Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR
MODULATING LEPTIN ACTIVITY
RELATED APPLICATIONS
This application is a continuation-in-part of and claims priority to
s 09/04.1.278 filed March 19, 1998 and claims the benefit of U.S. Provisional
Application No. 60/074.320 filed February I 1, 1998, the teachings of which
are
incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
Leptin, the adipocyte derived hormone, acts on specific regions of the brain
10 to regulate food intake, energy expenditure and neuroendocrine function
(Zhang Y,
et al.. Nature 372:425-432, 1994; Halaas JL et al., Science ?b9:543-546, 1995;
Campfield LA, et al., Science 369:546-549,1995, and Pellymounter MA, et al..
Science 269:540-543, 1995.). Leptin is structurally related to cytokines
(Zhang F, et
ul.. Nature 387:206-209. 1997) and acts on receptors that belong to the
1 ~ cvtokine-receptor superfamily (Tartaglia LA, et al.. Cell 83:1263-1271:
Lee G-H, et
ul.. Nature 379:632-635, 1996).
In diet-induced obesity in rodents, and in most humans with obesity,
resistance to peripheral leptin exists. and has yet to be explained. Human
obesity
could be related to low levels of functional circulating leptin or to
decreased action
?0 at the target cells in the brain. Supporting the latter possibility are
data
demonstrating that while functional mutations in the leptin gene exist in
humans
(Montague CT, et al., Nature 387:903-908. 1997), they are extremely rare
(Maffei
M, et al.. Diabetes 45: 679-68?. 1996: Shigemoto M, et al.. Eur J
Errdocrirrol.
137:511-513. 1997; Carlsson B. et ul.. Obes Res 5:30. 1997). In addition.
serum
?5 leptin levels are increased in human obesity and correlate positively with
body
weight (Maffei M et al.. Nut ATcd. 1:11 ~~-1161. 1995: Considine RV ct ul..
Errgl J
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Med. 334:292-295, 1996). Furthermore, some (Widdowson PS et al.. Diabetes
46:1782-1785. 1997), but not all studies (Van Heek M et al., JClin Invest
99:385-
390, 1997) of diet-induced obesity in rodents show that these animals develop
both
peripheral and central resistance to recombinant leptin. Together, these data
are
5 consistent with the possibility that the leptin-resistance which
characterizes human
obesity may be due to defects in leptin signal-transduction in the brain. Two
potential mechanisms for leptin resistance are defects at the level of the
blood brain
burner, and defects in the pathway of leptin signal transduction in target
cells.
Regarding the latter possibility, the leptin receptor (OBR) (mutation of which
causes
10 obesity in dbldb mice and falfa rats), is most closely related to the gp130
and LIFR
signal transducing subunits that are activated by cytokines such as IL-6, LIF
and
CNTF and hormone receptors for growth hormone such as erythropoietin
(Tartaglia,
1995). Several isoforms of the leptin receptor exist including a long form
that is
predominantly expressed in specific cell bodies in the hypothalamus. Potential
1 S mechanisms for inhibiting or enhancing leptin signaling are a matter of
considerable
interest.
Recently, a new family of cytokine-inducible inhibitors of signaling has been
identified including CIS (cytokine-inducible sequence), SOCS-1 (suppressor of
cytokine signaling), SOCS-2 and SOCS-3 (Start R et al, Nature 387:917-921,
1997;
20 Endo TA, et al. Nature 387: 921-924, 1997; Naka T et al. Nature. 387: 924-
929,
1997; Masuhara M et al, Biochem Biophvs Res Commurr. 239:439-446, 1997). A
number of different cytokines including IL-6, LIF, growth hormone (GH) and
erythropoietin (EPO) induce transcriptional activation of one or more of the
CIS or
SOCS genes irr vivo and in vitro, through activation of the JAK-STAT pathway
25 (Start, 1997; Endo, 1997; Naka, 1997; Yoshimura et al. Embo J. 14:2816-
2826,
1995, Masuhara, 1997). The results suggest that the CIS and SOCS proteins may
act
in a classic negative feedback loop by inhibiting JAK activity, thereby
switching off
cytokine signal transduction.
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SUMMARY OF THE IIWENTION
The present invention encompasses methods and compositions for altering,
or modulating, leptin activity by altering, or modulating, cytokine inhibitor
activity.
Specifically encompassed in the present invention are methods and compositions
to
S alter activity of the cytokine inhibitor, SOCS-3. SOCS-3 expression is
rapidly
induced by leptin treatment in regions of the hypothalamus that are known to
be
involved in the regulation of body weight. As demonstrated herein, it has now
been
determined that a SOCS-3-mediated leptin cell-signaling inhibitory pathway
exists.
Thus, this suggests that SOCS-3 is a negative regulator of leptin signal-
transduction.
10 Also as described herein, it is believed that excessive SOCS-3 activity is
an
important factor in the leptin resistance that characterizes most syndromes of
rodent
and human obesity. Inhibition of SOCS-3 expression or function is therefore a
potential target for the development of drugs aimed at improving leptin
sensitivity in
a mammal and hence inducing weight loss. Furthermore, inappropriately
increased
15 SOCS-3 activities in leptin-responsive neurons is a potential mechanism for
the
leptin resistance observed in various syndromes of obesity. Thus, as described
herein, altering SOCS-3 activity provides a means for modulating leptin-
induced
cell signaling and therefore modulating bodvweight.
The present invention also relates to methods of treating delayed onset of
20 puberty in mammals by increasing SOCS-3-mediated ieptin cell signaling and
to
methods of treating reproductive dysfunction, or infertility, such as
anovulation or
decreased spermatogenesis associated with low serum/plasma levels of leptin,
inactive leptin, leptin resistance or ineffective production of ieptin. The
methods of
the present invention can be used in either male or female mammals.
25 The present invention also relates to the methods of treating affective
mood
disurders in an individual associated with elevated leptin levels, such as
atypical
depression, wherein atypical depression is characterized by elevated leptin
levels in
an individual such that treatment resulting in decreased leptin-induced cell
signaling
would result in prevention or alleviation of symptoms of atypical depression.
The
30 present invention also relates to methods of treating affective mood
disorders
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associated with decreased leptin levels, such as melancholic depression,
wherein
melancholic depression is characterized by decreased leptin levels in an
individual,
such that treatment resulting in increased leptin-induced cell signaling would
result
in prevention or alleviation of symptoms of melancholic depression.
The present invention therefore pertains to methods of modulating leptin
cell signaling by altering SOCS-3 activity in a mammal. As defined herein,
modulating (also referred to herein as altering, adjusting or regulating)
leptin activity
means inhibiting or enhancing the biological activity of leptin or SOCS-3.
Inhibiting leptin activity encompasses partial inhibition as well as complete
10 abrogation of leptin activity.
The biological activity of leptin is defined herein as the ability of leptin
to
actW ate one, or more signal transduction pathways in a cell as a result of
interaction
between (e.g., binding) leptin and a leptin receptor associated with the cell.
The
signal transduction pathway includes for example, the activation of Janus
Kinase 2
15 (referred to herein as JAK2), thereby activating other pathways, such as
signal
transducers and activators of transcription (STAT), phosphoinositide-3 kinase
rasl
mitogen-activated protein kinase pathways ultimately leading to activation or
inactivation of gene transcription as well as other, non-transcriptional
effects. For
example, leptin can bind its cognate receptor, which is associated with JAK2;
JAK2
20 is activated, and phosphorylates the receptor, JAK2 and STAT3 proteins
(among
others). Phosphorylated STAT3 dimerizes and translocates to the nucleus, where
it
serves as a transcriptional activator. Therefore, leptin activity can be
measured as
the level of phosphorylation of the receptor, JAK2 or STAT3. Further, leptin
activity can be measured by the amount of gene transcription from STAT3
25 responsive genes.
As defined herein, SOCS-3 activity, or SOCS-3 mediated leptin cell
signaling, is the inhibition or inactivation (completely or partially) of
leptin induced
cell signaling. As demonstrated by the present invention, SOCS-3 mediates the
down regulation of leptin signaling as measured by lack of phosphorylation of
leptin
30 receptor, JAK2 or STAT3, as weal as by the association of JAK2 and SOCS-3.
As
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described herein, SOCS-3 transcription is part of a negative feedback loop
triggered
by leptin activation of the leptin receptor.
SOCS-3 activity can be inhibited by inhibiting or reducing the amount of
SOCS-3 protein expressed in a cell, or by introducing a polvnucleotide
encoding a
5 modified SOCS-3 protein into a cell, wherein the modified SOCS-3 protein
comprises a mutant, variant, derivative, or analog of the SOCS-3 protein.
SOCS-3 expression can be inhibited or reduced by transfecting a cell with a
polynucleotide construct encoding SOCS-3 antisense DNA or RNA. For example,
the antisense RNA can hybridize to the endogenous SOCS-3 mRNA and prevent
10 translation of SOCS-3 mRNA, thereby inhibiting or reducing expression of
SOCS-3
protein. SOCS-3 expression can also be inhibited or reduced by transfecting
the cell
with a polynucleotide construct encoding a transcriptional inhibitor such that
transcription of SOCS-3 is inhibited or reduced. Such a transcriptional
inhibitor
would interact specifically with SOCS-3 promoter sequences, resulting in
decreased
15 transcription of SOCS-3, decreased SOCS-3 protein expression and thus
decreased
SOCS-3 activity.
