs
designed to LMP-1, Smurfl, Smurf2 and Jabl.
Pharmaceutical compositions
The invention also provides for pharmaceutical compositions in the form of an
osteogenic composition. As used herein an "osteogenic composition" is a
composition
comprising a therapeutically effective amount of at least one BMP agent
combined with at
least one other agent and optionally, at least one pharmaceutically acceptable
diluent,
carrier, solubilizer, emulsifier, preservative and/or adjuvant. In a preferred
embodiment,
the osteogenic composition comprises a therapeutically effective amount of at
least one
LMP agent and at least one BMP agent and optionally, at least one
pharmaceutically
acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or
adjuvant. It is
understood that the phrase "at least one" includes one and more than one; for
example,
two, three, four or more.
In another embodiment an osteogenic composition comprises at least one Smurf
binding agent; and at least one BMP agent; and optionally, at least one
pharmaceutically
acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or
adjuvant. In the
present invention, Smurf binding agents include LMP-1 protein, and LMP-1
protein
fragments, variants or derivatives, and siRNA specific for Smurf mRNA. The
Smurf is
selected from the group consisting of Smad Ubiquitin Regulatory Factor-1
(Smurfl) and
Smurf 2.
In yet another embodiment, the osteogenic composition comprises at least one
phosphorylated Smad 1 competitive binding agent and at least one BMP agent. In
the
present invention, the phosphorylated Smad 1 competitive binding agent
includes but is
not limited to an LMP-1 protein, and LMP-1 protein fragment, variant or
derivative, and
siRNA specific for phosphorylated Smad 1.
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In another embodiment, the osteogenic composition comprises at least one
phosphorylated Smad 5 competitive binding agent and at least one BMP agent. In
the
present invention, the phosphorylated Smad 5 competitive binding agent
includes but is
not limited to an LMP-1 protein, and LMP-1 protein fragment, variant or
derivative, and
siRNA specific for phosphorylated Smad 5.
Also disclosed is an osteogenic composition comprising at least one
phosphorylated Smad 4 competitive binding agent and at least one BMP agent. In
the
present invention, the phosphorylated Smad 4 competitive binding agent
includes but is
not limited to an LMP-1 protein, and LMP-1 protein fragment, variant or
derivative,
siRNA specific for phosphorylated Smad 4.
Pharmaceutically acceptable diluents, carriers, solubilizers, emulsifiers,
preservatives and/or adjuvants are known to those sleilled in the art. These
include but are
not limited to cells, vectors, gels, microspheres, macromolecules,
biocompatible foams,
biocompatible matrices, and implants. Compositions may also comprise
incorporation of
any of the therapeutic molecules or agents into liposomes, microemulsions,
micelles or
vesicles for controlled delivery over an extended period of time. The term
"therapeutically
effective amount" means an amount which provides a therapeutic effect for a
specified
condition and route of administration. Whether an amount is therapeutically
effective may
be determined on a stand alone or combinatorial basis. Consequently, what
might
represent a therapeutically effective amount of one agent may change when that
agent is
combined with a further agent. In a preferred embodiment of the invention, an
LMP agent
is administered in combination with a BMP agent, wherein the dose or amount of
BMP
agent is subtherapeutic as compared to conventional rhBMP-2 therapy alone. As
a stand
alone therapy, a BMP agent at subtherapeutic doses or amounts would not,
therefore, be
therapeutically effective but when combined with the LMP agents of the present
invention
would represent a therapeutic dose or amount.
Administration and Dose
Depending on dosage form, the pharmaceutical compositions of the present
invention may be administered in different ways, i.e., intrathecal injection,
subcutaneous,
intravenous, intraperitoneal, intramuscular injection, in an implant or
combinations
thereof. The administration of the LMP agents of the present invention may
occur before,
after or simultaneously with the BMP agent and may be to a single targeted
site or
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separate sites. Sites for administration include, but are not limited to an
intervertebral
space, a facet joint, site of a bone fracture, bones of the mouth, chin and
jaw, and an
implant site.
In yet another embodiment, the therapeutic methods of the invention further
comprises a co-therapeutic treatment regimen comprising administering a
therapeutically
effective amount of an LMP agent in combination with a therapeutically
effective amount
of a BMP agent to treat disease in a patient. As used herein a "co-therapeutic
treatment
regimen" means a treatment regimen wherein two agents are administered
simultaneously,
in either separate or combined formulations, or sequentially at different
times separated by
minutes, hours or days, but in some way act together to provide the desired
therapeutic
response.
Dosages of the LMP agent, BMP agent or compositions of the present invention
may range from 1nM to 200nM if delivered as a recombinant fusion protein with
a PTD
attached or from 0.1 to 100 MOI (multiplicity of infection, i.e. number of
infectious viral
particles per cell) if delivered by an adenovirus or similar vector. In
combination, it is
understood that doses of one agent may be lowered when the dose of a co-
administered
agent is raised. For example, it is contemplated that on raising the dose of
an LMP agent,
the dose of the BMP agent administered may be lowered. It is also contemplated
with
synergistic compositions, that administration of one component of a
combination will
mitigate the need for an equal dose of a second component as is demonstrated
herein with
the LMP agent synergistically increasing the responsiveness of cells to a BMP
agent.
In one embodiment, therapeutically effective dose of BMP agent is less than
the
currently acceptable therapeutically effective amount. Currently, 1.5mg/mL of
bone
formed is the therapeutic concentration of rhBMP-2 in primates in vivo with
smaller doses
effective in cell culture and rodents. The therapeutically effective dose of
said at least one
BMP agent is at least 10-fold less than the dose required in conventional
therapy. The
dose required in conventional therapy can be 20 mg rhBMP-2 per site of 10 cc
of bone
formation. In other embodiments, the therapeutically effective dose of BMP is
at least 20-
fold, 50-fold, 100-fold, 1000-fold, 5000-fold or 10,000-fold less than the
dose required in
conventional BMP therapy when administered in combination with an LMP agent.
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Treatment outcomes
The therapeutic agents and compositions of the present invention are useful in
treating subjects having compromised bone conditions. The invention provides
for a
method of treating a bone disorder using a therapeutically effective amount of
an LMP
agent in combination with a BMP agent. The bone disorder or compromised bone
condition may be any disorder characterized by bone loss (osteopenia or
osteolysis) or by
bone damage or injury. Such bone conditions include but are not limited to
broken bones,
bone defects, bone transplant, bone grafts, bone cancer, joint replacements,
joint repair,
fusion, facet repair, bone degeneration, dental implants and repair, bone
marrow deficits
and other conditions associated with bone and boney tissue.
Examples of bone defects include but are not limited to a gap, deformation or
a
non-union fracture in a bone.
Examples of bone degeneration include but are not limited to osteopenia or
osteoporosis. In one embodiment, the bone defect is due to dwarfism.
The invention is especially useful for joint replacement or repair wherein the
joint
is vertebral, knee, hip, tarsal, phalangeal, elbow, anlcle, sacroiliac or
other articulating/non-
articulating joint.
Bone tissue engineering
Furthermore, the engineering and use of cell- and animal-based models of bone
disease in which the compounds of the invention may be,useful are also
described. The
invention includes a process for engineering bone tissue comprising combining
at least
one LMP agent and at least one BMP agent with a cell selected from the group
consisting
of osteogenic cells, pluripotent stem cells, mesenchymal cells, and embryonic
stem cells.
Also, disclosed is the engineered bone tissue produced by the above process. A
method
for inducing bone formation in a subject comprising administering the
engineered bone
tissue of the present invention is contemplated.
Further, the invention includes a process for engineering bone tissue
comprising
combining at least one phosphorylated Smad 4 competitive binding agent and at
least one
BMP agent with a cell selected from the group consisting of osteogenic cells,
pluripotent
stem cells, mesenchymal cells, and embryonic stem cells. Also included is
engineered
bone tissue produced by this process. In another aspect, the invention
includes a method
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for inducing bone foimation in a subject comprising administering the
engineered bone
tissue as described in this paragraph.
Also included in the invention is a method for inducing deposition and
maturation
of bone in a subject having compromised bone conditions comprising
administering to the
subject at least one Jabl-inhibiting agent and a therapeutically effective
dose of at least
one BMP agent.
Methods and Procedures
Cell culture: Mesenchymal stem cells, (MSCs) at passage 2 are purchased from
Cambrex
Bio Sciences. Cells are grown at 37 C in 5% COz in MSCBM media supplemented
with
MSCGM Singlequots (Cambrex Bio Sciences), split at confluence, and plated at 3
x 104
cells/well in 6-well dishes at passage 4 in these studies. The next day
treatments are
applied in the presence of 50 uM L-Ascorbic Acid 2-Phosphate and 5 mM (3-
glycerol
phosphate (Sigma-Aldrich). Medium is changed every 3-4 days with re-
application of
treatments where appropriate. Cells are transduced for 30 min with adenoviral
constructs
in 0.5 ml serum free medium. rhBMP-2 will continue to be supplied as a gift
from Wyeth
(Genetics Institute) courtesy of John Wozney.
Preparation of nuclear and cZoplasmic protein fractions: Cell pellets are
suspended in
buffer A (20 mM HEPES, pH 7.9, 10 mM KCI, 1 mM EGTA, 1 mM EDTA, 0.2 %
Nonidet P-40, 10% Glycerol, 1 mM PMSF and lug/ml protease inhibitor mix
(Sigma)),
incubated on ice for 10 min, and centrifuged. Supernatants (cytoplasmic
fraction) are
collected and nuclear pellets are suspended in high salt buffer B (buffer A
plus 600 mM
KCI, 20% glycerol), incubated on ice for 30 min and centrifuged. Supernatants
are
collected as the nuclear fraction. After protein determination, fractions are
subjected to
SDS-PAGE.
Measurement ofphosphorylated Smadl and Smad5 in the nucleus on overexpression
of
LMP-1: To show that increased levels of LMP-1 result in increased levels of
phosphorylated Smadl and Smad5, hMSCs are treated with the doses of LMP-1 that
successfully synergize with BMP-2 to determine the timecourse of increased of
phosphorylated Smadl in the nucleus when the agents are applied alone or
together.
Human MSCs are plated at 3 x 104 cells/well in 6-well plates, grown overnight,
and treated with Ad5F35-LMP-1 (0, 5, 10 pfu/cell), BMP-2 (100 ng/ml) or both
agents
(control = Ad5F35-GFP). After 1, 2, 4, 8, 12, 24 and 48 hrs, cells are
harvested and
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nuclear proteins are analyzed by Western blot for Smad proteins. Both Smadl
and Smad5
are measured. Antibody to phosphorylated Smadl is available and is used for
Western
analysis. Antibody to phosphorylated Smad5 is not available so an antibody
that detects
both phosphorylated and unphosphorylated Smad5 is used; and a phosphoserine
antibody
on a separate blot is used to determine the phosphorylation state. Although
there will be
several molecules having phosphoserine, comparison of the two blots should
allow
determination of whetlzer P-Smad5 also increases in the nucleus. Treating
cells with the
same adenoviral vector carrying GFP cDNA as a control is not expected to have
an effect
on nuclear levels of P-Smads. Increased P-Smad levels are expected to occur
rapidly in
response to BMP-2, since that involves phosphorylation of existing Smad
proteins to
activate the intracellular signaling cascade. In contrast, LMP-1 cDNA must be
transcribed
and translated into an intracellular protein, a process requiring several
hours. Thus a delay
in increased nuclear levels of P-Smads is expected in response to LMP-1 as
compared to
the response to BMP-2.
SDS-PAGE and Westen blotting: SDS-PAGE is performed using 10% gels and
transferred
to nitrocellulose membrane. The membrane is blocked with milk protein,
incubated with
specific antibody, washed with Tris Buffered Saline containing 0.1% Tween 20
(TBST),
incubated with anti-rabbit goat IgG-linked to horseradish peroxidase (NEN),
and again
washed with TBST. Chemiluminescent substrates are applied to the membrane and
the
signal is detected by exposing the membrane to X-ray film 30 seconds.
RNA extraction: RNA is isolated from cells grown in 6-well plates using RNeasy
Mini
Kits as specified by the manufacturer (Qiagen). Briefly, cells are harvested
and disrupted
in RLT buffer. The lysate is passed over QiaShredder columns, and the
resulting eluate
brought to 35% EtOH and passed over RNeasy columns to bind the RNA to the
silica-gel
membrane. After washing the bound RNA with RW 1 buffer and then RPE buffer,
the
RNA is eluted from the membrane with water. All RNA samples are DNase treated
either
using the Qiagen RNase-Free DNase Set during the RNeasy procedure or after
fmal
harvest of the RNA using the Ambion DNA-free Kit. After completion of the
digestion, 5
l of DNase Inactivation Buffer is added, the solution incubated for 2 minutes
at RT, and
the samples centrifuged for 1 min in a microfuge. The RNA containing
supernatant is
removed and stored at -70 C. In addition to the above, if the RNA is being
isolated from
transfected cells, the initial RNA prep is digested for 1 h at 37 C with the
restriction
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enzyme RsaI to cleave any contaminating plasmid DNA, the RNA reisolated using
an
RNeasy kit, and DNase treated with the Ambion reagents. Each sample consists
of RNA
isolated from 2 wells of a 6-well plate and at least three samples are
isolated for each
treatment/time point.
Real-time Reverse Transcription-Polymerase Chain Reaction (PCR) of RNA: Two g
of
total RNA is reverse transcribed in a 100 1 total volume containing 50 mM KC1,
10 mM
Tris, pH 8.3, 5.5 mM MgC12, 0.5 mM each dNTPs, 0.125 M random hexamer, 40
units
RNase Inhibitor, and 125 units MultiScribe (Applied Biosystems). In control
samples the
RNase inhibitor and MultiScribe are omitted. Samples are incubated for 10 min.
at 25 C,
30 minutes at 48 C, and then 5 min. at 95 C to inactivate the enzyme. Real-
time PCR is
then performed on 5 l of the resulting cDNA in a total volume of 25 l
containing 12.5 l
of 2X SYBR Green PCR Master Mix (Applied Biosystems), and 0.8 gM each primer.
The
PCR parameters used are 2 min. at 50 C, 10 min. at 95 C, and 45 cycles of 95 C
for 15
sec. followed by 1 min. at 62 C. PCR is also performed as described on a 1/800
dilution
of the cDNA with 18S primers for normalization of the samples. Relative RNA
levels
were calculated using the A A Ct method (Applied Biosystems).
The primers listed in Table 1 have been synthesized and successfully measured
mRNA levels of gene expression in human MSCs.
Table 1
RT PCR Primer/Probe Sets
Primer Name Primer Sequence (5' to 3') SEQ ID
NO
Ad35LMP-1 TTCTGAGCTTCGATGTGTGTGA 1
Forward
Ad35LMP-1 CATCATGGATTCCTTCAAGGTAGTG 2
Reverse
Ad35LMP-1 Probe 6FAM-CATCGATGCTCAGCACCCAGTCACC-TAMRA 3
bznrSMAD-1 ACCCTGTCTGAGGAGCGTGTA 4
Forward
bmrSMAD-1 ACCAAAGCGTCCACAGCTTT 5
Reverse
hmrSMAD-5 CACCAAGATGTGTACCATTCGAA 6
Forward
bmrSMAD-5 GAAGAGCCCATCTGAGTAAGGA 7
Reverse
bmrSMAD-6 GGATCTGTCCGATTCTACATTGTCT 8
Forward
hmrSMAD-6 TGTCCGGTGCTCCCAGTAC 9
Reverse
hmrDLX5 Forward GGAGTTGGCCGCCTCTCTAG 10
bmrDLX5 Reverse TGGCGAGTTACACGCCATAG 11
bmrNoggin TGCCGAGCGAGATCAAAGG 12
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Forward
hmrNoggin GTAGCGCGGCCAAAAGC 13
Reverse
hBMP-2 Forward TCCAAGAGACATGTTAGGATAAGCA 14
hBMP-2 Reverse TCCACGTACAAAGGGTGTCTCTTAC 15
hmrSMURF-1 CCCAGAGACCTTAACAGTGTGAACT 16
Forward
hmrSMURF-1 TTGAGTTGGCACTGGTGATTCA 17
Reverse
hmrSMURF-2 TCTCGGTTGTGTTCGTCTTCTTT 18
Forward
hmrSMURF-2 GCCTATTCGGTCTCTGGACTGAA 19
Reverse
Osterix Forward TCAGACGCCCCGACCTT 20
Osterix Reverse ATTGGCAAGCAGTGGTCTAGAGA 21
siRNA treatment of cells: MSCs are transfected with Lipofectamine 2000
(Invitrogen) or
Oligofectainine (Invitrogen) transfection reagent and either irrelevant siRNA
or specific
siRNA sequences (see Table 2). Silencing of the genes and specificity is
confirmed by
real-time RT-PCR analysis of specific mRNA levels and Western analysis of
protein
levels.
Table 2
siRNA sequences
siRNA Sense Sequence (5'-3') SEQ ID NO:
SMURFI CCUUGCAAAGAAAGACUUCtt 22
SMURF2 GGUGGUGGUUGAUGGAUCUtt 23
Jab -1 GCUCAGAGUAUCGAUGAAAtt 24
LMP-1 AGACCUUCUACUCCAAGAAtt 25
Mechanistic studies using Smurfl siRNA
1. Interaction of LMP-1 with Smurfl not Smurf2: To confirm the specificity of
the
Smurfl/LMP-1 interactivity, siRNA designed to selectively target Smurfl mRNA
are
utilized. Conversely, selectively inhibiting Smurf2 levels and showing that
LMP-1
interaction with its candidate binding protein is not affected may also be
employed.
To this end, human MSCs are plated at 3 x 104 cells/well in 6-well plates,
grown
overnight, and treated witli an irrelevant siRNA or siRNA specific for Smurfl
or Smurf2.
Twenty-four hours after siRNA treatment, cells are treated with Ad5F35-LMP-1
(0, 5, 10
pfu/cell) or Ad5F35-GFP. Cells are harvested 2, 4, 8, 12, 24, or 48 hr later
and both
cytoplasmic and nuclear proteins subjected to immunoprecipitation using LMP-1
antibody
and Western analysis of co-localizing proteins using the WW domain antibody.
In
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addition cellular total RNA and protein fractions are harvested at the same
timepoints and
the levels of Smurfl and Smurf2 mRNA measured by real-time RT-PCR using
primers
specific for Smurfl or Smurf2. Reduction of protein levels by siRNA is
confirmed by
Western blots using newly acquired antibodies thought to be specific to Smurfl
and
Smurf2.
Expected Results: Reduced Smurfl levels are expected to reduce the amount of
the WW
domain immunoreactive binding protein observed by Western analysis after
immunoprecipitation using LMP- 1 antibody compared with controls that have
normal
levels of Smurfl. In contrast, reduced Smurf2 levels are expected to have no
effect on the
amount of WW domain iminunoreactive binding protein as we do not expect LMP-1
to
bind Smurf2. These results would confirm that the interaction of LMP-1 is
entirely with
Smurfl.
2. Effects of decreasing Smurfl on phosphoMlated Smadl and Smad5 -Nuclear
studies:
The hypothesis is that LMP-1 binding to Smurfl blocks Smurfl from binding to P-
Smadl
and P-Smad5, and, thus, reduces P-Smad proteasomal degradation. The expected
overall
effect is increased nuclear levels of P-Smadl and P-Smad5. While it is
possible that
LMP-1 may have several modes of action, it is believed the interaction with
Smurfl is
responsible for the osteoinductive properties of LMP-1. If the hypothesis is
correct,
decreasing Smurfl levels should produce the same effect as LMP-1 to increase
bone
formation in vitro and responsiveness to BMP-2.
To this end human MSCs are plated at 3 x 104 cells/well in 6-well plates,
grown overnight,
and treated with a control irrelevant siRNA or siRNA specific for Smurfl. BMP-
2 (100
ng/ml) is applied to some cultures when the siRNA is removed. Cells are
harvested 4, 8,
12, 24, 36, or 48 hr later and nuclear proteins are analyzed for the presence
of P-Smads by
Western blot. The effectiveness of the siRNA applied to reduce the RNA and
protein
levels is monitored as described herein. Another group of MSCs plated as above
are
grown in differentiation medium for 21 days and stained with Alizarin Red to
assess
matrix mineralization.
Expected Results: It is expected that there will be an increase in nuclear
levels of P-
Smadl and P-Smad5 in cells treated with siRNA to Smurfl as these proteins
should not be
targeted for proteasomal degradation in the absence of Smurfl. It is
hypothesized that the
preliminary results showing increased nuclear levels of Smads in response to
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overexpression of LMP- 1 (see example 3), was caused by LMP- 1 blocking
proteasomal
degradation of the P-Smads. Thus, it is expected that a reduction in Smurfl
levels to
mimic LMP-1 bloclcing of Smurfl, will result in targeting P-Smads for
degradation. It is
also expected that an increased responsiveness to BMP-2 in the presence of
Smurfl
siRNA as evidenced by increased levels of nuclear P-Smads and increased
extracellular
matrix mineralization will be observed. Consequently, siRNA to Smurfl would
represent
a therapeutic opportunity for matrix mineralization.
3. Effects of decreasing Smurfl on phosphorvlated Smadl and Smad5-C~toplasmic
studies: The hypothesis is that LMP-1 binding to Smurfl blocks Smurfl from
binding to
Smadl, reduces proteasomal degradation of Smadl, and increases the
responsiveness of
cells to activation of the BMP-2 pathway. The expected overall effect is
increased
cytoplasmic levels of Smadl. While it is possible that LMP=1 may have several
modes of
action, it is believe the interaction witli Smurfl is important for the
osteoinductive
properties of LMP-1. If the hypothesis is correct, decreasing Smurfl levels
should
produce a similar effect as LMP-1 in increasing responsiveness to BMP-2 as
evidenced by
the osteogenic response in vitro. To this end, human MSCs are plated at 3 x
104 cells/well
in 6-well plates, grown overnight, and treated with a control irrelevant siRNA
or siRNA
specific for Smurfl. Cells are harvested 4, 8, 12, 24, 36, or 48 hr later,
cytoplasmic
proteins are analyzed for the presence of total Smadl and phospho-Smadl by
Western blot
and ELISA. The effectiveness of the siRNA to reduce the RNA and protein levels
of
Smurfl are monitored as described herein. Another group of MSCs plated as
above are
grown in differentiation medium for 21 days and stained with Alizarin Red to
assess
matrix mineralization.
Expected Results: It is expected that treatment will result in increased
cytoplasmic Smadl
in cells treated with siRNA to Smurfl as this protein should not be targeted
for
proteasomal degradation in the absence of Smurfl. It has been shown, in other
examples
herein, that increased cytoplasmic levels of phospho-Smadl in response to
overexpression
of LMP-1 and BMP-2 treatment. This result is believed to have been caused by
LMP-1
blocking proteasomal degradation of Smadl. Thus, we expect reduction in Smurfl
levels
to mimic LMP-1 in blocking Smurfl targeting of Smadl for degradation.
4. Effects of decreasing Smurfl expression on BMP-2 responsiveness: As stated
above, it
is expected that directly reducing the level of Smurfl will mimic the effect
of LMP-1
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overexpression. To test this hypothesis human MSCs are plated at 3 x 10~
cells/well in 6-
well plates, grown overnight, and treated witli a control irrelevant siRNA or
siRNA
specific for Smurfl. BMP-2 is applied to some cultures when the siRNA is
removed. After
1, 2, 4, 8, 24, 36, 48, and 72 hrs cells are harvested for analysis of total
RNA or secreted,
cytoplasmic and nuclear proteins including D1x5, Smad6 and BMP-2 as previously
described. Analysis by ELISA allows more accurate quantitation than can be
achieved by
Western blotting.
Expected Results: While not wishing to be bound by theory, it is expected that
expression
of Dlx5, Smad6, and BMP-2 will be increased at both the protein and mRNA
levels when
the Smurfl level is reduced using specific siRNA. Cells having reduced Smurfl
levels
also are expected to show an increased responsiveness to BMP-2 with regard to
expression
of these genes. An increased levels of expression of these genes is
interpreted as a critical
step for the synergistic response to BMP-2 observed in MSCs with reduced
levels of
Smurfl.
Biotin transfer Assay for detection of LMP-1 interacting proteins: Sulfo-SBED
(Pierce), a
trifunctional cross-linking agent, contains three functional groups (a
photoactivatable aryl
azide, a sulfonated N-hydroxy succinimide active ester with a cleavable
disulfide group
and a biotin moiety) and is widely used to identify interacting proteins
(Neely KE, Hassan
AH, Brown CE, Howe L, and Workman JL. Mol.Cell Biol. 2002;22:1615-25). LMP-1
is
labeled using this reagent, incubated as bait with nuclear proteins and cross-
linked to
interacting proteins by UV (365 nm). Proteins that physically interact with
LMP- 1 retain
the biotin group when suspended in SDS-PAGE reducing buffer. Biotin-containing
target
proteins are separated using neutravadin beads, detected by Western blotting
with
neutraviddin-HRP and the signal is developed with chemiluminescent substrate.
Corresponding protein bands are in-gel digested with trypsin. Tryptic peptides
are
recovered, concentrated and their mass profile is analysed by MALDI-TOF at the
Emory
University Microchemical Facility.
LMP-lt: A 223aa osteoinductive truncated LMP-1 variant (missing LIM domains):
Although LMP-1 is a LIM domain protein, it has been shown that a truncated
223aa
variant of LMP-1 which lacks the LIM domains still makes bone in vitro and in
vivo (Liu
Y, Hair GA, Boden SD, Viggeswarapu M, and Titus L., J.Bone Min.Res.
2002;17:406-
14). If binding to Smurfl and activation of the BMP-2 signaling pathway is
critical for
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LMP-1 action and induction of bone foimation, then it would be expected that
the
truncated LMP-1 also binds Smurfl and mimics the downstream effects of the
full length
protein in the presence or absence of BMP-2. To this end truncated LMP-1 (LMP-
lt)
fusion protein containing a Protein Transduction Domain (PTD) that readily
enters cells
can be designed. The suitability of this PTD for use in these and subsequent
experiments
is validated by showing that the PTD-LMP-It fusion protein retains the same
ability of
overexpressed full length LMP- 1 to compete with WW domain antibody binding,
to
induce increased nuclear levels of P-Smadl and P-Smad5, and to increase
expression of
BMP/Smad regulated genes. Other data herein demonstrate the use of a full
length TAT-
LMP-1 fusion protein to enter cells readily and induce bone formation. The PTD
domain
proposed for use in these studies been shown to be a more effective in protein
transduction
than the TAT protein transduction domain used in earlier studies (Mi Z, Mai J,
Lu X, and
Robbins PD., Mol.Ther. 2000; 2:339-47).
Experimental Design: Initial studies will be required to determine a dose of
PTD-LMP-lt
that enhances the effect of 100 ng/ml BMP-2 on mineralization of MSCs. Human
MSCs
are plated at 3 x 104 cells/well in 6-well plates, grown overnight, and
treated with PTD-
LMP-lt (0.3-30 nM), BMP-2 (100 ng/ml) or both agents. Cells are grown in
mineralizing
medium as described in the Examples herein for overexpressed full length LMP-1
and the
mineral stained with Alizarin Red. Two doses that synergize with BMP-2 are
selected for
use in subsequent studies.
Following dose selection, human MSCs are plated at 3 x 104 cells/well in 6-
well
plates, grown overnight, and treated with nothing or 2 doses of PTD-LMP-lt,
BMP-1 (100
ng/ml) or both agents (control = PTD-(iGal). After 1, 2, 4, 8, 12, 24, 36 and
48 hrs, cells
are harvested and total RNA plus cytoplasmic and nuclear protein fractions are
prepared.
The nuclear protein fraction from the untreated sample is analyzed for the
ability of
purified LMP-1 protein to compete with Smurfl antibody binding to Smurfl on a
Western
blot. All protein samples are analyzed for the presence of phosphorylated
Smadl and
Smad5 by Western blot using previously described appropriate antibody. All RNA
samples are analyzed by real-time RT-PCR for mRNA levels of Dlx5, Smad6, and
BMP-
2. Similarly secreted, cytoplasmic and nuclear proteins are analyzed by ELISA
using
commercially available antibodies to Dlx5, Smad 6, and BMP-2.
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Expected Results: It is expected that LMP-lt protein will enhance the
osteoinductive
responsiveness of MSCs to BMP-2 as seen with overexpression of the full length
LMP-1
protein. It is also expected that truncated LMP-1 will bind to Smurfl and
prevent WW
domain antibody binding. In addition, it is expected that there will be
increased nuclear
Smad levels and activation of Smad-regulated genes to exactly mimic the
outcome
observed with overexpression of full length LMP-1 in the presence or absence
of BMP-2.
This result would support the hypothesis that the LIM domains are not required
for LMP-1
to induce bone formation or for LMP-1 to enhance responsiveness to BMP-2.
Smurfl -WW domain and LMP- 1 interaction assay: Nuclear proteins are separated
by
SDS-PAGE and blotted onto nitrocellulose membrane. Protein blots are bloclced
with 5%
milk protein and pre-incubated with purified LMP-1 protein (10 uM) or TBST
buffer.
Blots are incubated with Smurf antibody at 1:5000 dilution (Rabbit antibody
raised to
WW-domain peptide). After washes, blots are incubated with HRP-labeled Anti-
rabbit
second antibody. The washed blots are then incubated with ECL substrate
solution and the
membranes are exposed to X-ray film for signal detection.
Protein A-based immunoprecipitation assay: Protein A-agarose beads are
incubated with
LMP-1 antibody, washed 3 times, incubated with nuclear proteins, and washed
again to
remove unbound protein. Bound proteins are eluted by 2 washes in 0.1 M citric
acid, pH
2.7. The eluates are neutralized with Tris base and concentrated by centricon
tubes
(Ambicon) prior to SDS-PAGE and Western blotting.
Pulse labeling of Smads: Cells are incubated for 1 h in the presence of
(35S)Methionine,
washed extensively, incubated 30 min with Ad5F35LMP-1 (control = Ad5F35GFP, or
nothing), rinsed and returned to normal medium in the presence of BMP-2 (100
ng/nll). 1,
2, 3, 4 h after transduction, cells are harvested, nuclear fractions prepared,
and
radiolabeled P-Smadl or P-Smad5 immunoprecipitated as above.
Immunoprecipitates are
subjected to SDS-PAGE, visualized and quantitated by autoradiography.
Modified pulse chase assay for nuclear and c~toplasmic analyses of proteins.
Nuclear analysis: It has been shown herein that phosphorylated Smadl is
increased
in the nucleus within 4 hrs in response to LMP-1 overexpression and is further
increased at
8 hours. Possible explanations include: 1) increased gene expression of Smadl;
2)
increased phosphorylation of the cytoplasmic pool of unphosphorylated Smadl
with
subsequent translocation to the nucleus; and 3) reduced degradation of Smadl.