SOCS-3 activity can also be inhibited by transfecting the cell with a
polynucleotide construct encoding an altered, or modified SOCS-3 protein,
polypeptide or peptide. In one embodiment, the modified SOCS-3 polypeptide is
a
20 competitive inhibitor (e.g., antagonist) of endogenous SOCS-3. The modified
SOCS-3 can interact with a SOCS-3 target protein (e.g., JAK2), without
interfering
with the activity of the target protein. Because the modified SOCS-3 protein
interacts with the intended SOCS-3 target, endogenous SOCS-3 could not
interact
with its intended target, thereby inhibiting or reducing the level of SOCS-3
mediated
25 leptin cell signaling. In another embodiment, SOCS-3 activity can be
inhibited or
reduced by introducing a SOi.S-3 inhibitor into the cell. Such an inhibitor
can be a
peptide or small organic molecule that interferes with SOCS-3 activity. Such
an
inhibitor can interact specifically with SOCS-3, or to its intended target, to
inhibit
SOCS-3 activity. For example, the inhibitor can interact with downstream
targets of
30 SOCS-3 such as JAK2.
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The present invention further encompasses methods of increasing or
enhancing SOCS-3 activity in a cell. Increased SOCS-3 activity in a cell can
inhibit
or reduce leptin-induced cell signaling. A reduction or inhibition of leptin-
induced
cell signaling can be useful to prevent, inhibit or alleviate atypical
depression in an
S individual, or to promote weight gain in an individual. SOCS-3 activity can
be
increased by transfecting a cell with a polynucleotide construct encoding a
biologically active form of SOCS-3 protein, or a biologically active fragment
thereof. In another embodiment, SOCS-3 activity can be increased by
transfecting a
cell with a nucleic acid encoding a modified SOCS-3 protein that has increased
10 biological activity.
The present invention also pertains to cell lines that can be used to evaluate
SOCS-3 mediated leptin activity and to screen candidate SOCS-3 inhibitors,
antagonists and agonists for activity. For example, a cell line can be
produced that
expresses SOCS-3, a cytokine receptor and a reporter gene construct wherein
15 transcription of the reporter gene construct is inhibited by SOCS-3. In one
embodiment, the cytokine receptor is the leptin receptor. In another
embodiment,
the reporter gene construct is a leptin responsive promoter attached to a
reporter
gene. The reporter gene can be the CAT gene, the luciferase gene or the
(3-galactosidase gene. Another cell line suitable for use in the present
invention is a
20 cvtoicine dependent cell line wherein SOCS-3 and the leptin receptor are
stably
expressed. In one embodiment, the cytokine receptor is the IL-3 receptor.
The cell lines of the present invention can be used to screen libraries such
as
organic molecule libraries or cDNA libraries to select and identify molecules
that
inhibit (or enhance) SOCS-3 activity. In one embodiment, cells expressing the
25 leptin receptor, SOCS-3 and a reporter gene construct are contacted with an
organic
molecule library or transfected with a cDNA expression library. These cells
are then
stimulated with leptin. Cells having increased reporter gene activity are
selected and
the organic molecule or cDNA is identified. In another embodiment, IL-3
dependent cells expressing leptin receptor and SOCS-3 are removed from IL-3,
30 contacted with a member of an organic molecule library or transfected with
a
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member of a cDNA expression library. Cells capable of proliferating in leptin
are
selected and the organic molecule or cDNA is identified.
Thus, as a result of the discovery described herein, methods and
compositions are now available to modulate leptin activity, specifically by
modulating the activity of the cytokine inhibitor, SOCS-3, thereby resulting
in either
an increase or decrease of leptin-induced cell signaling.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 A shows the results of a'zP-RT-PCR assay demonstrating the in vivo
effects of leptin on CIS-I, SOCS-1, SOCS-2, and SOCS-3 mRNA levels in the
10 hypothalamus from oblob mice.
Figure 1 B is the quantification of the data in Figure lA.
Figure 1 C is a plot of quantitative'zP-RT-PCR of SOCS-mRNA from
hypothalami of dbldb mice and lean (+/?) controls upon leptin treatment.
Figures 2A and 2B show the results of in situ hybridization with 35S-labeled
15 antisense SOCS-3 probes to brain sections from normal rats treated with
saline or
leptin.
Figure 3A is a graphic representation of leptin-induced erg-1 promoter
activation by SOCS-3 in CHO cells.
Figure 3B shows a Western blot demonstrating inhibition of leptin induced
20 leptin receptor tyrosine phosphorylation by SOCS-3 in COS-1 cells.
DETAILED DESCRIPTION OF THE INVENTION
The present invention encompasses the regulation of leptin activity in the
brain. Specif cally encompassed by the present invention is the regulation of
the
leptin- induced cell signaling pathway in the hypothalamus via regulation of
25 SOCS-3 activity.
Leptin is a hormone which has been shown to interact with its cognate
receptor. thereby initiating its cell-signaling pathway. Several different
leptin
receptor isoforms are predicted to exist, including a long form which has the
highest
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_g_
level of expression in regions of the hypothalamus, and specific regions
therein,
including the arcuate nucleus and the dorso-medial hypothalamus. In vitro and
in
vivo studies demonstrate that leptin activates cytokine-like signal
transduction by
stimulating the classic JAK-STAT pathway via the long receptor isofonn
(Ghilardi
5 N et al, Proc Natl Acad Sci USA. 93:6231-6235, 1997; Baumann H et al, Proc
Natl
Acad Sci USA. 93:8374-8378, 1996; Vaisse C, et al, Nature Genetics 14:95-97,
1996). Lack of functional leptin or of long form leptin receptors in the oblob
and
dbldb mice, respectively, causes severe obesity (Zhang et al, Nature 372:425-
432,
1994; Lee G-H, et al, Nature 379:632-635, 1996; Chen H et al, Cell 84:491-495,
10 1996).
The cloning of the genes encoding leptin (Zhang, 1994,) and the leptin
receptor (Tartaglia et al., 1995, 61183;1263-1271 ), and the study of these
proteins in
vivo and in virro, have dramatically demonstrated the importance of this
ligand-receptor system in the normal regulation of body weight and energy
balance.
15 In addition to this role which has been proposed to be its primary
function,
circulating leptin also appears to play an important role in the
neuroendocrine axis
(Ahima. R.S., et al., Nature 382:250-252, 1996), including the regulation of
reproduction. Administration of exogenous leptin has been shown to induce the
onset of puberty in mice (U.S. Patent Application No. 08/749,534, the
teachings of
20 which are incorporated herein by reference in its entirety). Treatment of
ad lib. fed
female mice with a dose of leptin which did not significantly alter body
weight,
resulted in an earlier onset of puberty.
Recently, a new family of cytokine inhibitors has been described, including
CIS-1 and SOCS-1, 2, and 3. CIS-1, an inhibitor of cytokine receptor
signaling, is
25 thought to bind directly to the cytokine receptors and possibly block key
phosphotvrosine residues of the receptor (Yoshimura, 1995). SOCS-1, 2 and 3
are
cytokine inducible inhibitors that were found to be active in hematopoietic
cells
(Stan. 1997; Endo, 1997; and Naka,1997) where it is thought to abrogate
cytokine
mediated cell proliferation. As demonstrated herein for the first time, leptin
30 specifically induces expression of SOCS-3 mRNA in hypothalamic nuclei known
to
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express high levels of the long form of the leptin receptor. No effect on CIS,
SOCS-1 or SOCS-2 could be detected. (See Example 1).
In situ hybridization experiments with SOCS-3 antisense RNA probes to
brain sections from both oblob mice and normal rats demonstrated a leptin
dependent specific increase of SOCS-3 mRNA in the arcuate nucleus and
dorso-medial hypothalamus (Example 2). These regions express the highest level
of
the long form leptin receptor mRNA in the hypothalamus, strongly suggesting
that
the effect by leptin on SOCS-3 mRNA levels is a direct effect in specific
neurons
expressing long form leptin receptors. The regions of the arcuate nucleus that
show
10 stimulation of SOCS-3 mRNA after leptin treatment are regions known to
express
NPY. POMC and AGRP, all of which are regulated by leptin in vivo. suggesting
that
cells expressing these neuropeptides may be direct targets of leptin.
Fos is often used as a marker for activated neurons. However, its use is
limited due to the inability to distinguish between direct and indirect
actions of a
15 given agent. Furthermore, NPY-expressing neurons in the arcuate nucleus,
which are
negatively regulated by leptin, are not positive for Fos activation after
leptin
treatment. However, as demonstrated in Example 2, herein, SOCS-3 mRNA is
increased in the region of the arcuate nucleus which express NPY, suggesting
that
SOCS-3 is a better marker than Fos for neurons that are regulated by leptin.
20 The lethal yellow (Ay'la) mouse develops obesity due to ectopic and
unregulated overexpression of the agouti protein (Dickie, J. Hered. 60:20-25,
1969;
Bultman et al., Cell 71:1195-1204, 1992; Miller et al., Genes Dev. 7:454-467,
1993), a potent melanocortin receptor (MCR) antagonist. The obesity in this
model
is characterized by hyperleptinemia and by resistance to both central and
peripheral
25 leptin administration (Halaas, 1997). Ultimately, the leptin resistance in
this model
must result from melanocortin antagonism induced by agouti, but the molecular
basis for the leptin resistance is unknown. It has been hypothesized that the
leptin
resistance of A'%a mice may be due to the blockade of MC4 receptors at a site
in the
brain downstream of leptin signaling (Seeley et al., Nature 390:349, 1997).
30 However, it has recently been demonstrated that A'la mice that are also
deficient in
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leptin, e.g. A''la, lep°bllep°°, are normally responsive
to leptin (Boston et al., Science
278:1641-1644, 1997). The latter observation suggests that leptin resistance
in A~%a
mice is a result of. or at least requires, in addition to MC4 receptor
blockade, chronic
exposure to high circulating levels of leptin. As described in Example 4, in
situ
hybridization histochemistry revealed that SOCS-3 mRNA is elevated in ad
libitu»r
fed Ayla mice as compared to lean control litter mates and in particular, in
the
dorsomedial hypothalamic nucleus, a site in which leptin induces SOCS-3 gene
expression. A similar region of the dorsomedial hypothalamic nucleus contains
Fos-like immunoreactivity following intravenous (Elmquist et al.,
Endocrinology
138:839-842, 1998) or central leptin administration to normal rats (Van Dijk,
et al.,
Am JPlrvsiol., ?71:81096-81100, 1996). This site has also been demonstrated to
have increased levels of NPY mRNA in Ay%a mice (Kesterson et al., Mol
Endocrinol. 11:360-637, 1997). Thus it is reasonable to believe that leptin
resistance in this model is a consequence of hyperleptinemia; serum leptin
levels in
the A'la mice employed here were 40 ng/ml compared to 7 ng/ml in control mice.
In
this model, increased leptin could drive SOCS-3 expression in key hypothalamic
nuclei involved in body weight regulation, thereby inhibiting the weight
reducing
effects of leptin.
In mammalian cell lines, SOCS-3, but not CIS-1, or SOCS-2, completely
blocked leptin induced signal- transduction (described in Example 5),
suggesting
that leptin-receptor signaling is also negatively regulated by SOCS-3 in vivo.
It is thought that the leptin receptor long form may exert a signaling action
similar to that of granulocyte colony stimulating factor, leukemia inhibitory
factor
receptor and gp 130. Ligand binding to these receptors leads to activation of
receptor-bound JAK kinases, which phosphorylate tvrosines in the cytoplasmic
domain of the receptor as well as in other cytoplasmic target proteins.
Several
pathways can be activated by JAK kinases, including the signal transducers and
activators of transcription (STAT), ras/mitogen-activated protein kinase, and
phospho-inositide-3 kinase pathways. As shown in Example 7, leptin stimulation
irr
vitro results in the induction of SOCS-3 mRNA. In addition. when SOCS-3 is
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present, leptin- induced phosphorylation of STAT3, JAK2 as well as the leptin
receptor is inhibited. Furthermore, leptin pretreatment of CHO cells
expressing the
leptin receptor resulted in a prolonged inhibition (longer than 24 hours) of
subsequent ieptin induced signaling. For example, as described in Example 8,
~ SOCS-3 mRNA was not induced, STAT3 DNA binding activity was not increased
nor was the leptin receptor phosphorylated in response to subsequent leptin
treatment. This inhibition lasted for at least 24 hours after leptin
pretreatment.
Based on the results described herein, it is reasonable to believe that SOCS-3
antagonizes leptin induced cell-signaling by interacting with JAK2 and
competing
10 with binding between JAK2 and its substrate (leptin receptor or STAT) or by
acting
as a JAK2 pseudo substrate, thereby preventing phosphorylation of the intended
target. SOCS-3 can inhibit JAK2 kinase activity thereby preventing
phosphorylation of downstream elements. Thus, it has now been determined that
a
SOCS-3-mediated leptin cell-signaling inhibitory pathway exists. SOCS-3
1 ~ therefore, can negatively regulate cell signaling via the leptin receptor.
SOCS-3 might antagonize leptin-induced cell signaling by recruitment of
tyrosine-phosphatases, which have been shown to be involved in
dephosphorylation
of the cytokine receptors. Two candidates are SHP-1 and SHP-2 (also known as
SYP). Both SHP-l and 2 have been shown to regulate cytokine signaling.
20 Recruitment of SHP-1 has been associated with
dephosphorylation/inactivation of
JAK2 and subsequent termination of erythropoietin signal transduction
(Klingmiiller, et al, Cell 80:729-738, 1995). A similar role for SHP-1 in
mediating
the down-regulation of JAK2 following stimulation of cells with growth hormone
has been proposed.
25 The present invention encompasses methods and compositions for
modulating leptin activity comprising altering SOCS-3 activity in a cell, for
example, a hypothalamic cell. Cells encompassed by the present invention can
be
found in all vertebrates including mammals and humans. The cells could also be
cells maintained in a cell line, e.g., transformed cells which are suitable
for use in
30 testing leptin or SOCS-3 activity.
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In one embodiment of the present invention, it is desirable to increase, or
up-regulate leptin activity via inhibition of SOCS-3 activity. Increasing
Ieptin
activity results in the increase of the leptin cell-signaling pathway,
resulting in, inter
alia, weight loss, restoration of reproductive function and/or alleviation of
the
5 symptoms of melancholic depression. In this embodiment, SOCS-3 activity is
inhibited by interfering with the interaction (e.g., binding) of SOCS-3 to its
intended
target. For example, the intended target of SOCS-3 interacts with JAK2
(Example
6). SOCS-3 may act as a pseudo-substrate of JAK2, or may recruit phosphatases
to
the JAK2-receptor complex. Thus, a polypeptide or peptide inhibitor/antagonist
10 comprising the SOCS-3 amino acid sequence of GenBank Accession Number
U88328, or a modified SOCS-3 amino acid sequence, or an active fragment
thereof,
can competitively interact with SOCS-3 and/or its intended target, e.g. by
binding to
JAK2, resulting in the increase or up-regulation of leptin activity.
As defined herein, modified SOCS-3 encompasses SOCS-3 molecules
15 comprising fragments, derivatives, analogs, variants and mutants of the
SOCS-3
protein. These modified SOCS-3 molecules possess SOCS-3 inhibitor/antagonist
activity, thereby inhibiting the activity of endogenous SOCS-3 present in a
cell,
resulting in an increase of leptin activity. Another activity of modified SOCS-
3
molecules can be the antigenic property of the modified SOCS-3 molecule
20 comprising the ability of the modified SOCS-3 to bind to SOCS-3-specific
antibodies. The modified SOCS-3 molecule can also possess immunogenic
properties whereby the modified SOCS-3 molecule induces an immunogenic
response, e.g., the production of antibodies that specifically bind to
endogenous
(native) SOCS-3.
25 A fragment of SOCS-3 encompasses polvpeptides that comprise only a part
of the lull-length SOCS-3 protein and inhibit endogenous SOCS-3 activity. Such
fragments can be produced by amino and/or carboxyl terminal deletions, as well
as
internal deletions. Fragments can also be produced by enzymatic digestion.
Such
modified SOCS-3 molecules can be tested for inhibitory activity as described
herein.
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"Derivatives" and "variants" of SOCS-3 can include truncated and hybrid
forms of SOCS-3. "Truncated" forms are shortened forms of SOCS-3, typically
with internal deletions of regions of the protein. "Hybrid" forms of SOCS-3
are
SOCS-3 molecules comprising a portion of a SOCS-3 amino acid sequence with
5 non-SOCS-3 amino acid sequence, e.g., SOCS-1 or SOCS-2 sequence.
"Variants" and "mutants" of SOCS-3 can be produced using in vitro and in
vivo techniques well-known to those of skill in the art, for example, site-
specific
mutagenesis and oligonucieotide mutagenesis. Manipulations of the SOCS-3
protein sequence can be made a the protein level as well. Any numerous
chemical
10 modifications can be carried out by known techniques including, but not
limited to,
specific chemical cleavage by cyanogen bromide, trypsin and papain. SOCS-3 can
also be structurally modified or denatured, for example, by heat. In general,
mutations can be conservative or non-conservative amino acid substitutions,
amino
acid insertions or amino acid deletions. The mutations can be at or near SOCS-
3
15 binding sites.
For example, DNA encoding a SOCS-3 mutant is prepared by site-directed
mutagenesis ofDNA that encodes endogenous SOCS-3. Site-directed (site-specifc)
mutagenesis allows the production of SOCS-3 variants through the use of
specific
oligonucleotide sequences that encode the DNA sequence of the desired
mutation, as
20 well as a sufficient number of adjacent nucleotides, to provide a primer
sequence of
sufficient size and sequence complexity to form a stable duplex on both sides
of the
deletion junction being traversed. Typically, a primer of about 20 to 25
nucleotides
in length is preferred, with about 5 to 10 residues on both sides of the
junction of the
sequence being altered. In general, the techniques of site-specific
mutagenesis are
25 well known in the art, as exemplified by publications such as EdeIman et
al., DNA
?:183, 1983. The site-specific mutagenesis technique typically employs a phage
vector that exists in both a single-stranded and double-stranded form. Typical
vectors useful in site-directed mutagenesis include vectors such as the M13
phage,
for example, as disclosed by Messing et al.. Third Cleveland Svmposiun: on
30 .'Macromolecules and Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam,
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1981. This and other phage vectors are commercially available and their use is
well-
known to those skilled in the art. A versatile and efficient procedure for the
construction of oligonucleotide directed site-specific mutations in DNA
fragments
using M 13-derived vectors was published by Zoller, M.J. and Smith, M.,
Nztcleic
5 Acids Res. 10:6487-6500, 1982. Also, plasmid vectors that contain a single-
stranded
phage origin of replication can be employed to obtain single-stranded DNA,
Veira et
al., Meth Enzvmol. 153:3 1987.