The short
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time required for the P-Smad increase to occur does not rule out increased
transcription
and translation of Smadl. However, the 4 hr time frame makes it less likely
that LMP-1
can be transcribed and translated and also cause increased subsequent
transcription and
translation of Smadl. The hypothesis for the increased nuclear level of P-
Smadl in
response to LMP overexpression is that LMP-1 binds Smurfl, preventing Smurfl
from
targeting P-Smadl for proteasomal degradation. Similar accumulation of P-Smad5
is
expected but has not yet been measured. The increased levels of P-Smadl and P-
Smad5
are expected to result in activation of BMP signaling, a requirement of which
is that they
localize in the nucleus in order to alter gene expression.
To this end, a modified pulse-chase experiment using 35 S-Methionine to label
newly synthesized Smad proteins and determine their rate of degradation in the
presence
or absence of overexpressed LMP-1 can be performed. This approach allows one
to
distinguish between Sinad synthesis in response to LMP-1 fiom P-Smad
accumulation in
response to LMP-1.
For the pulse-chase experiment, human MSCs are plated at 3 x 104 cells/well in
6-
well plates and grown overnight. 35S-Methionine (35S-Express, NEN) are applied
for 1 hr
to pulse label the pool of newly synthesized proteins. Upon removal of the
radiolabel and
subsequent washing, cells are incubated 30 min with Ad5F35-LMP-1 (0, 5, 10
pfu/cell),
Ad5F35-GFP, or nothing. BMP-2 (100 ng/ml) is applied to some cultures at the
end of the
transduction incubation to ensure phosphorylation of the Smadl. One, 2, 3, or
4 hr after
transduction, cells are harvested and nuclear protein fractions prepared.
Radiolabeled P-
Smadl or P-Smad5 are immunoprecipitated from the nuclear fraction using
specific
antibodies and analyzed by SDS-PAGE and fluorography.
Expected Results: Without wishing to be bound by theory, it is expected that
Smadl and
Smad5 proteins will be synthesized during the labeling period and that
treatment of cells
with BMP-2 will result in rapid phosphorylation of some of the newly
synthesized
molecules. The labeled P-Smadl and P-Smad5 would then bind Smad4 and move into
the
nucleus. LMP-1 and Smurfl are predominately found in the nucleus. As
overexpressed
LMP-1 protein is translated it is expected to move into the nucleus and bind
Smurfl. If
the hypothesis is correct, this event should block subsequent degradation of P-
Smadl and
P-Smad5 and result in a reduced rate of degradation compared with P-Smads in
cells not
overexpressing LMP-1.
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Cytoplasmic analysis: Human MSCs are plated at 3 x 104 cells/well in 6-well
plates, grown overnight and incubated 30 min with Ad5F35-LMP-1 (0, 5, 10
pfu/cell),
Ad5F35-GFP, or nothing. To determine stability of unphosphorylated Smadl, 35S-
Methionine (35S-Express, NEN) is applied for 30 min on the next day to pulse
label the
pool of newly synthesized proteins and the cells subsequently washed. Cells
are
incubated in chase medium containing cold methionine with or without
cycloheximide (10
ug/mL) for 2-3 hrs during which cells are harvested at various time points.
Cells are
harvested in the presence of protease inhibitors and cytoplasmic protein
fractions
prepared. Radiolabeled total Smadl are immunoprecipitated from the cytoplasmic
fraction
using specific antibody and analyzed by SDS-PAGE and auto-fluorography. To
measure
stability of phospho-Smadl, similar experiments are performed in control MSCs
or MSCs
overexpressing LMP-1, as above. Cells are labeled with 32P-orthophosphate for
2 hr in the
presence of the nuclear transport inhibitor, leptomycin B (25uM), to assure
that only decay
of the labeled phospho-Smad, not the net amount of decay and transport into
the nucleus is
measured. 32P is removed and cells are treated for 1 hr with BMP-2 (100
ng/ml).
Cytoplasmic fractions are prepared in the presence of protease and phosphatase
inhibitors
(to prevent loss of the 32P label). Radiolabeled 32P-Smadl are
immunoprecipitated from
cytoplasmic fractions and analyzed as above.
Expected Results: Without wishing to be bound by theory, in the absence of BMP-
2, it is
expect that unphosphorylated Smadl protein will be synthesized and incorporate
355-
Methionine during the labeling period. As overexpressed LMP-1 protein is
translated it is
expected to bind cytoplasmic Smurfl. If the hypothesis is correct, this event
should block
subsequent degradation of Smadl and result in a reduced rate of degradation of
Smadl
compared with cells not overexpressing LMP-1. It is expected that this block
to be true of
both.the unphosphorylated and phosphorylated form of Smad. To determine the
relative
susceptibility of unphosphorylated Smadl and phospho-Smadl to Smurfl-mediated
proteasomal degradation,comparisons of the kinetic curve for the decay of
unphosphorylated Smadl (35S-Methionine labeled) with that of hp ospho-Smadl
(32P-
orthophosphate labeled) can be made. It is expected that LMP-1 will impact
primarily the
unphosphorylated form.
Metabolic Pulse-Chase Analvsis for half life determination: To determine
metabolic half-
life of Smadl, MSCs are transfected with Ad5F35-LMP-1 or AD5F35-GFP and grown
for
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24 hr prior to metabolic labeling. Cells are washed in methionine-free medium
and
incubated for 15 min to deplete endogeiious methionine. Cells are then
incubated with the
pulse-labeling medium containing [35S]methionine (190 Ci/ml) for 30 min.
After washing
with chase medium (containing 150 mg/L unlabeled methionine). Cells are
incubated in
chase medium with or without cycloheximide (10 ug/mL) for 2-3 hr. Cells are
lysed in
buffer containing protease inhibitors (Sigma). Smadl is immunoprecipitated
from
cytoplasmic fractions using specific antibody and analyzed by SDS-PAGE and
auto-
fluorography.
To determine the half life of phospho-Smadl, cells are transfected with Ad5F35-
LMP- 1 or AD5F35-GFP and grown for 24hr prior to metabolic labeling. For in
vivo
[32P]orthophosphate labeling, cells are pre-incubated with phosphate-free
media for 1 h
and exposed to 1 mCi/ml [32P]orthophosphate for 2 h at 37 C. Leptomycin
B(lOng/ml) or
Ratjadone (lOng/ml) (Cal Biochem) is incubated with cells for 30 min to
inhibit nuclear
translocation of the labeled Smadl. Cells are then treated with BMP-2 (100
ng/ml) for 1
hr, lysed in buffer containing protease and phosphatase inhibitors, and the
cytoplasmic
fractions subjected to immunoprecipitation with phospho-Smadl specific
antibody.
Immunoprecipitates are visualized by SDS-PAGE followed by auto-fluorography.
Enzyme Linked Sorbent Assay (ELSA): Purified Smurfl is coated to individual
wells of
an Immulon 1B plate and the remaining surface blocked. Incubation of Smadl and
varying
concentrations of competing ligand (LMP-1) and/or vice versa are perfoimed
overnight at
4 C. Using appropriate primary and enzyme linked-secondary antibodies, optical
density
is monitored at specific wavelengths using the BioLumin 960 microtiter plate
reader or the
SpectraMax M2 microtiter plate reader. The assay can be adapted several ways
to suit
binding partner proteins. When biotin-labeled Smadl is assayed, streptavidin-
alkaline
phsophatase is used as the secondary reagent. After determining maximum
binding
between LMP-1/Smurfl and Smadl/Smurfl, mutual competition curves at various
concentrations will provide data for Scatchard plot analysis to obtain binding
affinity,
dissociation constant and number of binding sites.
Ubiquitin assay for determining extent of ubiquitination of Smadl/5 and/or LMP-
1: The
ELSA assay described above is modified for the ubiquitin assay as follows:
Microtiter
plates are coated with Smadl/5 or LMP-1 antibody and the remaining active
sites are
blocked by 1% BSA. Smadl/5 or LMP-1 are then captured by incubating nuclear
proteins
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in wells. Using specific enzyme/fluorescent-linlced Ubiquitin antibody and the
appropriate
substrate solution, the extent of ubiquitination can be assayed. The same
assay can be
adapted to study inhibitory effect of LMP-1 derived peptides on ubiquitination
of Smads
using an in vitro assay system with purified and commercially available
ubiquitination
assay reagents (Boston Biochemicals).
Transfection of MSCs for in vivo Bone Induction: MSCs are grown to confluence,
incubated with agents being tested, trypsinized, washed 2x with PBS, suspended
to 10-
20M/mL DMEM and 100 uL of the suspension applied to a sterile disc (2 x 5 mm)
of
bovine collagen. Implants are surgically placed subcutaneously on the chest of
4-6 wk
athymic rats (rnu /inu'). The animals are euthanized after 4 weeks; the
explants removed,
fixed in 70% ethanol, and analyzed by radiography and undecalcified histology.
Mechanistic Investigations of Ubiquitination
1. Ubiguitination of Smadl and Smad5.
It is hypothesized that the consequence of LMP-1 binding to Smurfl is a
reduction in the
number of ubiquitinated Smadl and Smad5 proteins. To this end, LMP-1 is
overexpressed in hMSCs, Smadl or Smad5 are captured on wells coated with the
specific
Smad antibody, and the level of ubiquitination of the Smads is quantitated by
ELISA.
Human MSCs are plated at 3 x 104 cells/well in 6-well plates, grown overnight
and treated
with Ad5F35-LMP-1 (0, 5, 10 pfu/cell), or Ad5F35GFP control plasmid. After 4,
8, 12,
24, 48, and 72 hr, cells are harvested and cytoplasmic and nuclear protein
fractions
prepared. The cell fractions are incubated with antibody to Smadl or Smad5 in
a 96 well
plate coated with the antibody. After washing to remove nuclear proteins not
associated
with Smadl or Smad5, fluorescence tagged-ubiquitin antibody is applied and the
fluorescence is detected using a Biolumin 960 microtiter plate reader. Use of
the
fluorescent tagged antibody increases sensitivity and allows quantitation of
the low level
of ubiquitin expected to be present in the sample. To validate the ELISA
results, another
aliquot of the cell fractions undergoes immunoprecipitation using anti-Smadl
or anti-
Smad5 antibody and the precipitated proteins are analyzed by Western blotting
using
antibody to ubiquitin as well as antibody to Smadl or Smad5.
Expected Results: It is expected that the Western analysis will show a smear
with botli the
ubiquitin and Smadl or Smad5 antibodies representing Smads conjugated with
ubiquitin
chains of various lengths. The Smadl antibody is expected to capture only
Smadl and the
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Smad5 antibody is expected to capture only Smad5. Free ubiquitin is removed
during the
ELISA washes. Additional ubiquitinated proteins are not expected to be
observed.
Compared with untreated or GFP treated cells, it is expected that lower levels
of Smad
ubiquitination will occur when LMP-1 is overexpressed due to interruption of
Smurfl
function. It should be noted that this method will not distinguish whether
decreased
ubiquitination is due to variation in the number of ubiquitin subunits linked
to Smads or
fewer Smad molecules that are conjugated to ubiquitin chains. Examination of
Western
blots performed for Smadl or Smad5 and ubiquitin may resolve this issue.
2. Effects of LMP-1 siRNA treatment on ubiguitination.
Upon demonstration that forced expression of LMP-1 can alter the level of
ubiquitinated
Smadl and Smad5, reduction of endogenous levels of LMP-1 using siRNA would be
expected to increase the rate of degradation of those Smad proteins.
Other examples herein have shown that siRNA targeting LMP- 1 are effective at
reducing
LMP-1 levels. Using these siRNA, studies investigating the relationship
between LMP-1
and ubiquitination of Smads can be performed. To show that application of
siRNA to
reduce the endogenous LMP-1 levels results in increased levels of
ubiquitinated Smadl/5
human MSCs are plated at 3 x 104 cells/well in 6-well plates, grown overnight,
and treated
with a control irrelevant siRNA or specific LMP-1 siRNA. Cells are harvested
4, 8, 12,
24, 48, or 72 hr later and both cytoplasmic and nuclear fractions prepared.
The cell
fractions are incubated with antibody to Smadl or Smad5 in a 96 well plate
coated with
the antibody. After washing to remove nuclear proteins not associated with
Smadl or
Smad5, fluorescence tagged-ubiquitin antibody are applied and the fluorescence
detected
as in otlier examples herein. This approach allows quantitation of the low
level of
ubiquitin that is expected to be present in the sample.
Expected Results: It is expected that reduced levels of endogenous LMP-1 will
increase
ubiquitination of both Smadl and Smad5. We would interpret this as being due
to greater
availability of Smurfl molecules for Smad binding.
3. Ubiquitination of Smadl by Smurfl
It is believed that LMP-1 prevents Smurfl interaction with Smadl, resulting in
decreased
ubiquitination of Smadl. Reduced ubiquitination is therefore expected to
result in reduced
proteasomal degradation of Smadl protein. To demonstrate that the interaction
of LMP-1
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with Smurfl inhibits ubiquitination of Smadl, in vitro ubiquitination assays
are
performed.
To this end, biotin labeled Smadl is prepared by the TNT-coupled reticulocyte
lysate
system (Promega) and impurities removed by capturing Smadl using neutravidin-
linked
resin. Smadl is mixed with a mixture of purified El and E2 ligases and
incubated with
the Smad ubiquitin E3 ligase, Smurfl, in the presence or absence of
recombinant LMP-1
protein. The reaction mixture also contains ubiquitin and the creatine kinase-
ATP
generating system. The reaction mixture is analyzed by SDS-PAGE and Western
blots
using specific antibody to ubiquitin or Strepavidin-HRP (to detect Biotin-
Smad1).
Expected Results: It is expected that there will exist a baseline
ubiquitination of Smad1 by
Smurfl under the conditions of the reaction. Further, it is expect that
addition of LMP-1
will inhibit this reaction. This will identify Smurfl as an E3 ligase with
which LMP-1
interacts to reduce Smurfl-induced ubiquitination of Smadl.
4. Effects of Smurfl siRNA treatment on ubiquitination.
It is possible that LMP-1 binding to Smurfl leads to its own ubiquitination
and subsequent
proteasomal degradation. However, there are examples of proteins that bind E3
ligases
without being degraded (Murillas R, Simms KS, Hatakeyama S, Weissman AM, and
K'uehn MR. J.Biol.Chem. 2002; 277:2897-907). Our empirical data that
relatively small
amounts of LMP-1 alone profoundly activate the BMP-2 pathway suggest that LMP-
1
itself may not be targeted for proteasomal degradation by Smurfl, but rather
occupy the
Smadl/5 binding site, preventing Smadl/5 targeting and degradation. To
determine
whether reducing the levels of Smurfl results in reduced LMP-1 ubiquitination
and
increased levels of endogenous LMP-1, human MSCs are plated at 3 x 104
cells/well in 6-
well plates, grown overnight, and Smurfl or control siRNA applied. Cells are
harvested 4,
8, 12, 24, 48, or 72 hr later and both cytoplasmic and nuclear fractions
prepared. The cell
fractions are incubated with antibody to LMP-1 in a 96 well plate coated with
the
antibody. After washing to remove nuclear proteins not associated with LMP-1,
fluorescence-tagged ubiquitin antibody is applied and fluorescence detected as
above. This
approach allows quantitation of the level of ubiquitin in the sample. In other
wells,
captured LMP-1 levels is quantitated by applying fluorescence tagged-LMP-1
antibody.
To validate that the ELISA measurements are specific to ubiquitinated LMP-1,
another
aliquot of the cell fractions undergoes immunoprecipitation using anti-LMP-1
antibody
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and the precipitated proteins are analyzed by Western blotting using antibody
to ubiquitin
as well as antibody to LMP-1.