Alternatively, nucleotide substitutions can be introduced by synthesizing the
appropriate DNA fragment in vitro, and amplifying it by PCR procedures known
in
10 the art.
In general, site-specific mutagenesis herewith can be performed by first
obtaining a single-stranded vector that includes within its sequence a DNA
sequence
that encodes the relevant protein. An oligonucleotide primer bearing the
desired
mutated sequence is prepared, generally synthetically, for example, by the
method of
15 Crea et al., Proc Natl Acad Sci USA. 75: 5765, 1978. This primer can then
be
annealed with the single-stranded protein sequence-containing vector, and
subjected
to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, to
complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is
formed wherein one strand encodes the original non-mutated sequence and the
20 second strand bears the desired mutation. This heteroduplex vector can then
be used
to transform appropriate host cells such as JM 101 cells, and clones can be
selected
that include recombinant vectors bearing the mutated sequence arrangement.
Thereafter, the mutated region can be removed and placed in an appropriate
expression vector for protein production.
25 The PCR technique can also be used in creating amino acid sequence
variants of SOCS-3. When small amounts of template DNA are used as starting
material in a PCR, primers that differ slightly in sequence from the
corresponding
region in a template DNA can be used to generate relatively large quantities
of a
specific DNA fragment that differs from the template sequence only at the
positions
30 where the primers differ from the template. For introduction of a mutation
into a
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plasmid DIVA, one of the primers can be designed to overlap the position of
the
mutation and to contain the mutation; the sequence of the other primer is
preferably
identical to a stretch of sequence of the opposite strand of the plasmid, but
this
sequence can be located anywhere along the plasmid DNA. It is preferred,
however,
that the sequence of the second primer is located within 500 nucleotides from
that of
the first, such that in the end the entire amplified region of DNA bounded by
the
primers can be easily sequenced. PCR amplification using a primer pair like
the one
just described results in a population of DNA fragments that differ at the end
position of the mutation specified by the primer.
10 The DNA fragments produced bearing the desired mutation can be used to
replace the corresponding region in the plasmid that served as PCR template
using
standard DNA technology. Mutations at separate positions can be introduced
simultaneously by either using a mutant second primer or performing a second
PCR
with different mutant primers and ligating the two resulting PCR fragments
15 simultaneously to the vector fragment in a three (or more) part ligation.
Another method for preparing variants, cassette mutagenesis, is based on the
technique described by Wells et al. Gene 34, 315, 1985. The starting material
can
be the plasmid (or vector) comprising the SOCS-3 DNA to be mutated. The
codon(s) within the SOCS-3 to be mutated are identified. There must be unique
?0 restriction endonuclease sites on each side of the identified mutation
site(s). If such
restriction sites do not exist, they can be generated using the above-
described
oligonucleotide-mediated mutagenesis method to introduce them at appropriate
locations in the SOCS-3 DNA. After the restriction sites have been introduced
into
the plasmid, the plasmid is cut at these sites to linearize it. A double
stranded
25 oligonucleotide encoding the sequence of the DNA between the restriction
sites but
containing the desired mutations) is synthesized using standard procedures.
The
two strands are synthesized separately and then hybridized together using
standard
techniques. This double-stranded oligonucleotide is referred to as the
cassette. This
cassette is designed to have 3' and 5' ends that are compatible with the ends
of the
30 linearized plasmid, such that it can be directly iigated to the plasmid.
The plasmid
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now contains the mutated SOCS-3 DNA sequence, that can be expressed to produce
SOCS-3 with altered binding activity.
The inhibitor compounds of the present invention include any molecule that
interacts with endogenous SOCS-3 or to SOCS-3 target molecules such as JAK2
5 such that upon interacting with said molecules, inhibitors the SOCS-3
mediated
inhibition of leptin cell-signaling activity. Encompassed by the present
invention
are inhibitor compounds that mimic the structure and confon~nation of the
substrate
moiety when interacting with the binding or active site. Molecular inhibitors
of the
present invention will typically have an inhibition constant (K;) of ten
micromolar,
10 or less. Specifically encompassed are organic molecules that mimic the
structure
and conformation of SH2 binding domains and interact with SOCS-3, thereby
inhibiting its activity. In one embodiment the inhibitor contains or mimics
phosphotyrosine.
Also encompassed by the present invention are small organic molecules that
I 5 mimic the structure of SOCS-3, or, alternatively, the binding site of the
SOCS-3
target, and therefore, interfere with the interaction of SOCS-3 with its
intended
target molecule.
Peptides suitable for use as SOCS-3 inhibitors can be produced in libraries.
The peptides of the library can be immobilized on a surface, for example the
20 peptides can be immobilized on a chip or on beads.
The libraries of peptides comprise a mixture of substantially equimolar
amounts of peptides. In one embodiment, the library can be designed to mimic
SOCS-3 target molecules, e.g., JAK2. In another embodiment, the library
comprises
peptides or phostyrosin containing peptides that interact with the SH2 domain
of
25 SOCS-3, thereby inhibiting the ability of SOCS-3 to bind target molecules.
The inhibitors of the present invention can be synthesized using standard
laboratory methods that are well known to those of skill in the art, including
standard solid phase techniques. Inhibitors comprising naturally occurring
amino
acids can also be produced by recombinant DNA techniques known to those of
skill,
30 and subsequently phosphorylated.
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The inhibitors of the present invention can comprise either the 20 naturally
occurring amino acids or other synthetic amino acids. Synthetic amino acids
encompassed by the present invention include, for example, naphthylalanine, L-
hydroxypropylglycine, L-3,4-dihydroxyphenylalanyl, a-amino acids such as L-a-
S hydroxylysyl and D-a-methylalanyl, L-a-methyl-alanyl, ~3 amino-acids such as
(3-
analine, and isoquinolyl.
D-amino acids and other non-naturally occurring synthetic amino acids can
also be incorporated into the inhibitors of the present invention. Such other
non-
naturally occurring synthetic amino acids include those where the naturally
10 occurring side chains of the 20 genetically encoded amino acids (or any L
or D
amino acid) are replaced with other side chains of the 20 genetically encoded
amino
acids (or any L or D amino acid) are replaced with other side chains, for
instance
with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered
alkyl, amide,
amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and
the
15 lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered
heterocyclic. In
particular, proline analogs in which the ring size of the proline residue is
changed
from 5 members to 4, 6, or 7 member can be employed.
As used herein, "lower alkyl" refers to straight and branched chain alkyl
groups having from 1 to 6 carbon atoms, such as methyl, ethyl propyl, butyl
and so
20 on. "Lower alkoxy" encompasses straight and branched chain alkoxy groups
having
from 1 to 6 carbon atoms, such as methoxy, ethoxy and so on.
Cyclic groups can be saturated or unsaturated, and if unsaturated, can be
aromatic or non-aromatic. Heterocyclic groups typically contain one or more
nitrogen, oxygen, and/or sulphur heteroatoms, e.g., furazanyl, furyl,
imidazolidinyl,
25 imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyI (e.g.
morpholino),
oxazolyl, piperazinyl (e.g., 1-piperazinyl, pyridyl, pyrimidinyl, pyn~olidinyl
(e.g. 1-
pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl,
thiomorpholinyl
(e.g. thiomorpholino), and triazolyl. The heterocyclic groups can be
substituted or
unsubstituted. Where a group is substituted, the substituent can be alkyl,
alkoxy,
30 halogen, oxygen. or substituted or unsubstituted phenyl. (See U.S. Patent
No.
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x,654,276 and U.S. Patent No. 5,643,873, the teachings of which are herein
incorporated by reference).
Peptide mimetics that mimic the SOCS-3 protein can also be designed to
inhibit SOCS-3 activity, thereby resulting in an increase of leptin activity.
These
~ mimetics can be designed and produced by techniques known to those of skill
in the
art. (See e.g., U.S. Patent Nos. 4.612,132; 5,643,873 and 5,654,276, the
teachings of
which are herein incorporated by reference). These mimetics are based on the
SOCS-3 sequence, and possess activity antagonistic to the biological activity
of the
corresponding peptide compound, but possess a "biological advantage" over the
10 corresponding peptide inhibitor with respect to one, or more, of the
following
properties: solubility, stability, and susceptibility to hydrolysis and
proteolysis.
Methods for preparing peptide mimetics include modifying the N-terminal
amino group, the C-terminal carboxyl group, and/or changing one or more of the
amino linkages in the peptide to a non-amino linkage. Two or more such
1 S modifications can be coupled in one peptide mimetic inhibitor. Examples of
modifications of peptides to produce peptide mimetics are described in U.S.
Patent
Nos: 5,643,873 and 5,654,276, the teachings of which are incorporated herein
by
reference. Peptide mimetic libraries can also be produced as described above.
Alternatively, the SOCS-3 inhibitor can be an antibody or antibody fragment
?0 that interacts with SOCS-3, thereby preventing SOCS-3 from interacting with
downstream target molecules such as JAK2, or such that SOCS-3 interacts with
JAK2 without interfering with JAK2 kinase activity. The term "antibody" is
meant
to encompass polyclonal antibodies, monoclonal antibodies (mAbs), chimeric
antibodies (e.g., humanized antibodies) and antibody fragments that retain the
2~ biological activity of specific binding to SOCS-3, such as Fab, Fab',
F(ab')2 and Fv.