Expected Results: It is not believed that LMP-1 undergoes ubiquitination by
Smurfl.
Thus, it is expected that decreased levels of Smurfl will not change the level
of
ubiquitinated LMP-1 compared with cells with normal levels of Smurfl. Further
it is
predicted that reduced levels of Smurfl will have no effect on the level of
LMP-1 protein
within cells. In concluding that Smurfl does not target LMP-1 for proteasomal
degradation, it is suggested that LMP-1 binds Smurfl at the site that also
binds Smadl or
Smad5, blocking Smad ubiquitination and targeting for proteasomal degradation.
Therefore, this interruption of Smurfl function would be a primary mechanism
by which
LMP-1 enhances signaling of the BMP-2 pathway.
5. Ubiquitination reaction in vitro:
Purified Smadl (100 ng) is buffer-exchanged to ubiquitination buffer (50 uM
Tris-HCl pH
7.8, 5 mM MgCIZ, 0.5 mM dithiothreitol (DTT), 2 mM NaF, and 3 M okadaic
acid).
Smadl is then combined with a mixture of purified El and E2 enzymes and
incubated
with Smurfl (E3 ligase) in the presence or absence of reconibinant LMP-1 or
LMP-2
protein. The reaction mixture also contains 2 mM ATP, ubiquitin (150 M),
ubiquitin
aldehyde (5 M), and creatine kinase-ATP generating system (Boston Biochem).
The
ubiquitin aldehyde is included to prevent hydrolysis of polyubiquitin chains.
The reaction
mixture (40 L) is incubated 4 hr at 37 C. Aliquots at various time points are
taken for
SDS-PAGE and western blotting using specific antibody for Smadl and/or
ubiquitin.
Mechanistic studies of the BMP-2 receptor BMPRIA)
1. Smurfl/Smad6 complexes
One hypothesis by which LMP-1 enhances cellular responsiveness to BMP-2 is by
increasing the BMPRIA (ALK3) level in the plasma membrane. Smurfl has been
shown
to regulate the proteasomal degradation of BMP-2 receptors through its
interaction with I-
Smads (Murakami, G., Watabe, T., Takaoka, K., Miyazono, K., and hnamura, T.,
Mol.
Biol. Cell. 2003, 14:2809-2817, 2003. It is suggested that I-Smad/Smurfl
complexes form
in the nucleus and translocate to the plasma membrane where I-Smad binding to
the
receptor occurs. Once bound to the receptor complex, Smurfl ubiquitinates the
I-Smad
and BMPRIA receptor, targeting them for proteasomal degradation (Ebisawa, T.,
Fukuchi,
M., Murakami, G., Chiba, T., Tanaka, K., Imamura, T., and Miyazono, K., J.
Biol. Chem.
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2001, 276:12477-12480; Izzi, L. and Attisano, L., Oncogene. 2004, 23:2071-
2078). To
test the hypothesis that LMP-1/Smurfl interaction prevents formation of
Smurfl/I-Smad
(Smad6) complexes, recombinant Smurfl, LMP-1 and biotin-Smad6 are purified and
tested in an ELSA binding competition assay. The assay is described fully
herein.
Briefly, wells of a 96-well plate are coated with Smurfl protein and
preincubated in the
presence or absence of different amounts of LMP-1. After washing, biotin-Smad6
is
incubated with the Smurfl complexes and binding detected using strepavadin-
alkaline
phosphatase. Absorbance is monitored at 405 nm using the SpectraMax M2
microplate
reader.
Expected Results: It is expected that, in the absence of LMP-1, Smurfl/Smad6
complexes
will be detected. In the presence of increasing concentrations of LMP-1,
decreasing
ainounts of Smurfl/Smad6 complex are expected to be formed until complete
inhibition of
Smad6 binding is achieved.
2. Smurf- 1 mediated export of Smad6 from the nucleus.
In the absence of receptor activation, I-Smads have been shown to primarily
reside in the
nucleus, but activation of the receptor results in translocation of I-Smads to
the cytoplasm
(Itoh, F., Asao, H., Sugamura, K., Heldin, C. H., ten Dijke, P., and Itoh, S.,
EMBO J.
2001, 20:4132-4142; Nakayama, T., Gardner, H., Berg, L. K., and Christian, J.
L., Genes
Cells, 1998, 3:387-394). It has been suggested that the export of Smad6 from
the nucleus
is facilitated by Smurfl (Izzi, L. and Attisano, L., Oncogene 2004, 23:2071-
2078); Suzuki,
C., Murakami, G., Fukuchi, M., Shimanulei, T., Shikauchi, Y., Imamura, T., and
Miyazono, K., J. Biol. Chem., 2002, 277:39919-39925). Since the hypothesis is
that LMP-
1 binds Smurfl in the same site as Smurfl binds Smads, it is expected that the
consequence of LMP-1/Smurfl interaction will be that less Smad6 moves to the
cytoplasmic compartment as a result of BMP-2 activation of its receptor. To
this end,
Flag-Smad6 is overexpressed to show that LMP-1 blocks the BMP-2-mediated
translocation of Smad6 into the cytoplasm. Overexpressed Flag-Smad6 is used
rather than
endogenous Smad6 because of the specificity of the Flag antibody and cross-
reactivity of
Smad6 antibodies with other Smads.
Experimental Design: Human MSCs are plated at 3 x 104 cells/well in 6-well
plates,
grown overnight, and transfected with 3 ug empty vector or plasmid containing
Flag-
Smad6. After the transfection, cells are allowed to recover for 1 hr prior to
transduction
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with Ad5F35-LMP-1 or Ad5F35-GFP (0, 5, 10 pfu/cell). Twenty-four hours after
the
transduction is complete, BMP-2 (100 ng/mL) is applied to the cells. Cells are
harvested
1, 2, 4, 8, and 12 hrs after BMP-2 treatment is initiated and cytoplasmic and
nuclear
fractions prepared. Fractions are subjected to SDS-PAGE and Western analysis
using
Flag specific antibody.
Expected Results: An increase in Smad6 in the cytoplasm upon BMP-2 receptor
activation is expected. However, in cells overexpressing LMP-1 this increase
is not
expected. These results would suggest LMP-1/Smurfl complexes form and
interrupt the
formation of Smurfl/Smad6 complexes that move Smad6 into the cytoplasm. It is
believed that disrupting movement of Smad6 from the nucleus to the cytoplasm
is a
critical first step that is required for LMP-1 disruption of Smurfl/Smad6-
mediated
proteasomal degradation of BMPRlA.
This finding would also be iinportant in elucidation of the overall mechanism
of
LMP-1 enhancement of responsiveness of MSCs to BMP-2. If LMP-1 blocks Smurfl
from interacting with Smad6, it would seem that the free Smad6 would still be
available in
the cytoplasm to oppose the other effects of LMP-1 on BMP action. However, it
is
expected that Smad6 is not exported from the nucleus to the cytoplasm in the
presence of
LMP- 1. Thus, it is important to demonstrate that Smad6 levels do not increase
in the
cytoplasm.
3. LMP-1 effects on the amount of BMPRlA in the plasma membrane of MSCs.
It is believed that the consequence of reducing Smurfl/Smad6 interaction will
be an
increase in the level of BMPRIA in the plasma membrane. Results disclosed
herein
suggest that BMP-2 increases BMPRIA in cells overexpressing LMP-1 more than in
control cells. This fmding would represent the sunl of BMP-2 action to
increase receptor
number and the action of LMP-1 to reduce proteasomal degradation of the
receptor. This
experiment allows for the elucidation of the effect of LMP-1 alone on this
increase in
receptor. Increasing receptor number is an extremely powerful mechanism for
increasing
the responsiveness of MSCs to BMP-2.
Experimental Design: Human MSCs are plated at 3 x 104 cells/well in 6-well
plates,
grown overnight, and treated with Ad5F35-LMP-1 or Ad5F35-GFP (0, 5, 10
pfu/cell).
After 1, 2, 4, 8, 12, 24 and 48 hrs, cells are harvested and plasma membrane
enriched
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fractions prepared. Fractions are subjected to SDS-PAGE and Western analysis
using
BMPRlA specific antibody.
Expected Results: Increased levels of BMPRlA in the plasma membranes of MSCs
overexpressing LMP-1 are expected. This increase would likely be due to
decreased
proteasomal degradation of the receptor by Smurfl/Smad6 complexes in the
presence of
increased LMP-1. Consequently, this finding would represent a second
significant
mechanism by which LMP-1 enhances the osteoinductive efficacy of BMP in MSCs.
4. Effects of LMP-1 on ubiquitinated BMPRIA.
It is hypothesized that the receptor increase shown above would be due to
decreased
ubiquitination and proteasomal degradation because of reduced interaction with
the
Smurfl/Smad6 complex. To test that hypothesis, the levels of ubiquitinated
BMPRIA are
measured. Past experience suggests that detection of ubiquitinated receptors
by ELISA
will be difficult because receptor proteins do not adhere to plastic
consistently. Thus,
Western blot analysis is used to determine changes in the level of
ubiquitination.
Experimental Design: Human MSCs are plated at 3 x 104 cells/well in 6-well
plates,
grown overnight and treated with Ad5F35-LMP-1 (0, 5, 10 pfu/cell), or
Ad5F35GFP
control plasmid. After 4, 8, 12, 24, and 48 hr, cells are harvested and plasma
membrane
enriched fractions prepared. BMPRIA are immunoprecipitated from the cell
fractions
using beads coated with specific antibody. The beads are washed and the
precipitated
proteins are analyzed by Western blotting using antibody to ubiquitin as well
as antibody
to BMPRIA.
Expected Results: Western analysis is expected to show a smear with both the
ubiquitin
and BMPRIA antibodies representing BMPRIA conjugated with ubiquitin chains of
various lengths. Additional ubiquitinated proteins are not expected. Compared
with
untreated or GFP treated cells, lower levels of BMPRIA ubiquitination are
expected when
LMP-1 is overexpressed due to interruption of Smurfl/Smad6 function. It should
be noted
that this method may not quantitatively distinguish whether decreased
ubiquitination is
due to variation in the number of ubiquitin subunits linked to BMPRIA or fewer
BMPRIA molecules that are conjugated to ubiquitin chains. Nevertheless, a
comparison
between treatments can be made, reflecting different amounts of ubiquitinated
BMPRIA
among various treatments.
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If ubiquitinated BMPRIA is not detectable it is lilcely to be caused by rapid
degradation of the ubiquitinated receptor. In that case, the experiment would
be performed
in the presence of the proteasome inhibitor, lactacystin, to accumulate
ubiquitinated
BMPRIA for detection in Western blots.
This method may not be sensitive enough to detect the low levels of
ubiquitinated
BMPRIA that are present, but it is felt that ELISAs are lilcely to be
inconsistent using
hydrophobic receptor proteins. Therefore, if there are not enough
ubiquitinated receptors,
Smurfl and Smad6 can be overexpressed in the presence or absence of LMP-1 and
the
experiment can be repeated. Alternatively, if there are too few receptors to
detect by
Western analysis, Flag-BMPRIA can be overexpressed the experiment repeated
using
Flag antibody to immunoprecipitate proteins for Western blot analysis.
Mechanistic investigations of LMP- 1 and Jab1
1. Effect on BMP-2 responsiveness on interrupting Jab 1 -mediated proteasomal
degradation of Smad4
One hypothesis is that LMP-1 enhances responsiveness of MSCs to BMP-2 by
binding to
Jab 1 and preventing Jab 1-induced proteasomal degradation of Smad4. Jab 1 is
an adapter
protein that targets Smad4 for ubiquitination and regulates the rate of Smad4
degradation,
an important role in controlling the cytoplasmic levels of Smad4. Smad4 is
required for
translocation of phospho-Smadl/5 into the nucleus and has been shown to be
critical in
regulating the responsiveness of cells to BMP-2 (Hata, A., Lagna, G.,
Massague, J., and
Hemmati-Brivanlou, A., Genes Dev. 1998, 12:186-197; Moren, A., Hellman, U.,
Inada,
Y., Imamura, T., Heldin, C. H., and Moustakas, A., J. Biol. Chem. 2003,
278:33571-
33582).
Other studies herein have demonstrated that LMP- 1 overexpression increases
Smad4 levels. Thus, reduction of endogenous levels of LMP-1 using siRNA would
be
expected to decrease Smad4. It is hypothesized that this decrease would be
caused by
increased proteasomal degradation of Smad4 and that it would not occur in the
presence of
the proteasomal inhibitor, lactacystin. More specific evidence that reducing
LMP-1
enhances proteasomal degradation of Smad4 requires demonstration of increased
ubiquitination of Smad4 protein when LMP-1 levels are reduced. To this end,
studies are
performed that demonstrate that reduction of endogenous LMP-1 by siRNA
increases
proteasomal degradation of Smad4.
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Experimental Design: Human MSCs are plated at 3 x 104 cells/well in 6-well
plates,
grown overnight, and treated with a control irrelevant siRNA or specific LMP-1
siRNA in
the presence or absence of lactacystin. Cells are harvested 4, 8, 12, 24, 48,
or 72 hr later
and total RNA or cytoplasmic protein fractions prepared. An aliquot of the
protein
fraction is analyzed by Western blot using antibodies to Smad4. Another
aliquot of
cytoplasmic proteins is incubated with antibody to Smad4 in a 96 well plate
coated with
the antibody. After washing to remove proteins not associated with Smad4,
fluorescence
tagged-ubiquitin antibody is applied and the fluorescence detected using a
SpectraMax M2
combined fluorescence and absorbance microplate reader. This approach allows
quantitation of the low level of ubiquitin that is expected to be present in
the sample.
Expected Results: An increase in ubiquitination would likely be due to greater
availability
of functional Jab 1 molecules for Smad4 binding. Talcen together these
findings would
suggest that LMP-1 could block proteasonial degradation of Smad4. A less
likely
alternative is that the LMP-1/Jabl interaction results in increased
sumoylation of Smad4
which is an alternative mechanism of enhanced stability of Smad4 (Lee, P. S.,
Chang, C.,
Liu, D., and Derynek, R., J. Biol. Chem. 2003, 278:27853-27863). Another
possibility is
that the interaction of Jab 1 with Smad7, a TGF-13 inhibitory Smad, could
augment the
BMP-2 responsiveness portion of these experiments by decreasing availability
of TGF-B
phospho-R-Smads (Kim, B. C., Lee, H. J., Park, S. H., Lee, S. R., Karpova, T.
S.,
McNally, J. G., Felici, A., Lee, D. K., and Kim, S. J., Mol. Cell Biol. 2004,
24:2251-
2262).
2. Investigation of the requirement of Jabl for LMP-1 induced effects.
Studies herein have demonstrated a direct interaction between Jab 1 and LMP- 1
and it is
well lcnown that ectopic expression of Jabl in certain cell lines can decrease
Smad4
steady-state levels (Wan, M., Cao, X., Wu, Y., Bai, S., Wu, L., Shi, X., Wang,
N., and
Cao, X., EMBO Rep. 2002, 3:171-176).
Experimental Design: To demonstrate that Jabl is required for LMP-1-induced
effects on
Smad4, human MSCs are plated at 3 x 104 cells/well in 6-well plates, grown
overnight,
and treated with a control irrelevant siRNA or specific Jab 1 siRNA in the
presence or
absence of rhBMP-2 (100 ng/ml). Cells are harvested 4, 8, 12, 24, 48, or 72 hr
later and
total RNA or cytoplasmic and nuclear protein fractions prepared. Cell
fractions are
analyzed by Western blots using specific antibodies to Smad4 and phospho-
Smadl. The
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efficacy of Jab 1 -specific siRNA to lower Jab 1 levels are assessed by real
time RT-PCR
and Western analysis using Jab-1 specific antibody.
Expected Results: It is expected that reducing endogenous Jabl will increase
cytoplasmic
Smad4 levels as has been shown herein with overexpression of LMP-1. Further,
in cells
treated with rhBMP-2, it is expected that reducing Jab 1 will increase nuclear
phospho-
Smadl, as a result of the increased pool of cytoplasmic Smad4 available to
translocate
activated R-Smads to the nucleus. These results would indicate that Jab 1
regulates
responsiveness of BMP-2 in MSCs by regulating Smad4 proteasomal degradation.
This
would complement worlc by others demonstrating this relationship in
transformed cell
lines or cancer cells (Wan, M., Cao, X., Wu, Y., Bai, S., Wu, L., Shi, X.,
Wang, N., and
Cao, X., EMBO Rep., 2002 3:171-176).
3. Effect of LMP-1 on Jab l -mediated decrease in Smad4
If decreasing endogenous Jabl as above increases Smad4, mimicking the effect
of LMP, it
would be expected that overexpression of Jabl would decrease cytoplasmic
Smad4.
Further, if LMP-1 exerts its effects on Smad4 levels by decreasing its Jab1-
induced
degradation, then overexpressed LMP-1 should reverse the effect of
overexpressing Jabl.
Experimental DesigYn: To test this hypothesis, human MSCs are plated at 3 x
104
cells/well in 6-well plates, grown overnight, and transfected with plasmid
containing Jabl
or empty vector. After a 1 hour recovery period cells are transduced with
Ad5F35-LMP-1
or Ad5F35-GFP (0, 5, 10, 25, 50, 100 pfu/cell). After 1, 2, 4, 8, 12, 24 and
48 hr, cells are
harvested and cytoplasmic fractions prepared. Cytoplasmic fractions are
analyzed for
Smad4 and Jabl by Western blot using specific Smad4 or Jabl antibody.
Expected Results: It is expected that increasing Jabl levels will result in
decreased
cytoplasmic Smad4 in the absence of overexpressed LMP-1. If the hypothesis
that LMP-1
increases Smad4 through its interaction with Jabl is correct, then
overexpression of LMP-
1 should overcome the effect of Jabl overexpression. These results would
confirm that
LMP-1 blocks Jabl-induced proteasomal degradation of Smad4. The LMP-1/Jabl
interaction may either prevent Jabl binding to Smad4 or block the ability of
Jabl to serve
as an adapter protein that increases targeting of Smad4 for ubiquitination and
subsequent
proteasomal degradation. This question could be resolved by competition
binding studies.
In either case, however, the overall effect of a LMP-1/Jabl interaction would
be to
increase Smad4 levels.
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If LMP-1 overexpression fails to overcome the effect of Jabl overexpression on
Smad4 levels there may be insufficient LMP-1 levels to overcome the high level
of Jabl
expression. To resolve this issue, the experiment would be repeated with
higher doses of
Ad5F35-LMP-I. While it has been demonstrated in other studies herein that the
doses of
Ad5F35-LMP-1 that are applied synergize with BMP-2 in MSCs, the doses to
overcome
the effect of Jabl overexpression have not been tested. If toxicity is
observed as a result of
"double overexpression," a recombinant LMP-1 fusion protein (TAT-LMP-1) could
be
used instead of Ad5F35-LMP-1 as demonstrated in other studies herein.
4. Enhancement of physiolo ig cally relevant markers of BMP-2 responsiveness
To investigate the combined effect on BMP-2 responsiveness, a series of
experiments are
performed to demonstrate the effect of LMP-1 overexpression. First, a Smad-
responsive
luciferase reporter construct (9xGCCG) is first used to determine the effect
of LMP-1
overexpression on luciferase activity. Next, expression of the BMP-2 regulated
gene,
Dlx5 is measured. Other studies disclosed herein suggest Dlx5 is important for
the
synergistic effects of LMP-1 and BMP-2 to induce the osteoblast phenotype in
MSCs.
Experimental Design: Human MSCs are plated at 3 x 104 cells/well in 6-well
plates,
grown overnight transfected with 3ug 9xGCCG/luciferase reporter construct and
treated
with Ad5F35-LMP-1 (0, 5, 10 pfu/cell), (control = Ad5F35GFP), BMP-2 (100
ng/mL), or
both. After 4, 8, 12, 24, 48, and 72 hr, cells are harvested and luciferase
activity
determined (Promega).
A second set of experiments is performed without transfection of luciferase
reporter in
which RNA is harvested at the same time points for detection of D1x5
expression using
real time RT-PCR.
Expected Results: LMP-1 overexpression is expected to greatly enhance the
reporter
construct activity in response to BMP-2. In addition, a smaller increase in
activity is
expected with either LMP-1 alone or BMP-2 alone. Since LMP-1 increases the
pool of R-
Smads and the Co-Smad, this could result in some increase in activated R-
Smad/CoSmad
complexes in the nucleus (as suggested by other data herein). However it is
expected that
the largest effect observed in enhancing BMP-2 efficacy would be due to the
effect of
LMP-1 to reduce degradation of many important proteins in the BMP-2 signaling
cascade.
LMP-1 is further expected to enhance the BMP-2 increase in the D1x5 gene
expression as suggested by results disclosed herein. Therefore, if the results
are as
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expected, one mechanisms by which LMP-1 increases responsiveness to BMP-2 may
likely involve reduced degradation of BMPRlA, R-Smads, and Co-Smad4.
Alkaline Phosphatase mRNA levels: Alkaline phosphatase activity is an early
marlcer of
the osteoblast phenotype. Human MSCs treated with Ad5/35LMP-1 (0, 1, 5, 10
pfu/cell)
with and without BMP-2 (100ng/ml) are harvested for RNA at day 8. Cells are
washed
with PBS once and cell lysates are prepared by sonication. RNA is isolated and
alkaline
phosphatase mRNA was quantified by RT-PCR with the alkaline phosphatase
specific
primers. Alkaline phosphatase activity is measured using p-nitrophenyl
phosphate as
substrate where nzyme activity is expressed as p-nitrophenol produced
(nmoles/ml). Data
is normalized to 18S.
Osterix Message in Human MSCs: Osterix is a novel zinc finger-containing
transcription
factor required for osteoblast differentiation and bone formation. Human MSCs
are
treated with Ad5/35LMP-1 with and without BMP-2 and harvested for RNA at day
8.
RNA is isolated and osterix mRNA is quantified by RT-PCR with the osterix
primers.
Data is normalized to 18S.
Bacterial Strains and Cloning of cDNAs in bacterial expression vectors: All
cloning
methods are performed according to standard protocols. Escherichia coli XL1
blue and BL
21-codon plus (DE3)-RP (Stratagene) hosts are maintained on LB agar plates and
grown at
37 C in the presence of ampicillin at 100 mg /L. LMP-1, LMP-lt, LMP-2, LMP-3,
Smadl
and Smad5 cDNAs were cloned into TAT-HA vector. LMP-1 mutants were generated
using the primers in Table 3.
Table 3
Primers for LMP-1 mutants
LMP primer name Primer Sequence (5'-3') SEQ ID
NO:
hLMPlMutant A forward cgcccccgccgcggacgcagcacggtacacctttg 26
primer cac
hLMPlMutant A reverse gtgcaaaggtgtaccgtgctgcgtcogcggcgggg 27
primer gcg
hLMPlMutant B forward ggcccggccctttggggcggcagcagcagctgaca 28
primer gcgccccgcaac
hLMPlMutant B reverse gttgcggggcgctgtcagctgctgctgccgcccca 29
primer aagggccgggcc
Smurfl cDNA is cloned into pTrcHis vector (Invitrogen). For generation of
SmurflAWW2 mutant, the primers in Table 4 are used. Mutagenesis is performed
with
Quikchange site-directed mutagenesis kit (Stratagene).
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Table 4
Primers for Smurfl mutants
Smurfl primer name Primer Sequenoe (5'-3') SEQ ID NO:
hSMURFIWW2 forward gtgtgaactgtgatgaacttaatcaccagtg 30
primer ccaactc
hSMt7RF1WW2 reverse gagttggcactggtgattaagttcatcacag 31
primer ttcacac
Expression and purification of recombinant proteins: Bacterial cultures are
grown at 37 C
until O.D. 600 reaches 0.8. IPTG is added to 200 M and the culture grown for
another 8
hr. Cells are harvested and pellets are suspended in ice-cold lysis buffer (20
mM
phosphate buffer, pH 7.0 containing 50 mM Tris-HCI, pH 7.5 and 0.5 M NaCI).
The
uniform cell suspension is sonicated (Sonicator, Model W-3 85, Heat systems-
Ultrasonics,
Inc.) using 4 x 15 sec bursts at minimuni power-output settings in ice with a
2 min interval
between each burst. The lysate is centrifuged at 10,000 g at 4 C and the
supernatant
applied to Sephacryl S-100/S-200 columns (HiPrep 16 X 60) using AKTA FPLC
system
with Unicorn 4.0 software (Amersham Pharmacia Biotech) at a flow rate of
lml/min.
Fractions (2-4 ml) are collected invnediately after the void volume (35 ml).
Aliquots, from
each fraction are assayed by slot blotting, SDS-PAGE and western blotting. The
fractions,
identified by westein blots are pooled, dialyzed against 20 mM phosphate
buffer, pH 7.5
containing NaCI (50 mM) and imidazole (20 mM) and applied to Ni++ affinity
resin
(Probond, Invitrogen) previously equilibrated with 4 x 10 ml of buffer. Non-
specific
proteins are washed off the column with 3 x 10 ml of 20 mM phosphate buffer,
pH 6.0
containing NaCI (50 mM) and imidazole (20 mM). Affinity-bound proteins are
eluted
using 3 x10 ml washes with 20 mM phosphate buffer, pH 4.0 containing NaC1(50
mM).
Fractions containing the desired protein are pooled (based on western blot)
and then
concentrated and de-salted using the centriprep devices (Amicon). Proteins are
quantitated
using BioRad protein assay reagent. The yield of recombinant protein is
routinely 0.5 to 1
mg of pure protein from every 2-liter culture.
Biotinylation of protein ligands: Purified protein ligands are prepared at lp
mg/ml in 50
mM sodium borate buffer, pH 8.5; 0.5 M NaCI. Various amounts of sulfo-NHS-
biotin
(100 mM stock in DMSO) are mixed with protein ligand to achieve a molar ratio
of Sulfo-
NHS-biotin/protein ligand of 10.0 in a 100 l reaction volume. After 2 hr on
ice with
occasional shaking the reaction is terminated with the addition of lysine to a
fmal
concentration of 20 mM. The unreacted free biotin is removed by gel filtration
and the
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concentrated labeled ligand stored at -200 C until use. Labeled or unlabeled
LMP-1,
Smadl, Smurfl and Smad5 are prepared by using TnT coupled in vitro
transcription/translation system (Promega).
Slot-blot assay: 20 ul of purified Smurfl (50 g/ml) is blotted onto
nitrocellulose in slot
blot wells and the wells are blocked with 0.5% Tween 20 in TBST for 30 min.
Biotinylated ligand (LMP-1, LMP-2, LMP-3, Smadl or Smad5) is mixed with
varying
concentrations of competing proteins or peptides and incubated in slot blot
wells with
Smurfl for 90 min. Wells are washed and the blots were blocked with TBST
containing
0.5% Tween 20. The blots are then incubated with HRP-labeled avidin for 1 hr.
After
washes the blots are incubated with ECL substrate solution and the membranes
are
exposed to X-ray film for signal detection.
Preparation of peptides having a protein transduction domain (PTD): Peptides
are
synthesized with a protein transduction domain (PTD) at the c-terminal end,
(rrqrrtsldmlcr,
herein incorporated as SEQ ID NO: 32) according to Mi Z, et. al.;Mol.Tlier=.
2000;2:339-
47.
Osteogenic differentiation of hMSCs: hMSCs at passage 4 are seeded at 3 x 104
cells/well
in a 6-well plate. The next day, the cells are infected with Ad35LMP-1 (1-10
pfu/cell) and
incubated with and without BMP-2 (100ng/ml). The medium is replaced every 3-4
days
and deposition of mineral observed after 2 weeks. To assess mineralization,
cultures are
washed with PBS and fixed in a solution of ice-cold 70% ethanol for 2-3 hours.
Cultures
are rinsed with water and stained for 10 minutes with lml of 40 mM Alizarin
red (pH 4.1).
Cultures are rinsed 2-3 times with PBS to reduce non-specific staining, air
dried and
photographed.
Ectopic Bone Formation Experiments
Ninety 4-6 week old male athymic rats (rnu /rnu , Harlan; housed in sterile
cages (2
rats/cage) and observed daily) are used to test LMP-1-mimetic compounds for
their ability
to enllance responsiveness of MSCs treated with sub-optimal doses of BMP-2
(2.5 ug/ml)
to induce bone formation in the rat model of ectopic bone formation. Athymic
rats are
used as they have no immune response to implanted foreign materials. After
approval by
the IACUC, 4-6 week old rats are anesthetized using 1.5% Isoflurane and the
chest
scrubbed with Chlorohexiderm spray. This method is consistent with the
recommendations
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of the Panel on Euthansia of the AVMA is selected because it is a rapid and
painless
method of euthanasia for rats.
Chest implants: The surgical area is draped using sterile drapes with a hole
cut to allow
access to the surgical area. Surgery is performed on a draped circulating
water heating
pad. A 1 cm skin incision is made in 4 locations on the chest, the skin
separated from the
muscle by blunt dissection and the discs loaded with BMP-2 + cells positioned
in separate
poclcets. Incisions are closed using resorbable sutures. Rats typcially
survive 4 weeks after
which they are euthanized using canister COZ consistent with the
recommendation of the
AVMA.
Controls: Because of the narrow efficacious dose range for LMP-l it is
necessary to test
multiple doses of each compound and to include positive control (i.e.TAT-LMP-
1) and
negative (i.e. cells alone or cells + BMP-2 only) controls. The positive
control chosen
should not induce bone formation when given at the chosen dose without BMP-2.
Each
dose or control is tested on multiple sites and there are multiple sites/rat.
If compounds
are postive, more testing can be performed.
Anesthesia: Anesthesia is adminsitered using 1.5% isoflurane prior to the
surgical
procedure. Prior to making incisions Bupivicaine 0.1-0.3 ml is given by
subcutaneous or
intramuscular injection around the surgical site. Buprenorphine (Buprenex)
(0.05mg/100g) is injected subcutaneously immediately post-operatively and
every 8 hrs
for 3 days post-op to relieve pain.
Ectopic experiments of Smurfl bindingpeptides that induce bone formation in
vivo
With the main hypothesis that interaction of LMP-1 with Smurfl results in
increased BMP-2 signaling activity and bone formation, it is expected that
peptides that
bind Smurfl and activate the BMP/Smad signaling pathway in vitro can also
induce bone
in vivo in the rat model of ectopic bone formation. Once peptides that induce
bone
formation have been identified, these peptides are tested in combination with
low doses of
BMP-2 to determine whether there are synergistic effects (as seen in vitro)
that might
lower the required dose of either agent. To evaluate potential synergy,
identified herein is
a dose of BMP-2 (2.5 ug) that induces bone formation in only 50% of the
implants and a
lower dose (1 ug) that consistently fails to induce bone formation in the rat
ectopic model.
These doses are known as the "suboptimal doses."
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Experimental Design: In these studies, multiple doses of each of several
peptides are
studied. The positive control are MSCs treated with Ad5F35-LMP-1 (5 pfu/cell).