Also encompassed are single-chain antibodies (sFvs). These antibody fragments
lack
the Fc portion of an intact antibody, clear more rapidly from the circulation
and can
have less non-specific tissue binding than an intact antibody. These fragments
are
produced by well-known methods in the art, for example by proteolytic cleavage
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with enzymes such as papain (to produce Fab fragments) or pepsin (to produce
F(ab')2 fragments.
Polyclonal antibodies are heterogeneous populations of antibody molecules
derived from the sera of animals immunized with an antigen. A monoclonal
antibody (mAb) contains a substantially homogenous population of antibodies
specific to antigens, which population contains substantially similar epitope
binding
sites. MAbs may be obtained by methods known to those skilled in the art. See,
for
example Kohler and Milstein, Nature 256:495-497, 1975; U.S. Patent No.
4,376,110; Ausubel et al, eds., Current Protocols in Molecular Biology, Green
Publishing Assoc. and Wiley Interscience, N.Y., 1987, 1992; and Harlow and
Lane
Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, 1988; Colligan
et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and
Wiley
Interscience, N.Y., 1992, 1993; the contents of which references are
incorporated
entirely herein by reference. Such antibodies can be of any immunoglobulin
class
1 S including IgG, IgM, IgE, IgA, and any subclass thereof. A hybridoma
producing a
mAb of the present invention can be cultivated in vitro, in situ, or in vivo.
Production of high titers of mAbs in vivo or in sitar makes this the presently
preferred method of production.
Chimeric antibodies which include humanized antibodies, are molecules
?0 wherein different portions of which are derived from different animal
species, such
as those having variable regions derived from a murine mAb and a human
immunoglobulin constant region. Chimeric antibodies are primarily used to
reduce
immunogenicity in application and/or to increase yields in production, for
example.
Chimeric antibodies and methods for their production are known in the art
(Cabilly
25 et al., Proc Natl Acad Sci USA 81: 3273-3277, 1984; Mornson et al., Proc
Natl Acad
Sci USA 81:6851-6855, 1984; Bouiianne et al., Nature 31?:643-646, 1984;
Cabilly
et al., European Patent Application 125023 (published November 14, 1984);
Neuberger et al., Nature 314:268-270, 1985; Tani~uchi et al., European Patent
Application 171496 (published February 19. 1985); Morrison et al., European
Patent
30 Application 1739494 (published March 5, 1986); Neuberger er al., PCT
Application
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-20-
WO 86/01533, (published March 13, 1986); Kudos et al., European Patent
Application 184187 (published June 11, 1986); Sahagan et al., Jlmmunol.
137:1066-1074, 1986; Robinson et al., International Patent Publication #
PCT/US86/02269 (published 7 May 1987); Liu et al., Proc Natl Acad Sci USA
5 84:3439-3443, 1987; Sun et al., Proc Natl Acad Sci USA 84:214-218, 1987;
Better
et al., Science 240:1041-1043, 1988; and Harlow and Lane Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1988. These references are
entirely incorporated herein by reference.
Typically, antibodies of the present invention are high affinity anti-SOCS-3
10 antibodies. and fragments or regions thereof, that have potent inhibiting
and/or
neutralizing activity in vivo against SOCS-3. Such antibodies can include
those
generated by immunization using purified recombinant SOCS-3 or peptide
fragments thereof.
Methods for determining antibody specificity and affinity can be found in
15 Harlow, et al., Antibodies: A Laboratory Manual, Cold spring Harbor
Laboratory
Press, Cold Spring Harbor, NY, 1988; Colligan et al., eds., Current Protocols
in
Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., 1992, 1993;
and Muller, Meth. Enrymol., 92:589-601 1983; which references are entirely
incorporated herein by reference.
20 Further, SOCS-3 inhibitors/antagonists can function at the genetic level.
Such antagonists include agents which decrease, inhibit, block or abrogate
SOCS-3
expression, production or activity. Such an agent can be an antisense nucleic
acid or
sequence specific peptide nucleic acid. In addition, such an antagonist may
interfere
with SOCS-3 promoter activity. Further, such an antagonist can be a SOCS-3
25 mutant such as a mutant that functions as a competitive inhibitor which can
be
introduced and expressed in the cell where SOCS-3 activity is to he reduced.
The
mutant can be a full length derivative of SOCS-3 or fragments or derivatives
of
SOCS-3 as described above, such that expression of the mutant in a cell,
inhibits the
endogenous SOCS-3 activity. Such antagonists can be introduced into a cell by
30 transfection, for example calcium phosphate precipitation or lipofection;
or by
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infection with a virus or pseudovirus containing the desired construct. or by
electroproration. Methods of introducing nucleic acid into a cell are well
known in
the art.
In another embodiment. the present invention encompasses introducing into
5 a cell a nucleotide expression construct, wherein said construct encodes a
modified
form of SOCS-3. A modified form of SOCS-3 can include a dominant negative
SOCS-3. Such a molecule can competitively bind the SOCS-3 target molecule
without inactivating said target molecule (e.g., a dominant negative SOCS-3
would
bind its target molecule, such as JAK2 and prevent endogenous SOCS-3 from
10 binding, such that JAK2 remains phosphorylated, and/or such that JAK2
remains
capable of phosphorlyating the appropriate downstream molecules, such as the
cytokine receptor or STAT molecule.
Several vectors for use in such constructs are well known in the art.
Furthermore, mechanisms of delivery of said constructs to an individual are
well
15 known in the art. For example, recombinant expression vectors which include
synthetic or cDNA-derived DNA fragments encoding modified SOCS-3 molecules
comprising DNA encoding a modified SOCS-3 protein operably linked to suitable
transcriptional or translational regulatory elements derived from mammalian,
microbial, viral or insect genes. Such regulatory elements include a
transcriptional
20 promoter, an optional operator sequence to control transcription, a
sequence
encoding suitable mRNA ribosomal binding sites, and sequences which control
the
termination of transcription and translation, as described in detail below.
The ability
to replicate in a host, usually conferred by an origin of replication, and a
selection
gene to facilitate recognition of transformants may additionally be
incorporated.
25 Operably linked indicates that components are linked in such a manner that
expression of the DNA encoding a fusion protein is controlled by the
regulatory
elements. Generally, operably linked means contiguous.
Mammalian expression vectors may comprise non-transcribed elements such
as an origin of replication, a suitable promoter and enhancer linked to the
gene to be
30 expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' to
3'
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nontranslated sequences, such as necessary ribosome binding sites, a poly-
adenylation site, splice donor and acceptor sites. and transcriptional
termination
sequences.
The transcriptional and translational control sequences in expression vectors
5 to be used in transforming vertebrate cells may be provided by viral
sources. For
example, commonly used promoters and enhancers are derived from Polyoma,
Adenovirus 2, Simian V irus 40 (SV40), and human cytomegalovirus. DNA
sequence derived from the SV40 viral genome, for example, SV40 origin, early
and
late promoter, enhancer, splice, and polyadenylation sites may be used to
provide the
10 other genetic elements required for expression of a heterologous DNA
sequence.
The early and late promoters are particularly useful because both are obtained
easily
from the virus as a fragment which also contains the SV40 viral origin or
replication
(Fiers et al., Nature 273:113, 1978. Smaller or larger SV40 fragments may also
be
used, provided the approximately 250 by sequence extending from the Hind III
site
15 toward the BgII site located in the viral origin or replication is
included. Exemplary
vectors can be constructed as disclosed by Okayama and Berg (Mol Cell Biol
3:280,
1983.
Preferred eukaryotic vectors for expression of mammalian DNA include
pIXY321 and pIXY344, both of which are yeast expression vectors derived from
20 pBC102.K22 (ATCC 67,255) and yeast.
In a further embodiment of the present invention, a method is provided to
increase leptin induced signaling, wherein leptin-induced signaling results in
the
phosphorylation of STAT molecules, thereby increasing the amount of gene
transcription of STAT-responsive genes.
35 In another embodiment of the present invention it is desirable to decrease
or
down-regulate leptin activity via increasing the activity of the SOCS-3
mediated
leptin cell signaling pathway, thereby resulting in weight gain or
prevention/alieviation of symptoms of atypical depression. SOCS-3 activity can
be
increased by introducing into a cell a nucleic acid construct expressing SOCS-
3 or a
30 biologically active fragment thereof. In this embodiment the SOCS-3
protein, or
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biologically active fragment of SOCS-3, comprises a SOCS-3 protein or fragment
with biological activity comparable to the activity of endogenous SOCS-3,
resulting
in the negative regulation of leptin activity.
The present invention further provides methods to identify molecules that
~ modulate the SOCS-3-mediated leptin cell-signaling pathway. Specifically
encompassed by the present invention are methods to identify inhibitors/
antagonistslagonists of SOCS-3 activity. Inhibitors of SOCS-3 activity can be
identified and tested in in vitro assays and in ex vivo cell-based assays, as
described
herein. Candidates exhibiting the desired activity in vitro or ex vivo can be
further
10 evaluated in art-accepted animal models.