MSCs
(1 -2M) are mixed with appropriate doses of peptides in a 100 uL total volume
and placed
on a collagen disc. The disc is implanted subcutaneously on the chest of
athymic rats and
explanted after 4 weeks. Bone formation is evaluated by palpation, x-ray and
semi-
quantitative scoring of non-decalcified histologic sections (Edwards JT,
Diegmann MH,
and Scarborough NL. Clin.Orthop. 1998;219-28). It has been previously found
that 1-2
million cells transduced with TAT-LMP can induce bone formation in this model,
although not consistently. Hence, these studies will attempt to achieve more
consistent in
vivo bone formation results which will be required for clinical translation.
In addition the
ability of the successful peptides to enhance the ability of suboptimal doses
of BMP-2 to
induce ectopic bone formation in this model may also be investigated.
Expected Results: It is expected that the peptides that are able to bind
Smurfl and
enhance BMP-2 signaling will also induce bone in implants containing
transduced MSCs
in the rat model of ectopic bone formation. Further it is predicted that MSCs
treated with
lower doses of the same peptides will improve the bone induction by suboptimal
doses of
BMP-2. Both findings are inteipreted as extremely promising strategies for
inducing bone
in a clinical setting and these strategies would be moved to the more
challenging rabbit
and non-human primate models.
Abbreviations: MSCs, mesenchymal stem cells; hMSC, human mesenchymal stem
cells;
P-Smad, phosphorylated Smad; R-Smad, receptor Smad: I-Smad, inhibitor Smad;
PTD,
protein transduction domain; siRNA, small interfering RNA; LMP, LIM
mineralization
protein; Smurf, Smad Ubiquitin Regulatory Factor; BMP-2, bone morphogenic
protein-2;
rhBMP-2, recombinant human bone morphogenic protein-2; Jab1, Jun Activation
Domain
Binding Protein; pfu, plaque forming units; MOI, multiplicity of infection.
As used herein, the when referring to treatment of cells, tissues or animals,
the terms
"BMP-2" and "rhBMP-2" are synonymous.
The present invention may be more fully understood by reference to the
following
non-limiting examples.
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EXAMPLES
Example 1: LMP-1 dramatically and syner isgtically increases the
responsiveness of
mesenchymal stem cells (MSCs) to BMP-2.
It is lcnown that LMP- 1 induces bone but produces very small amounts of BMPs.
In an effort to elucidate the mechanism behind this phenomena, the hypothesis
that LMP-1
increases the responsiveness of cells allowing them to respond to lower levels
of BMPs
with respect to osteoblastice differentiation, was tested.
MSC cultures transfected with the chimeric Ad5F35 vector overexpressiong LMP-1
were
treated with either LMP-1 (0, 1, 5, 10 pfu/cell) or BMP-2 (100 ng/mL) alone or
in
combination. Neither BMP-2 nor LMP-1 alone induce any bone nodule
mineralization in
human MSCs on day 21, but treating MSCs overexpressing LMP-1 (5-10 pfu/cell)
with
rhBMP-2 (100 ng/mL) induced dramatic bone nodule mineralization as shown by
alizarin
red staining. Thus, unexpectedly, concurrent exposure to LMP-1 enabled an
ineffective
dose of BMP-2 to facilitate bone formation suggesting that LMP-1 increases the
BMP
signaling pathway activity/sensitivity. The chimeric Ad5F35 vector, in which
the Ad5
fiber protein is replaced with the Ad35 fiber protein, was used to reduce
susceptibility to
neutralizing antibodies also tranduces human MSCs more effectively than Ad5
vectors.
(Yotnda P, Onishi H, Heslop HE et al. Gene Ther. 2001;8:930-7; Gugala Z,
Olmsted-
Davis EA, Gannon FH, Lindsey RW, and Davis AR.. Gene Ther. 2003;10:1289-96).
LMP-1 was also shown to increase the responsiveness of human MSCs to BMP-2
as evidenced by alkaline phosphatase mRNA levels and an increase in enzyme
activity.
Example 2: LMP Variant (LMP-2) does not induce nodule formation in rat
calvarial
osteoblast cultures.
Secondary rat calvarial osteoblasts do not spontaneously differentiate without
exposure to a stimulus such as glucocorticoid (GC). Cells were transfected
with plasmids
containing three LMP (hLMP-1; hLMP-2 and hLMP-3) variants and assessed for
multilayer mineralized nodule formation 14 days after treatment. Control cells
received
no treatment and all data were normalized to the control group. Overexpression
of hLMP-
1 resulted in nodule formation (225 nodules) comparable to that seen with GC
(275
nodules). hLMP-3, which is a truncated version of LMP-1 also induced nodule
formation
(290 nodules). However, hLMP-2 which lacks a 45aa region failed to induce
nodule
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fonnation (20 nodules), suggesting that the missing 45aa are required for
LMP's
osteoinductive properties.
Example 3: Detection of Smadl and phosphorylated Smadl (P-Smadl)
Measuring activation of the BMP signaling pathway requires the ability to
measure
Smadl and Smad5. SDS-PAGE separated cytoplasmic and nuclear protein blots were
probed with Smad-1 specific antibody. A comparison of cytoplasmic and nuclear
protein
extracts from untreated pleuripotent cells demonstrated that most of the Smadl
(54 kDa)
was detected in the cytoplasmic fraction.
For detection of P-Smadl (phosphorylated Smadl) in the nucleus, SDS-PAGE
blots of nuclear proteins were prepared from MSCs overexpressing LMP-1 at 4
and 8
hours following infection with Ad5F35-LMP-1. The blots were probed with
primary
antibody specific to P-Smadl. The binding of primary antibody was detected
using the
HRP-labeled second antibody after signal development by enhanced chemi-
luminescence
(ECL). A single band at the apparent size of 54 kDa showed an increase of
phosphorylated Smadl in the nuclear fraction as early as 4 hours in cells over-
expressing
LMP-1 compared to control MSCs not over-expressing LMP-1. Human MSCs treated
with BMP-2 also have increased levels of cytoplasmic phosphorylated Smadl in
the
presence of overexpressed LMP-1.
For detection of P-Smadl (phosphorylated Smadl), in the cytoplasm, SDS-PAGE
blots of cytoplasmic proteins were prepared from MSCs overexpressing LMP-1 at
4 and 8
hours following infection with LMP-1 delivered as Ad5F35-LMP-1. Cells were
treated
with rhBMP-2 at 100ng/mL or 200 ng/mL alone or in combination with LMP-1 (5
pfu/cell). rhBMP-2 treatment at either 100ng/mL or 200 ng/mL resulted in a
small
increase in cytoplasmic phosphorylated Smadl (64 kDa band). Addition of rhBMP-
2 (100
ng/mL) + LMP- 1 (5 pfu/cell) however, resulted in a significant increase (over
10 fold
increase) in the amount of phosphorylated Smadl protein which was not seen
with LMP-1
at 5pfu/cell alone. These data support the hypothesis that LMP-1 blocks the
Smurfl-
mediated degradation of unphosphorylated Smadl resulting in a larger pool of
Smadl and
therefore a greater amount of phosphorylated Smadl is produced for a given
amount of
BMP-2.
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Example 4: Investigation of increased Smadl induced by BMP and LMP-1.
To determine whether the increased level of Smadl protein might be due to
increased expression of Smadl, MSCs were treated with rhBMP-2 (100 ng/mL) or
Ad5F35-LMP-1 (5 pfu/cell). Control cells were untreated. After 4 or 8 hrs
total RNA was
harvested and Smadl mRNA was measured by real time RT-PCR. LMP-1 and BMP-2
each increased Smadl mRNA by 4 hours, 5 fold and 6.2 fold, respectively. At 8
hours
Smadl mRNA was increased by 4.5 fold and 2 fold for LMP-1 and BMP-2
respectively.
The data suggest that LMP-1 causes increased nuclear levels of P-Smadl via
increased
expression of Smadl mRNA, which results in increased Smadl protein that can be
phosphorylated by the BMP receptor kinase.
Given the short time frame of P-Smadl accumulation in the nucleus shown above
however, this mechanism is not the lilcely explanation for the.short term (4
hr) increase. It
is more likely a result of decreased degradation of P-Smads that is
responsible for the short
term regulatory mechanism.
Examnle 5: LMP-1 and BMP-2 syner istg ically increase expression of Smad-re
lated
genes.
In order to measure immediate downstream marlcers of BMP signaling, mRNA
levels of Dlx5, a gene lcnown to be induced by the BMP-Smads was measured.
MSCs
were untreated or treated with BMP-2 (100 ng/mL), Ad5F35-LMP-1 (5 pfu/cell),
or both.
After 24 hr total RNA was harvested and Dlx5 mRNA levels measured by real time
RT-
PCR. Data are expressed as fold change in Dlx5 mRNA. Untreated cells exhibited
1 fold
increase. Cells treated with BMP-2 demonstrated 6 fold increase, while cells
treated with
LMP-1 demonstrated 2 fold increase. A synergistic effect was seen on treatment
with both
LMP-1 (at 5 pfu/cell) and BMP-2 (at 100 ng/mL), showing 25 fold increase.
While protein levels were not measured in this study, methods are well known
and
include Western analysis and ELISA.
Example 6: Measurement of other Smad-regulated targets
Measurement of the mRNA and protein levels of Smad6, and BMP-2 can also be
performed. For these studies, human MSCs are plated at 3 x 10~ cells/well in 6-
well
plates, grown overnight, and treated with Ad5F35-LMP-1 (0, 5, 10 pfu/cell),
BMP-2 (100
ng/ml) or both agents in combination or in series. The control treatment would
be the
vector, Ad5F35-GFP. After 1, 2, 4, 8, 12, 18, 24, 36, 48, and 72 hrs, cells
are harvested
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for analysis of total RNA or cytoplasmic and nuclear protein. After reverse
transcription,
real-time PCR is performed using primers specific for human BMP-2 and Smad6.
Similarly, secreted, cytoplasmic and nuclear proteins can be analyzed by ELISA
using
commercially available antibodies to the proteins of interest, with alkaline
phosphatase
conjugated secondary antibodies. Specificity of the antibody can be determined
by
Western blot to assure that there is only one immunoreactive species.
Intracellular BMP-2 is expected to show several immunoreactive bands
corresponding to various cleavage steps during processing of pro-BMP-2. The
processed
BMP-2 protein will also likely be found largely in the medium, as it is
rapidly secreted.
Measurement of BMP-2 transcripts can be used for determining the relative
effect of
LMP-1 and BMP-2 on BMP-2 gene expression, as the majority of the protein
measured in
the medium from BMP-2 treated cells may be exogenously added as a treatment.
Example 7: LMP-1 is associated with an 85 kDa nuclear protein.
Recombinant LMP-1 was labeled with SBED-biotin transfer reagent and incubated
as bait with nuclear proteins. Biotin transfer to target proteins was
accomplished by photo-
activation and decoupling was performed by reduction of bound protein-
partners.
Enrichment of biotinylated proteins was performed using neutravidin-beads.
Biotinylated
proteins were separated by SDS-PAGE and detected on Western blots using HRP-
labeled
neutravidin and ECL. The corresponding coomassie stained bands were excised
for
tryptic digestion and MALDI-TOF analysis. LMP-1 was seen to associate with
three
protein bands; one 85 kDa band and two smaller bands. The smaller size bands
were
sequenced and represent cytoskeletal proteins likely involved with cellular
localization of
full length LMP-1 and not its osteoinductive properties. Based on partial
sequence
analysis, an 85 kDa band was investigated further (see below).
Example 8: Peptide mass profile analysis of the 85 kDa protein binding to LMP-
1.
The SDS-PAGE separated LMP-1 binding protein bands were in-gel digested by
5% (w/w) trypsin. Molecular mass of tryptic-peptides was obtained by MALDI-TOF
and
analyzed by Pepldent. The resulting 85 kDa candidate proteiu matches from the
data base
were analyzed. Sniurfl and its splice-isoform variant showed the best ranking
with peptide
mass profile. Although the tryptic digestion was done on doublet protein
bands, the unique
identity was still able to be determined due to identical peptide profiles of
both isoforms of
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Smurfl in tryptic digestion. By comparison the peptide profile obtained did
not match the
peptide mass profile of Smurf2.
Example 9: The 851cDa protein reacts with Smurfl/2 antibody.
Identity of the gel purified LMP-1 binding protein was verified by performing
a
Western blot using antibody that binds both Smurfl and Smurf2. The doublet
containing
two protein bands, one running at 86 kDa and the other running at 841cDa, were
both
immuno-reactive and probably represent two lcnown splice variants of Smurfl of
similar
molecular size rather than Smurfl and Smurf2. Based on molecular size, immuno-
reactivity with Smurfl/2 antibody in Western blots, and tryptic-peptide
profiles, the
putative LMP-binding protein was identified as Smurfl.
Example 10: Immunoprecipitation endogenous Smurfl and LMP-1 in MSCs.
Human MSCs were plated at 3 x 104 cells/well in 6-well plates, grown
overnight,
and treated with Ad5F35-LMP-1 (0, 5, 10 pfu/cell), or Ad5F35-GFP. After 2, 4,
8, 12, 24,
and 48 hrs, cells were harvested and nuclear proteins mixed with LMP-1 or
Smurfl
antibody and subjected to immunoprecipitation. Eluted proteins were separated
by SDS-
PAGE and Western analysis performed using LMP-1 or Smurfl antibodies.
Nuclear protein extracts of untreated MSCs were incubated with LMP-1 antibody
and immunoprecipitated using protein-A beads. The immunoprecipitated proteins
were
concentrated and analysed in Western blots with LMP-1 and Smurfl/2 antibody,
separately. Both endogenous LMP-1 and Smurfl were present in the complex
immunoprecipitated with the LMP-1 antibody from untreated MSCs. This
observation
was confirmed by detection of LMP-1 when Smurfl antibody was used for the
immunoprecipitation.
Other studies demonstrated interaction of these proteins in the nuclear
fraction. It is
unlcnown whether the Smurfl/LMP-1 interaction will also be observed in the
cytoplasm
but these studies can be performed according to the methods described herein.
Example 11: LMP-1 interacts with Smurfl via the Smurfl WW domain.
Blots of SDS-PAGE resolved nuclear proteins from MSCs showed a predominant
band at 85 kDa when probed with Smurfl WW-domain antibody in Western blots.
Pre-
incubation of LMP-1 (10 uM) with these blots inhibited Smurfl antibody
binding. Binding
competition between LMP-1 protein and WW-antibody towards the same target
sequence
suggests that the interaction with LMP-1 occurs at the WW-domain(s) of Smurfl.
The
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WW-domains are the region that enables Smurfl to bind to critical BMP
signaling Smadl
and Smad5 and target them for degradation. These data support the hypothesis
that LMP-
1 blocks Smurfl from interacting with and targeting Smadl and Smad5.
Not only did LMP-1 inhibit binding, truncated LMP variants also inhibit Smurf
WW-domain antibody binding to Smurfl. Blots of SDS-PAGE resolved nuclear
proteins
from untreated MSCs were incubated with the antibody specific for the Smurfl
WW-
domain. Both full length recombinant LMP-1 and recombinant LMP-lt (a C-
terminal
truncated version of LMP-1), when preincubated on the blots, were able to
prevent the
WW-domain antibody from binding to Smurfl. These data suggest that the LIM
domains
(located at the C-terminus of LMP-1) are not needed for the direct interaction
with
Smurfl.
Example 12: Characterization of the binding properties of Smurfl, LMP-1,
Smadl, and
Smad5 using purified proteins to confirm competitive binding of LMP-1.