Candidate inhibitors, such as peptides, small organic molecules or
derivatives of JAK2, can be evaluated for their ability to specifically
interact with
SOCS-3 in standard binding or capture assays known in the art. For example,
SOCS-3 can be immobilized to a suitable surface (such as wells of a plastic
1 ~ microtiter plate or on beads) and contacted under physiological conditions
to the
peptide library, organic molecule library or JAK2 derivatives that have been
labeled
for subsequent detection. In another embodiment, the peptide or small organic
molecule library; the antibody or antibody fragments or the target molecule or
target
molecule derivatives can be immobilized on a solid support and contacted with
20 SOCS-3.
Peptide libraries, such as an oriented peptide library (Z. Songyang et al.
Cell
71:767, 1993; can be screened for peptides that interact with SOCS-3. Peptide
libraries and other small organic molecule libraries can also be screened
using other
assays known in the art, such as proximity assays or Biospecific Interaction
Analysis
25 (BIA). Biospecific Interaction Analysis (BIA) in real time can be performed
to
evaluate candidate molecules fir their ability to bind SOCS-3. Surface plasmon
resonance (SPR), which is the basis for BIA measurements, is an optical
phenomenon arising in metal films under conditions of total internal
reflection. The
phenomenon produces a sharp dip in the intensity of reflected light at a
specific
30 angle. The position of this resonance angle depends on several factors,
including the
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refractive index of the medium close to the non-illuminated side of the metal
film.
Refractive index is directly related to the concentration of dissolved
material in the
medium. By keeping other factors constant, SPR is used to measure changes in
the
concentration of macromolecules in a surface layer of solution in contact with
a
dextran-coated gold film. Using the BIAcoreTM instrument from Pharmacia
Biosensor AB, the association and dissociation rate constants for a peptide or
organic molecule binding to SOCS-3 can be measured. Polypeptides peptides,
peptide mimics or small organic molecules exhibiting higher association
constants
(Ka) have the greatest potential for ability to interact with SOCS-3 and
inhibit
~10 SOCS-3 activity.
The present invention includes cell lines suitable for use in the screening
methods described herein. In one embodiment, the cell line is a mammalian cell
line
such as CHO cells, Ba/F3 cells, HepG2 cells or H35-hepatoma cells, wherein
said
cells stably express a cytokine receptor and a reporter gene construct wherein
the
reporter gene construct is active in the absence of SOCS-3. The cell line is
further
modified by the introduction of SOCS-3 whereby the reporter gene construct is
inhibited by SOCS-3 expression. In one embodiment the cytokine receptor is the
leptin receptor long form. In another embodiment, the reporter gene encodes
luciferase. In another embodiment, the reporter gene encodes ~-galactosidase.
In a
further embodiment, the reporter gene construct contains SOCS-'i promoter
elements.
In a preferred embodiment, the cell lines, cell signaling components {such as
leptin receptors, JAK2), SOCS-3 are of human origin.
Candidate antagonists/agonists can be assessed for their ability to
25 inhibit/enhance SOCS-3 activity, by their ability to allow reporter gene
expression
or cell proliferation of SOCS-3 expressing cells comprising the steps of
culturing
the cells described above under conditions suitable for maintenance and
growth;
contacting said cells with the candidate molecule or an organic molecule
library
comprising SOCS-3 inhibitors or transfecting the cells with a cDNA expressing
the
candidate molecule with a cDNA expression library comprising DNA encoding
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candidate SOCS-3 inhibitors; contacting the cells with leptin; selecting the
cells
having increased reporter gene activity and identifying the organic molecule
or
cDNA that had contacted the cells selected. Methods of measuring gene
transcript
and enhancing or inhibition thereof are well known to those of skill in the
art.
5 The present invention further encompasses a cytokine dependent cell line
wherein the cells also stably express SOCS-3 and the leptin receptor long
form. For
example. the cytokine can be IL-3, IL-6 and other closely related cvtokines.
In one
embodiment, the cytokine dependent cell line is Ba/F3 cells. In another
embodiment, the cytokine is IL-3. The invention further provides a method of
10 isolating and identifying inhibitors of SOCS-3, comprising the steps of
culturing the
cvtokine-dependent cells described above in the presence of said cvtokine
under
conditions suitable for maintenance and growth; removing said cells from the
cytokine (in the case of BA/F3, the cytokine would be IL-3), contacting the
cells
with a candidate organic molecule or with a library comprising SOCS-3
inhibitor
1 S molecules or transfecting said cells with a cDNA expressing a SOCS-3
candidate
inhibitor or a eDNA expression library comprising DNA encoding candidates
SOCS-3 inhibitors; contacting said cells with leptin under conditions suitable
for
growth and maintenance of the cells; selecting cells capable of proliferating
in the
presence of leptin and identifying the organic molecule of cDNA that contacted
the
20 cells selected as described. Methods to transfect cells with eDNA
expression
libraries and subsequently isolate the cDNA are well known in the art.
(Sambrook et
al, Molecular Cloning).
Candidate inhibitors/agonists can further be evaluated in animal models.
Animal models where SOCS-3 activity can be evaluated are known in the art, for
25 example see Leibel et al., J. Biol. Chen:. 272:319337-319340, 1997.
Inhibitors identified as described by the present invention can be useful to
treat obesity or prevent weight gain in a mammal. Such molecules may also be
useful to treat affective depression, such as melancholic depression. to
induce the
onset of puberty or to correct reproductive dysfunction. Alternatively, some
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affective disorders can be treated by decreasing leptin activity. For example
a
SOCS-3 agonist can be administered to an individual in need of such treatment.
The present invention further encompasses methods of reducing food intake
in a mammal comprising increasing leptin cell-signaling comprising inhibiting
5 SOCS-3 activity. In one embodiment, the mammal loses bodyweight.
The present invention further comprises a method of inducing puberty in a
mammal comprising increasing leptin cell signaling comprising inhibiting SOCS-
3
activity.
The present invention further comprises a method of treating a mood
10 affective disorder in a mammal comprising inhibiting SOCS-3 activity.
The antagonists/agonists of the present invention can be formulated into
compositions with an effective amount of the inhibitor/antagonist/agonist as
the
active ingredient. An effective amount of a SOCS-3 inhibitor/antagonist is an
amount effective to partially or completely inhibit SOCS-3 activity resulting
in
15 increased leptin activity. An effective amount of a SOCS-3 agonist is an
amount
effective to enhance SOCS-3 activity resulting in a decrease of leptin
activity.
Methods to evaluated leptin activity, such as monitoring food intake, energy
expenditure, weight gain/loss, reproductive function and neuroendocrine
function
are well-known to those of skill in the art. It will be appreciated that the
actual
20 effective amounts of the inhibitor/antagonist/agonist in a specific case
will vary
according to the specific compound being utilized, the particular composition
formulated, the mode of administration and the age, weight and condition of
the
mammal, for example. Dosages for a particular mammal can be determined by one
of ordinary skill in the art using conventional considerations, (e.g. by means
of an
25 appropriate, conventional pharmacological protocol).
Such compositions can also comprise a pharmaceutically acceptable cattier,
and are referred to herein as pharmaceutical compositions. The compositions of
the
present invention can be administered intravenously, parenterally, orally, by
transdetirtal patch, by inhalation or by suppository. The
inhibitoriantagonist/agonist
30 composition may be administered in a single dose or in more than one dose
over a
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period of time to achieve a level of inhibitoriantagonist/agonist which is
sufficient to
confer the desired effect.
Suitable pharmaceutical carriers include, but are not limited to water, salt
solutions, alcohols, polyethylene glycols, gelatin. carbohydrates such as
lactose,
amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin,
fatty acid
esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc. The pharmaceutical
preparations can be sterilized and desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing
osmotic pressure, buffers, coloring, and/or aromatic substances and the like
which
10 do not deleteriously react with the active compounds. They can also be
combined
where desired with other active agents, e.g., enzyme inhibitors, to reduce
metabolic
degradation.
For parenteral application, particularly suitable are injectable, sterile
solutions, preferably oily or aqueous solutions, as well as suspensions,
emulsions, or
1 ~ implants, including suppositories. Ampoules are convenient unit dosages.
The inhibitors/antagonists/agonists of the present invention can be
administered to an individual mammal in need of such treatment, in conjunction
with an agent or agents that allow the inhibitor to pass through the blood
brain
barrier. The inhibitor/antagonist/agonist and the agent can be administered
20 simultaneously or sequentially. Such agents are known in the art, such as
those
described in US Patents 5,112,596; 5,268,164; 5,686,416 and 5,506,206; the
teachings of which are incorporated herein by reference in their entirety.
The following Examples are offered for the purpose of illustrating the present
invention and are not to be construed to limit the scope of this invention.
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EXAMPLES
Example I : Quantification of CIS and SOCS mRNAs by RT-PCR in oblob mice
after leptin administration.
.-Id libitum fed male oblob mice (Jackson Laboratories, Bar Harbor, ME) aged
S 7-8 weeks were injected intraperitoneally with 100 pg recombinant mouse
leptin,
(from Eli Lilly, Indianapolis, IN) or saline. Two hours later, the mice were
decapitated, the skull was reflected from the brain, and hypothalami were
isolated by
snap freezing in liquid nitrogen. Samples of cerebellum, kidney and liver were
also
taken. Total RNA from the various tissues was isolated using the RNA-STAT-60
10 reaeent as described by the manufacturer (TEL-TEST, Inc., Friendswood, TX).
Total RNA purification and subsequent cDNA synthesis was done in parallel from
all tissue samples. The cDNA was synthesized from 1.0 pg of total RNA by using
dT- oIigonucleotides and the Advantage RT-PCR kit from Stratagene (La Jolla,
CA). The final volume of the cDNA samples was 100 ~1. The following primers
15 were used for specific PCR amplification of mouse CIS-1, mouse SOCS-l,
mouse
SOCS-2 and mouse SOCS-3:
CIS-lA: 5'-ctggagctgcccgggccagcc-3', 400 by {GenBank Acc. Number D31943),
SEQ ID NO:1;
CIS-1 B: 5'-caaggctgaccacatctgg~ 3', SEQ ID N0:2;
20 SOCS-IA: 5'-ccactccgattaccggcgcatc-3', 350 by (GenBank Accession Number
U88325), SEQ ID N0:3;
SOCS-1B: 5'-gctcctgcagcggccgcacg-3',SEQ ID N0:4;
SOCS-2A: 5'-aagacgtcagctggaccgac-3', 300 by (GenBank Acc. Number U588327),
SEQ ID NO:S; SOCS-2B:5'-tcttgttggtaaaggcagtccc-3', SEQ ID N0:6;
2~ SOCS-3A: 5'-accagcgcczcttcttcacg-3'. 450 by (GenBank Acc. Number U88328),
SEQ ID N0:7;
SOCS-3B:5'-gtggagcatcatactgatcc-3', SEQ ID N0:8.
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Each 50 ~1 PCR reaction was carried out with 5.0 pl of cDNA as template. The
assay conditions were: 10 rrilVl Tris-HCl (pH 8.8), 50 mM KCI, I.S mM MgCI,.
0.01% gelatin 0.2 mM dNTPs. 20 pmol of each primer. 2.~ units of Taq
polymerise
(Stratagene) and 1.0 ~1 of'-'P-dCTP (29.6 TBq/mmol. 370 MBq/ml)(NEN, Boston,
MA). The mixture was overlaid with 25 wl of mineral oil, and after initial
denaturation at 96°C for 3 min the samples were subjected to 24-32
cycles of
amplification: denaturation at 95°C for 1 min, annealing at 60°C
for I min, and
extension at 7?°C for 45 seconds. Ten ul of the reaction were then
combined with 5
~g of sequencing stop solution (Amersham International, Buckinghamshire, LTK)
10 and heated to 85°C for f ve minutes before loading ~ ~1 onto a 4%
urea-acrylamide
gel (38 x 31 x 0.03 cm). Electrophoresis was carried out at 60 W of constant
power
four hours, before the gels were transferred to filter paper, dried and
finally
subjected to'-P quantification by Phosphorimager analysis (Molecular
Dynamics).
Preliminary PCR experiments showed that the rate of amplification was linear
I S for CIS-1, SOCS-1 and SOCS-3 when applying less than 30 PCR-cycles. The
amplification rate of SOCS-2 was linear for 27 cycles, after which non-linear
amplification appeared. We chose 25 cycles of PCR amplification for
quantification
of CIS-1, SOCS-1, SOCS-2 and SOCS-3. PCR reactions were spiked with
'ZP-dCTP and assembled in parallel for each cDNA and subjected to PCR
20 amplification under the above conditions of limiting number of cycles. PCR
products were then separated on denaturing acrylamide gels and finally
subjected to
autoradiography.
Ad libitum fed male oblob mice aged 7-8 weeks were injected intraperitoneally
(ip) with 100 pg recombinant mouse leptin, or saline. Two hours later, total
RNA
25 was purified from hypothalami, and quantitative RT-PCR for CIS, SOCS-1,
SOCS-2
and SOCS-3 mRNAs was performed. Leptin treatment caused a 2.0 fold increase in
SOCS-3 mRNA, while no effect on CIS, SOCS-l, or SOCS-2 mRNA levels were
detected (Figure lA and 1B). A similar effect on SOCS-3 mRNA was seen I or 3
hours after leptin administration (data not shown). ~1o effect of leptin on
CIS,
30 SOCS-1, SOCS-2 or SOCS-3 mRNA was detected in cerebellum, kidney or liver
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(data not shown). To determine whether the effect of leptin on hypothalamic
SOCS-
3 mRNA was mediated by the long form of the leptin receptor. a similar
experiment
was performed in dbldb mice and control littermates. Leptin increased SOCS-3
mRNA 2.2 fold in hypothalamus of control mice (+/?), while no effect of leptin
was
~ detected in dbldb mice.
Example 2: Localization of SOCS-3 mRNA by in situ hybridization in the rodent
brain after in vivo leptin administration.
In order to localize the specific anatomic regions of the hypothalamus and
other
10 parts of the brain in which leptin affects SOCS-3 mRNA leveis,'sS-labeled
RNA
antisense probe was generated. The SOCS-3A and SOCS-3B primers from above
were used amplify a 450 base pair fragment of the mouse SOCS-3 cDNA. The PCR
products were cloned into pCR2.1 (Invitrogen, Carlsbad, CA) according to the
manufactures recommendations. The orientation of the cloned cDNA was verified
I S by sequencing using standard double-stranded plasmid techniques. For
generation of
sense'--'S-labeled RNA, the plasmid was linearized by digestion with BamHI,
and
subjected to in vitro transcription with T7 polymerase according to the
manufactures
protocols (Promega). In situ hybridization histochemistry was conducted
according
to methods well known in the art (Simmons). Tissue sections of mouse and rat
brain
20 were mounted onto slides, air dried, and stored in desiccated boxes at -
20°C. Prior to
hybridization, the slides were immersed in 10% neutral buffered formalin,
incubated
in 0.001% proteinase K (Boehringer Mannheim) for 30 min., then in 0.025%
acetic
anhydride for 10 min., and dehydrated in ascending concentrations ethanol. The
RNA probes were then diluted to 10°cpm/ml in hybridization solution
of 50%
25 formamide, 10 mM Tris-HCI, pH 8Ø ~ mg tRNA, 10 mM dithiothreitol.
10°,'°
dextran sulfate, 0.3 M NaCI, I mM EDTA, pH 8, and 1 x Denhardt's solution
(Sigma>. Hybridization solution and a glass coverslip was applied to each
slide and
sections were then incubated for 12-16 hours at ~6°C. The coverslips
were removed
and the slides washed 4 times with 4x SSC. Sections were then incubated in
0.002%
30 RNAase A (Boehringer Mannheim) with 0.5 M NaCI, 10 mM Tris-HCI, pH 8, and 1
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mM EDT A, for 30 min. at 37°C. Sections were rinsed in decreasing
concentrations
of SSC containing 0.25% DTT: 2x at 50°C for 1 hour, 0.2x at 55°C
for 1 hour, and
0.2x for 1 hour at 60°C. Sections were next dehydrated in graded
ethanol (50, 70,
80, and 90%) containing 0.3 M NH40Ac followed by 100% ethanol. Slides were air
5 dried and placed in X-ray film cassettes with BMR-2 film (Kodak) for 3-5
days.
Slides were then dipped in NTB2 photographic emulsion (Kodak), dried and
stored
with desiccant in foil-wrapped slide boxes at 4°C for 2-3 weeks. Slides
were
developed with D-19 developer (Kodak), counterstained with thionin, dehydrated
in
graded ethanol, cleared in xylene, and coverslipped with Permaslip. Sections
were
10 analyzed v~~ith a Zeiss Axioplan light microscope using brightfield and
darkfieId
optics. Photomicrographs were produced by capturing images with a digital
camera
(Kodak, DCS) mounted directly on the microscope and an Apple Macintosh Power
PC computer. Image editing software (Adobe Photoshop) was used to combine
photomicrographs into plates and figures were printed on a dye sublimation
printer
15 (Kodak 8600). Only the sharpness, contrast, and brightness were adjusted.
The
results are shown in Figure 2. In brain sections from normal rats fed ad
libiturn and
given a single intravenous injection of recombinant leptin ( 1 pg/g body
weight),
strong specific hybridization was detected in the arcuate nucleus (Arc) and
the
dorsomedial hypothalamic nucleus (DMH), as compared to saline injected rat
brain
20 sections (Figure 2B and 2A, respectively). In other regions of the brain,
including
the cerebellum, no specific hybridization signals were detected.
Example 3: SOCS-3 inhibits leptin induced transcriptional activation in CHO
cells.
SOCS-3 was tested for its ability to inhibit leptin induced transcriptional
activation. erg-1 is an immediate early gene induced upon leptin stimulation.
25 erg-1-luc is a reporter construct expressing the promoter elements of erg-1
fusrd to
the luciferase gene. CHO cells were transiently transfected the leptin
receptor erg-1-
luc together with either alone or with CIS-1, SOCS-2 or SOCS-3. As shown in
Figure 3A, SOCS-3, but not CIS-1 or SOCS-2 blocked leptin-induced activation
of
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the erg-1 luciferase reporter construct while serum-induced erg-1 gene
transcription
was unaffected by expression of SOCS-3.
Example 4: Localization of SOCS-3 mRNA by in situ hybridization in the
"Agouti"
mouse.