Recombinant Smadl and Smad5 are prepared by bacterial expression and
purification. Smads and LMP-1 are then fluorescently labeled. The Enzyme
Linked
Sorbent Assay (ELSA) described herein is performed in microtiter plates.
Binding curves
for evaluating the competitive binding to Smurfl of fluorescently labeled LMP-
1 with
unlabeled Smadl or Smad5 are then obtained. Conversely, the competitive
binding of
fluorescently labeled Smads with unlabeled LMP-1 may also be measured.
Fluorescence is
monitored using a Biolumin 960 combined fluorescence and absorbance microplate
reader. Scatchard analyses, well known in the art, can serve to assess the
binding affinity,
dissociation constants, number of binding sites, and stoichiometry for each
protein
involved in the interaction.
The results can then predict competition between LMP-1 and Smads for binding
Snlurfl at
one or two sites. Competitive binding at two sites might occur, as Smurfl
contains two
WW domains and it is uncertain whether all or some of the proteins bind one or
both sites.
If Smad5 does not accumulate in the nucleus it might not be expected to
compete with
LMP-1 for Smurfl binding because it might be assumed that LMP-1 binds Smurfl
at a
different WW domain than Smad5.
Example 13: Identification of two regions within LMP-1 with high affmity for
Smurfl
WW-domains
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On finding that fiill length LMP-1 binds Smurfl WW domains, comparative
sequence analysis of three LMP-1 variants was performed and identified a 45
amino acid
"osteoinductive region" that is present in the two LMP-1 isofonns that induce
bone
formation, but is not present in a third isoform that fails to induce bone.
These LMP
variants (LMP-1, LMPlt and LMP-3) were created which contain a unique peptide
sequence, (AADPPRYTFAPSVSLNKTARPFGAPPPADSAPQQNG; SEQ ID NO: 33)
within a larger osteoinductive region which includes two Smurfl WW-domain
interaction
sites (WW interacting site A comprising the sequence ADPPRYTFAP; herein
referred to
as SEQ ID NO: 34; and WW interacting site B comprising the sequence
GAPPPADSAP;
herein referred to as SEQ ID NO: 35). The WW domain interacting sites are
absent in a
non-osteogenic LMP variant (LMP-2). The 45 ainino acid osteoinductive region
appears
to be critical for the bone-forming activing of LMP. Two LMP variants (LMP lt
and
LMP-3) are truncated at the carboxy-terminus, and do not contain the LIM
domains, but
do induce bone formation. Thus, it is the 45 amino acid osteoinductive region
and not the
LIM domains that are required for bone formation (Liu, Y, et. al.; J. Bone
Min. Res.,
2002:17:406-414).
Slot blots prepared with recombinant Smurfl and hybridized with biotin-labeled
LMP variants demonstrated that only the LMP variants containing the WW-domain
interaction sites (LMP-1, LMP-lt and LMP-3) were able to bind to Smurfl.
To determine which of the two WW-domain interaction sites were required for
the
binding of LMP with Smurfl, two mutant LMP-1 proteins that are mutated in
either WW
interaction site A (Prolines at positions 100 and 101 of the 457 amino acid
parent protein
being converted to Alanine, herein incorporated as SEQ ID NO: 36; LMP-1AWWA)
or
site B (Prolines at positions 122, 123 and 124 of the 457 amino acid parent
protein being
converted to Alanine, herein incorporated as SEQ ID NO: 37 ; LMP-10WWB) were
prepared. The mutations remove proline residues that are required for
interaction with the
Smurfl WW domain and disrupt the PY motif in each of the two sites.
LMP-1AWWA-SEQ ID NO: 36;
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 36
<211> LENGTH: 457
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 36
Met Asp Ser Phe Lys Val Val Leu Glu Gly Pro Ala Pro Trp Gly Phe
1 5 10 15
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Arg Leu Gin Gly Gly Lys Asp Phe Asn Val Pro Leu Ser Ile Ser Arg
20 25 30
Leu Thr Pro Gly Gly Lys Ala Ala Gln Ala Gly Val Ala Val Gly Asp
35 40 45
Trp Val Leu Ser Ile Asp Gly Glu Asn Ala Gly Ser Leu Thr His Ile
50 55 60
Glu Ala Gln Asn Lys Ile Arg Ala Cys Gly Glu Arg Leu Ser Leu Gly
65 70 75 80
Leu Ser Arg Ala Gln Pro Val Gln Ser Lys Pro Gln Lys Ala Ser Ala
85 90 95
Pro Ala Ala Asp Ala Ala Arg Tyr Thr Phe Ala Pro Ser Val Ser Leu
100 105 110
Asn Lys Thr Ala Arg Pro Phe Gly Ala Pro Pro Pro Ala Asp Ser Ala
115 120 125
Pro Gln Gln Asn Gly Gln Pro Leu Arg Pro Leu Val Pro Asp Ala Ser
130 135 140
Lys Gln Arg Leu Met Glu Asn Thr Glu Asp Trp Arg Pro Arg Pro G1y
145 150 155 160
Thr Gly Gln Ser Arg Ser Phe Arg Ile Leu Ala His Leu Thr Gly Thr
165 170 175
Glu Phe Met Gln Asp Pro Asp Glu Glu His Leu Lys Lys Ser Ser Gln
180 185 190
Val Pro Arg Thr Glu Ala Pro Ala Pro Ala Ser Ser Thr Pro Gln Glu
195 200 205
Pro Trp Pro Gly Pro Thr Ala Pro Ser Pro Thr Ser Arg Pro Pro Trp
210 215 220
Ala Val Asp Pro Ala Phe Ala Glu Arg Tyr Ala Pro Asp Lys Thr Ser
225 230 235 240
Thr Val Leu Thr Arg His Ser Gln Pro Ala Thr Pro Thr Pro Leu Gln
245 250 255
Ser Arg Thr Ser Ile Val Gln Ala Ala Ala Gly Gly Val Pro Gly Gly
260 265 270
Gly Ser Asn Asn Gly Lys Thr Pro Val Cys His Gln Cys His Lys Val
275 280 285
Ile Arg Gly Arg Tyr Leu Val Ala Leu Gly His Ala Tyr His Pro Glu
290 295 300
Glu Phe Val Cys Ser Gln Cys Gly Lys Val Leu Glu Glu Gly Gly Phe
305 310 315 320
Phe Glu Glu Lys Gly Ala Ile Phe Cys Pro Pro Cys Tyr Asp Val Arg
325 330 335
Tyr Ala Pro Ser Cys Ala Lys Cys Lys Lys Lys Ile Thr Gly Glu Ile
340 345 350
Met His Ala Leu Lys Met Thr Trp His Val His Cys Phe Thr Cys Ala
355 360 365
Ala Cys Lys Thr Pro Ile Arg Asn Arg Ala Phe Tyr Met Glu Glu Gly
370 375 380
Val Pro Tyr Cys Glu Arg Asp Tyr Glu Lys Met Phe Gly Thr Lys Cys
385 390 395 400
His Gly Cys Asp Phe Lys Ile Asp Ala Gly Asp Arg Phe Leu Glu Ala
405 410 415
Leu Gly Phe Ser Trp His Asp Thr Cys Phe Val Cys Ala Ile Cys Gln
420 425 430
Ile Asn Leu Glu Gly Lys Thr Phe Tyr Ser Lys Lys Asp Arg Pro Leu
435 440 445
Cys Lys Ser His Ala Phe Ser His Val
450 455
LMP-1dWWB- SEQ ID NO: 37
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<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 37
<211> LENGTH: 457
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 37
Met Asp Ser Phe Lys Val Val Leu Glu Gly Pro Ala Pro Trp Gly Phe
1 5 10 15
Arg Leu Gln Gly Gly Lys Asp Phe Asn Va1 Pro Leu Ser I1e Ser Arg
20 25 30
Leu Thr Pro Gly Gly Lys Ala Ala Gln Ala Gly Val Ala Val Gly Asp
35 40 45
Trp Val Leu Ser Ile Asp Gly Glu Asn Ala Gly Ser Leu Thr His Ile
50 55 60
Glu Ala Gln Asn Lys Ile Arg Ala Cys Gly Glu Arg Leu Ser Leu Gly
65 70 75 80
Leu Ser Arg Ala Gln Pro Val Gln Ser Lys Pro Gln Lys Ala Ser Ala
85 90 95
Pro Ala Ala Asp Pro Pro Arg Tyr Thr Phe Ala Pro Ser Val Ser Leu
100 105 110
Asn Lys Thr Ala Arg Pro Phe Gly Ala Ala Ala Ala Ala Asp Ser Ala
115 120 125
Pro Gln Gln Asn Gly Gln Pro Leu Arg Pro Leu Val Pro Asp Ala Ser
130 135 140
Lys Gln Arg Leu Met Glu Asn Thr Glu Asp Trp Arg Pro Arg Pro Gly
145 150 155 160
Thr Gly Gln Ser Arg Ser Phe Arg Ile Leu Ala His Leu Thr Gly Thr
165 170 175
Glu Phe Met Gln Asp Pro Asp Glu Glu His Leu Lys Lys Ser Ser Gln
180 185 190
Val Pro Arg Thr Glu Ala Pro Ala Pro Ala Ser Ser Thr Pro Gln Glu
195 200 205
Pro Trp Pro Gly Pro Thr Ala Pro Ser Pro Thr Ser Arg Pro Pro Trp
210 215 220
Ala Val Asp Pro Ala Phe Ala Glu Arg Tyr Ala Pro Asp Lys Thr Ser
225 230 235 240
Thr Val Leu Thr Arg His Ser Gln Pro Ala Thr Pro Thr Pro Leu Gln
245 250 255
Ser Arg Thr Ser Ile Val Gln Ala Ala Ala Gly Gly Val Pro Gly Gly
260 265 270
Gly Ser Asn Asn Gly Lys Thr Pro Val Cys His Gln Cys His Lys Val
275 280 285
Ile Arg Gly Arg Tyr Leu Val Ala Leu Gly His Ala Tyr His Pro Glu
290 295 300
Glu Phe Val Cys Ser Gln Cys Gly Lys Val Leu Glu Glu Gly Gly Phe
305 310 315 320
Phe Glu Glu Lys Gly Ala Ile Phe Cys Pro Pro Cys Tyr Asp Val Arg
325 330 335
Tyr Ala Pro Ser Cys Ala Lys Cys Lys Lys Lys Ile Thr Gly Glu I1e
340 345 350
Met His Ala Leu Lys Met Thr Trp His Val His Cys Phe Thr Cys Ala
355 360 365
Ala Cys Lys Thr Pro Ile Arg Asn Arg Ala Phe Tyr Met Glu Glu Gly
370 375 380
Val Pro Tyr Cys Glu Arg Asp Tyr Glu Lys Met Phe Gly Thr Lys Cys
385 390 395 400
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His Gly Cys Asp Phe Lys Ile Asp Ala Gly Asp Arg Phe Leu Glu Ala
405 410 415
Leu Gly Phe Ser Trp His Asp Thr Cys Phe Val Cys Ala I1e Cys Gln
420 425 430
Ile Asn Leu Glu Gly Lys Thr Phe Tyr Ser Lys Lys Asp Arg Pro Leu
435 440 445
Cys Lys Ser His Ala Phe Ser His Val
450 455
Recombinant Smurfl was run on SDS-PAGE, transferred to membrane, and
incubated with buffer, LMP-1 protein, LMP-2 protein (negative control, no
osteoinductive
region), LMP-lAWWA or LMP-1AWWB. After washing, Smurfl WW domain antibody
was applied and then detected with HRP-labeled secondary antibody.
Slot blots of competitive binding assays were analyzed as before and it was
found that
only WW-domain interaction site B was required for the interaction with
Smurfl. The
specificity of these interaction sites was confirmed by demonstrating that LMP-
1 was no
longer able to bind to a mutant form of Smurfl which had its WW2 domain
deleted
(SmurflAWW2).
Example 14: Peptide mimics of LMP-1
To design and test molecules that mimic the LMP- 1 interaction with Smurfl,
known crystallography data and homology modeling of related homologues similar
to the
WW-2 domain in Smurfl and the two potential interacting motifs in LMP-1 (WW-A
and
WW-B) as defmed by iSPOT were used (Brannetti, B. and Helmer-Citterich, M.,
Nucleic
Acids Res. 2003; 31:3709-3711). The MODELLER program was used to assign
structure
to the two interacting elements and to model templates. The designed Smurfl WW-
2
template showed a close (up to 70%) match to the coordinates of
crystallographic data
available for homologous WW domains from the protein data bank (PDB) (Fiser,
A. and
Sali, A., Methods Enzymol. 2003; 374:461-491). DOCKING and SSA (Surface
Solvant
Accessability) programs were then used to defme the key residues in each
binding partner
(Morris, G. M., Goodsell, D. S., Huey, R., and Olson, A. J., J. Comput. Aided
Mol.Des.,
1996, 10:293-304). A directory of commercially available low molecular weight
and cell
penetrable chemicals were screened using the LUDI program with both
complimentary
screening (WW-2) and analogue screening (WW-A or WW-B) to identify candidate
compounds (Honma, T., Med. Res. Rev. 2003, 23:606-632).We used computational
mutagenesis to eliminate non-specific compounds and cross-matched the
complimentary
and analogue compound lists to arrive at 75 candidate compounds for each WW
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interacting domain of LMP-1. The specificity of the LMP motif that interacts
with Smurfl
was confirmed and it was determined that the activity of the full length LMP
protein could
be replicated by a small peptide. We synthesized small peptides comprising
various
portions of the osteoinductive region,
(KPQKASAPAADPPRYTFAPSVSLNKTARPFGAPPPADSAPQQNGQPLRPLVPDA
SKQRLM; herein referred to as SEQ ID NO: 38, BOLDED fragments represent the WW-
domain interacting sites, with ADPPRYTFAP representing Site A, SEQ ID NO: 34
and
GAPPPADSAP representing Site B, SEQ ID NO: 35) containing one, two, or none of
the
putative LMP WW-domain interacting sites. The Table below lists the sequences
of
Peptide 1, 3, 5, and 7 that were designed to the osteogenic region of LMP-1.
These
peptides were chenzically synthesized, HPLC purified for in vivo bone
formation studies.
Only peptide 7 is acetylated at N-terminus, and was therefore not expected to
alter the
peptide function.
Table 5
Peptide mimics
Peptid SEQ ID Amino Acid Sequence
# NO:
1 39 HzN-APSVSLNKTARPFGAPPPADSAGGRRQRRTSKLMKR-CONH,
3 0 H2N-KPQKASAPAADPPRYTFAPSVSGGRRQRRTSKL -CONH2
5 1 HzN-
SAPAADPPRYTFAPSVSLNKTARPFGAPPPADSAPQQNGGGRRQ
RRTSKLMKR-CONH2
7 2 cetyl-
GAPPPADSAPQQNGQPLRPLVPDASKQRLMGGRRQRRg KLMKR-
H2
Competition studies (across the range of 0.OOluM to 1.0uM, and including
0.001,
0.01, 0.1 and luM) of the peptides revealed that the two peptides that
contained intact
30 WW-domain interacting Site B were able to compete with full length LMP-1 in
a dose
dependent manner as well as with Smadl and Smad5 for binding with Smurfl at
concentrations of 1.0uM and lOuM.
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This result is consistent with the mutational analysis above establishing Site
B as
the more critical site in LMP. To determine if the ability of the small LMP-
derived
peptides to competitively bind Smurfl has physiologic significance, the
peptides were
linlced to a protein transduction domain (PTD) that facilitates entry into
cells (Sequence:
RRQRRTSKLMKR, herein incorporated as SEQ ID NO: 32). We found that only
peptide
7 had the ability to induce bone formation in a rat ectopic bone formation
assay.
Example 15: Osteoinductive region fusion peptides
To verify that the 36 amino acid sequence
(AADPPRYTFAPSVSLNKTARPFGAPPPADSAPQQNG; SEQ ID NO: 33) is
responsible for the LMP- 1 interaction with Smurfl, an "osteoinductive region-
fusion
peptide" (PTD-LMP-1/OR) containing a PTD that readily enters cells
(RRQRRTSKLMKR, herein incorporated as SEQ ID NO: 32) was designed. Initial
studies were performed to determine a dose of PTD-LMP-1/OR that enhanced the
effect of
100 ng/ml BMP-2 to mineralize hMSCs. Once two effective doses, 15nM and
17.5nM,
were established, hMSCs were plated at 3 x 104 cells/well in 6-well plates,
grown
overnight, and treated with PTD-LMP-1/OR, BMP-1 (100 ng/ml) or both agents.
The
control was PTD-PGa1. After 1, 2, 4, 8, 12, 24, 36, 48, and 72 hrs, cells were
harvested
and total RNA plus cytoplasmic and nuclear protein fractions prepared. The
nuclear
protein fraction from the untreated sample was analyzed for the ability of
purified PTD-
LMP-1/OR to compete with Smurfl antibody binding to Smurfl on a Westein blot.
All
protein samples were analyzed for the presence of P-Smadl and P-Smad5 by
Western blot
using appropriate antibody. All RNA samples were analyzed by real-time RT-PCR
for
mRNA levels of Dlx5, Smad6, and BMP-2. Similarly, protein fractions were
analyzed by
ELISA using connnercially available antibodies to D1x5, Smad6, and BMP-2.
The results illustrate that the 36aa peptide has all the ability of full
length LMP-1
to compete with WW domain antibody binding, to induce increased nuclear levels
of P-
Smadl and P-Smad5, and to increase expression of BMP/Smad regulated genes. It
is
noted that PTD-fusion peptide derivatives of one of the osteoinductive LMP
isoforms
(LMP-3) have been shown by others to induce bone formation (Pola E, Gao W,
Zhou Y et
al. Gene Ther. 2004;11:683-93).
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Example 16: Full length and truncated LMP-1 variants fonn bone in ectopic and
orthotopic models in vivo.
Bone marrow cells and leukocytes were transfected with the cDNA of full length
or truncated LMP-1 lacking LIM domains. Collagen discs containing the
engineered cells
were implanted subcutaneously into athymic rats. Histology of the implants
showed
ectopic and orthotopic bone formation. New bone trabeculae lined with
osteoblasts were
seen in the implants containing cells overexpressing truncated LMP-1 (LMP-lt)
and the
absence of new bone in implants containing control cells. This demonstrates
that
genetically engineered cells can form bone in vivo and confirms that the LIM
domains of
LMP-1 are not needed for bone formation.
Example 17: Bone marrow cells transfected with LMP-1 induce bone in the rat
model of
spine fusion
Spine fusion is a more challenging bone formation model than an ectopic site.
Rats were implanted with carrier soaked with bone marrow cells which had been
transfected with the cDNA of LMP- 1 in an active or inactive form. Lateral
radiogroaphy
of the animals at 4 weeks indicated solid spine fusion when the marrow cells
expressed
active LMP-1 protein but no evidence of bone formation was seen when the
marrow cells
had been transfected with an inactive form of LMP-1. These data indicate that
genetically
engineered bone marrow cells can induce rat spine fusion.
Example 18: A TAT-LMP-1 fusion protein enters the nucleus of cells and forms
bone in
vivo
Fusion proteins that contain an 11 aa sequence, found in the HIV protein Tat,
are
kiiown to readily enter cells within minutes (Nagahara H, Vocero-Alebani AM,
Snyder EL
et al. Nat.Med.1998; 4:1449-52). This method of protein delivery is superior
to adenoviral
gene transfer because it avoids immune response issues. To confirm that a LMP
fusion
protein would enter cells, a FITC labeled LMP-1 Tat fusion protein (TAT-LMP-1)
was
synthesized and fluroescence monitored for localization in the MSCs when
treated at
doses of 10, 25 and l00nM. The fusion protein readily entered nucleated blood
cells in a
dose dependent manner with 2, 25 and 90% of the cells positive for the label,
respectively.
Western blots from another experiment confirmed the ability of TAT-LMP-1 to
enter the
nucleus. X-rays showed good bone formed on collagen discs in rats implanted
with cells
treated for 30 min with TAT-LMP-1. The bone induction with TAT-LMP-1 was not
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always consistent between experiments and thus this was not an optimal "stand
alone"
strategy for bone formation. A better understanding of the mechanism of action
of LMP- 1
and a designer small molecule may allow it to perform more consistently as a
"stand
alone" initiator of bone formation or as a means of increasing the
responsiveness to BMPs.
Example 19: LMP-1 siRNA
In an effort to selectively inhibit expression of specific genes in order to
elucidate
aspects of the proposed LMP-1 mechanism of action, siRNA were designed to
target
LMP-1. Use of small inhibitory RNAs (siRNAs) to destroy specific mRNAs has
become
the method of choice for specifically silencing expression of genes (Martinez
J,
Patlcaniowska A, Urlaub H, Luhrmann R, and Tuschl T. Cell 2002;110:563-74;
Maeda S,
Hayashi M, Komiya S, Imamura T, and Miyazono K. EMBO J. 2004; 23:1-12).
Conditions for siRNA treatment were first optimized and MC3T3-E1 cells were
transfected with siRNA (10pmo1) to LMP- 1 disclosed herein and according the
methods
disclosed herein. LMP- 1 mRNA levels were reduced by 90% 48 hrs after siRNA
treatment
as compared to no treatment. Reduced bone nodule mineralization was also
observed in
addition to the decrease in LMP-1 mRNA levels.
Example 20: Smurfl siRNA
In an effort to demonstrate the effects of reducing the level of functional
Smurfl to
support the hypothesis that LMP-1 acts by decreasing the amount of Smurfl
available to
bind to Smadl/5, siRNA to Smurf were designed and tested for target reduction
in TE85
human osteosarcoma cells. Cells were treated according the methods taught
herein and
total RNA was harvested and Smurfl mRNA levels measured by RT-PCR using
primers
specific for Smurfl. Smurf2 mRNA levels were also measured using specific
primers and
these levels did not change. There was a single product of each primer set
which was
sequenced to confirm its identity. The results indicate that Smurfl siRNA
produced a
dose-dependent decrease in Smurfl mRNA levels.
Example 21: Osterix Message in Human MSCs
Osterix is a novel zinc fmger-containing transcription factor required for
osteoblast
differentiation and bone formation. Human MSCs were treated with Ad5/35LMP-1
(0, 1,
5, 10 pfu/cell) with and without BMP-2 (100ng/mL) and harvested for RNA at day
8.
RNA was isolated and osterix mRNA quantified by RT-PCR with the osterix
primers.
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Data was normalized to 18S. These data illustrate that LMP-1 increased the
responsiveness of human MSCs to BMP-2 as evidenced by increase in osterix
message.
The data are summarized in Table 6.
Table 6
Increase in Osterix mRNA
Treatment No BMP- LMP-1 LMP-1 LMP-1 LMP-1
treatment 2 (5pfu/cell) (10 (5pfu/cell) (10
pfu/cell) +BMP-2 pfu/cell)
+BMP-2
Fold 1.0 88.3 0.4 0.4 178.6 424.6
Increase
in
Osterix
mRNA
Example 22: Overexpression of LMP-1 increases BMPRIA (ALK3) levels in human
MSCs.
Human MSCs were treated for 8 hours with rhBMP-2 (100 ng/mL) alone or with
Ad5F35-LMP-1 (5 pfu/cell) and cytoplasmic proteins were enriched for the
plasma
membrane fraction and resolved by SDS-PAGE separation. The blot was then
probed
with antibody specific for the Type IA BMP receptor (BMPRIA/ALK3) and a
predominant band at the expected size for BMPRIA (55 kDa) was observed. rhBMP-
2
treatment (100ng/mL) resulted in an expected increase in BMPRIA over untreated
control
cells. However, when rhBMP-2 was given in the presence of LMP-1 a
significantly
greater increase in the BMPRIA levels was observed. These data support the
hypothesis
that LMP-1 interrupts the Smurfl/Smad6 mediated degradation of BMP receptors.
Example 23: LMP-1 Interacts with Jun Activation Domain Binding Protein (Jabl)_
The yeast-two-hybrid (Y2H) system (Clontech) was used to identify other
proteins
which could interact with LMP-1. Positive clones were selected based on Y2H
screening
of a bone marrow library. The sequencing and database matching of 10 putative
positive
clones identified Jab 1 as a likely candidate binding partner for LMP-l. It
was then
determined if this association occurred in cells. Immunoprecipitation of
cytoplasmic
proteins using LMP-1 antibody beads demonstrated that Jabl was found in
complexes
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with LMP-1. Although these data demonstrate that an association between Jabl
and
LMP-1 occurs in cells, they do not establish direct binding of the two
proteins.
Example 24: LMP-1 binds to Jabl
To demonstrate that LMP-1 binds Jabl directly, cytoplasmic proteins from human
osteoblastic TE-85 cells were separated by SDS-PAGE and blots were probed with
Biotin-
LMP-1. The bound biotin-LMP-1 was detected using neutravadin-HRP. Two bands
were
present on the blot and demonstrated that LMP-1 is capable of binding directly
to both
Smurfl (851cDa) and Jab1 (381cDa). The identity of these two bands were
confirmed by
staining with antibody specific to Smurfl and Jabl. These blots provide
evidence that
LMP-1 interacts directly with Jabl supporting the hypothesis that LMP-1 may
interrupt
the binding of Jabl to one of its targets (Smad4).
Example 25: Identification of bindinginteractions with Jab 1.
While not identified herein, the sites or protein domains necessary for
interaction
between LMP- 1 and Jab 1 can be determined in the same manner as those for the
interaction between LMP-1 and Smurfl (see Examples 13 and 14). Once
identified,
peptides containing those sites or domains may be designed modulate the
interaction of the
proteins.
Example 26: LMP-1 overexpression increases nuclear Smad4 levels in human MSCs.
To demonstreate the effects of LMP-1 overexpression and rhBMP-2 treatment on
Smad4, human MSC's were treated with rhBMP-2 (100 ng/mL), or LMP-1 (Ad5F35,
MOI=5) for 8 hours. SDS-PAGE separated nuclear protein blots were probed with
Smad4
specific antibody. A 66 kDa band representing nuclear Smad4 was seen to
increase 8
hours after LMP-1 treatment. A nonspecific band (running above the 66kDa band)
was
also seen. These data support the hypothesis that LMP-1, presumably via its
interaction
with Jabl, decreases the targeting of Smad4 for proteasomal degradation
thereby resulting
in increased Smad4 levels. As was expected, rhBMP-2 alone did not affect the
nuclear
Smad4 levels as it does not interact with Jabl. Collectively these data
support the direct
interaction of LMP-1 and Jabl in cells and the fact that LMP-1 can increase
the levels of
Smad4 in cells. This may represent a third regulatory point for LMP-1 to
modulate
cellular responsiveness to BMPs since Smad4 is required for nuclear transport
of activated
R-Smads.
Example 27: Screening of LMP-1 mimics
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To identify small molecules that mimic the effect of LMP-1 on induction of
bone
formation, and have properties that make it more clinically convenient,
multiple
compounds may be screened which contain or mimic the more important LMP-1
interacting domain (B). These compounds are tested for their ability to
compete with
LMP-1 for Smurfl binding.
In these studies, recombinant Smurfl is applied to wells of a 96 well plate.
After
removing excess Smurfl, test compounds (in excess) are pre-incubated with
Smurfl
followed by incubation of the Smurfl complexes with recombinant biotin-LMP-1.
Biotin-
LMP-1/Smurfl is detected using Strepavidin-alkaline phosphatase. Absorbance is
determined at 405 nm using the SpectraMax M2 microtiter plate reader.
Compounds that
block Biotin-LMP-1 binding may then be re-screened using appropriate lower
amounts of
the compound to determine the IC50 (dose that prevents 50% of the maximum LMP-
1
binding). Those compounds with the lowest IC50 are considered the most
efficacious and
can then be screened for cellular effects. The IC50 is used to determine the
dose of each
compound that we will screen.
Example 28: Screening compounds in vitro
Candidate compounds that most efficiently inhibit binding of LMP-1 to Smurfl
in
binding competition assays are further evaluated for their ability to mimic
LMP-1 in cells.
One appropriate cell line are mesenchymal stem cells (MSCs) and appropriate
endpoints
include an increase BMPRIA levels and an increase in luciferase production
from a Smad-
activated reporter construct.
In these studies, human MSCs are plated at 3 x 104 cells/well in 6-well
plates,
grown overnight, and treated with candidate compounds at the IC50 dose and two
doses
above and below that dose. Ad5F35LMP-1 and Ad5F35GFP are applied as positive
and
negative controls. After 1, 2, 4, 8, 12, 24 and 48 hrs, cells are haivested
and plasma
membrane enriched fractions prepared. Fractions are subjected to SDS-PAGE and
Western analysis using BMPRIA specific antibody. In the second set of
experiments
MSCs plated as above and transfected with a 9xGCCG/Smad-activated luciferase
reporter
construct are incubated with all compounds (at the successful dose) that
increased
BMPRIA in the first experiment. These studies are performed in the presence or
absence
of 100 ng/mL BMP-2.
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The results will allow for identification of compounds and doses of compounds
that will mimic the effect of LMP-1 on the particular endpoint. It is also
expected that
doses of candidate compounds that successfully increase BMPRIA will increase
luciferase
activity somewhat when applied alone, but will greatly enhance the effect of
BMP-2 on
the luciferase activity.
Example 29: Screening compounds in vivo
Compounds found to activate the BMP/Smad signaling pathway in vitro can then
be screened in vivo for effects on induction of bone in the rat model of
ectopic bone
formation described herein.
Example 30: Synergy screening
Once compounds that induce bone formation have been identified, those
compounds are then tested in combination with low doses of BMP-2 to determine
whether
there are synergistic effects (as demonstrated in other examples herein) that
might lower
the required dose of either agent.
To evaluate potential synergy, we have identified herein a dose of BMP-2 (2.5
ug)
that induces bone formation in only 50% of the implants and a lower dose (1
ug) that fails
to induce bone formation in the rat model.
In the synergy studies, multiple doses of each of several candidate compounds
are
tested in cell culture studies of enhancement of BMP-2 signaling in MSCs.
These studies
test the ability of each compound to enhance the efficacy of a suboptimal dose
of BMP-2
(2.5 ug) to make bone in more than 50% of the implants. The positive control
is MSCs
treated with TAT-LMP-1 (0.625 nM); negative controls include MSCs alone and
BMP-2
(2.5 ug) alone. MSCs (1-2M) are mixed with appropriate doses of compounds in a
100 uL
total volume and placed on a collagen disc. The disc is implanted
subcutaneously on the
chest of athymic rats and explanted after 4 weeks. Bone formation is evaluated
by
palpation, x-ray and semi-quantitative scoring of non-decalcified histologic
sections
(Edwards, J. T., Diegmann, M. H., and Scarborough, N. L., Clin. Orthop., 1998,
219-228).
The most promising compounds are tested twice more to determine the best one
or two
compounds to be used in future experiments in higher animals.
If the LMP-1 enhancement of BMP-2 efficacy to induce bone cannot be emulated
by the chemical compounds that are screened, the need to also mimic the LMP-
1/Jabl
interaction can be examined as the control of Smurf4 levels could be a rate-
limiting step.