5 The Agouti (or lethal yellow, Ay/a) mouse is an autosomal dominant marine
obesity model. Obesity in these mice is accompanied by increased linear growth
and altered hair pigmentation. Like other non ob or db mouse models of
obesity,
Agouti mice have elevated levels of leptin and are refactory to leptin
treatment either
intravenously or injected directly into brain tissue. The disorder is caused
by ectopic
10 and unregulated expression of agouti, a protein normally restricted to hair
follicles,
where it affects pigmentation by antagonizing melanocyte stimulating hormone
(-MSH). Agouti also antagonizes MC4 receptors, whose expression is largely
restricted to the brain. SOCS-3 expression was localized in brain tissue of
Agouti
mice following the methods described in Example 2. Specific hybridization was
15 detected in the arcuate nucleus, while no specific hybridization signals
were detected
in other regions of the brain. This data supports the theory that expression
of
SOCS-3 plays a role in the desensitization of these animals to leptin
signaling and
hence is an important factor in the loss of weight control in these animals.
Example 5: Suppression of leptin receptor signaling by SOCS-3 in mammalian
cell
20 lines.
SOCS-3 was tested for its effect on leptin receptor signaling in mammalian
cell
lines. COS-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM.
low glucose) supplemented with 10% fetal calf serum (FCS1, 100 units/ml
penicillin
and 10 pgiml streptomycin at 37°C in 5% CO~. CHO cells were grown in
HAM's
25 F12 medium supplemented with 10% FCS, 100 units/ml penicillin, and 10 ~g/ml
streptomycin. In all experiments including JAK cDNA, the amount of transfected
JAK cDNA was 1/10 of the total amount of DNA transfected. For Western blotting
experiments, cells were grown in 10 cm dishes and transfected using 80 pl of
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Lipofectamine and a total of 20 pg of plasmid DNA. Cells were serum-deprived
for
12-15 h prior to stimulation with hormones. Cells were harvested 48 hours post
transfection. For Western blotting experiments, cells were rinsed in ice-cold
phosphate-buffered saline, and scraped into 1000 pl of ice-cold Iysis buffer B
(1%
~ Nonidet P-40. 0.5% Triton X-100, 10% glycerol, 150 mM NaCI; 2 mNI Na,VO,, 20
mM NaF, 1 mM phenylmethylsulfonyl fluoride, 5 pg/ml leupeptin, 5 p/ml
aprotinin,
50 mM Tris-HCI, pH 7.4). Lysates were finally clarified by centrifugation at
23,000
g for 15 min. and the supernatant immunoprecipitated as described below.
Immunoprecipitations were performed at 4°C by incubating clarified cell
extracts
10 with the 12CA5 or OBR antibodies and protein A-agarose beads (1:15 dilution
of a
50% slurry in 1% Nonidet P-40, 0.5% Triton X-100, 10%, glycerol, 150 mM NaCI,
50 mM Tris-HCI, pH 7.4) on a rotating wheel overnight. The agarose beads were
pelleted by low speed centrifugation and washed 3 times with 1 ml of ice-cold
lysis
buffer B. For immunoblotting, proteins were boiled for 5 min. and subjected to
15 SDS-PAGE, followed by transfer of the resolved polypeptides to
nitrocellulose
membranes. The membranes were blocked with 10% nonfat dried milk in Towbin
buffer (20 mM Tris-HC1, pH 7.4, 150 mM NaCI, 0.05% Tween 20) for 2 h at room
temperature and then incubated with antibodies in 5% milk for 12-15 h at
4°C. After
removal of unbound antibodies by three washes each for 20 min in Towbin
buffer,
20 membranes were incubated with horseradish peroxidase-conjugated anti-rabbit
or
anti-mouse immunoglobulin (1:1000) in 2.5% milk for 1.5 h at room temperature
and washed five times in Towbin buffer. The targeted proteins were detected
using
enhanced chemiluminescence (ECL) as described by the manufacturer (Amersham
International, Buckinghamshire, CTK). Stripping of nitrocellulose membranes
was
25 done by soaking membranes in 1% SDS, 70 mM Tris-HC1, pH 6.8 and 0.1%
mercaptoethanol at 50°C for 30 minutes with slow agitation.
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Leptin Receptor and Stat3 Phosphorylation.
COS-1 cells were transiently co-transfected with expression vectors for mouse
OBR1 and JAK2. together with either pcDNA3, or HA-tagged CIS-1, SOCS-2,
SOCS-3 in PCDNA3 (Invitrogen).
5 The intact coding region of CIS was cloned by combining an EST (TIGR clone
ID 104844, provided by Damien Dunnington, SmithKline Beecham
Pharmaceuticals) with a 5'RACE product derived from human skeletal muscle
mRNA (Clontech, Palo Alto, CA). The intact coding region of SOCS-2 was cloned
by combining an EST (IMAGE clone ID 131550, WashU-Merck EST project} with
10 a 5'RACE product derived from human skeletal muscle Marathon cDNA
(Clontech,
Palo Alto, CA). The intact coding region of SOCS-3 was cloned by PCR
amplification from Balb/c mouse genomic DNA (Sigma). A tandem hemagglutinin
tag (HA) as fused to the C-terminal ends of all clones, before subcioning into
the
mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA). All clones were
15 verified by sequencing.
Forty-eight hours post transfection including 15 hours of serum starvation,
cells
were either treated or not with 1.00 nM leptin for 10 minutes. Western
blotting of
leptin receptor immunoprecipitates with anti-pY antibodies demonstrated that
SOCS-3 completely blocked leptin induced leptin receptor tyrosine
phosphorylation,
20 while CIS-1 or SOCS-2 had no effect (Figure 3B, top panel). SOCS-3 was
tested
for its ability to block downstream signaling by measuring STAT3 tyrosine
phosphorylation in transfected COS-1 cells. SOCS-3 also completely blocked
leptin
induced STAT3 phosphorylation, while CIS-1 and SOCS-2 were without effect.
JAK2 Phosphorylation
25 CHO cells were transfected with OBR1 and JAK2 together with either empty
vector, CIS-HA. SOCS-2-HA, or SOCS-3-HA expression vectors. Forty-eight
hours post transfection, including 15 hours of serum starvation, cells were
either
treated or not with 100 nNI mouse leptin for 5 min. J AK2 immunoprecipitates
(anti-JAK2 antibodies were from UBI) were subjected to SDS-PAGE and Western
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-3 S-
blotting with antiphosphotyrosine antibodies (4610, UBI) or anti-JAK2
antibodies
(Santa Cruz).
SOCS-3, but not CIS or SOCS-2, inhibited leptin tyrosine phosphorylation of
JAK2. Expression of CIS-HA, SOCS-2-HA or SOCS-3-HA has no effect on JAK2
S protein expression in these cells.
Example 6: Leptin-dependent Co-immunoprecipitation of JAK2 with SOCS-3.
COS-1 cells were transfected with expression vectors encoding OBRI and
JAK2, together with either SOCS-2-HA or SOCS-3-HA expression vectors as
described in Example S. Forty-eight hours post transfection, including 1 S h
of
10 serum starvation, cells were stimulated or not with 100 nM leptin for S
minutes.
HA-immunoprecipitates (anti-HA antibodies were from Babco) were subjected to
SDS-PAGE and Western blotting with anti-JAK2 antibodies (Santa Cruz).
JAK2 was co-immunoprecipitated with SOCS-3, but not SOCS-2, in a
leptin-dependent manner in transfected COS-1 cells. These data are consistent
with
1 S results published earlier on SOCS-1 in transfected 293 cells (Endo et al.,
1997).
These results, together with the results from above (Figure 3), are consistent
with the
possibility that SOCS-3 inhibits leptin-receptor signal transduction by
interacting
with JAK2 and subsequently inhibiting its tyrosine-kinase activity as proposed
for
SOCS-1 by Endo et al., 1997.
20 Example 7: Activation of SOCS-3 mRNA by leptin in CHO cells stably
expressing
leptin receptor.
CHO cells stably expressing either then long OBRI or short (OBRs) form of the
leptin receptor were grown and serum-deprived as described in Example 4. Total
RNA was isolated from confluent cells grown in 10 cm dishes using the RNA-STAT
2S method as described in Example 1. Northern blotting was performed according
to
standard procedures (Sambrook et al., 1989), and probed with a j=P-labelled
DNA
probe (Gibco-BRL random labelling kit) encompassing the coding region of the
mouse SOCS-3 gene.
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Serum induces SOCS-3 mRNA in both CHO-OBR and CHO-OBRs cells.
Leptin induced SOCS-3 mRNA levels after 1 hour of treatment in CHO-OBRI, but
not in CHO-OBRs. After 2 and 4 hours of leptin treatment, SOCS-3 mRNA levels
return to baseline in the CHO-OBRI cells. Therefore leptin_has the capability
to
activate endogenous SOCS-3 gene-expression in cells expressing the long form
of
the leptin receptor consistent with leptin directly activating SOCS-3 mRNA in
neurons expressing OBRI. CIS and SOCS-2 mRNA were not induced by leptin in
the two cell lines.
Example 8: Leptin pretreatment of CHO-OBRI cells causes leptin-resistance in
proximal leptin-receptor signaling.
CHO-OBRI cells were tested for leptin resistance under conditions where
endogenous SOCS-3 protein levels are elevated. CHO-OBRI cells were stimulated
with leptin for 1 hour and then washed to remove leptin from the medium. At
different times after the leptin pretreatment, freshly applied leptin was
tested for the
1 S ability to induce intracellular signaling. As demonstrated by Northern
blotting,
leptin was unable to induce SOCS-3 mRNA for up to 24 hours after leptin
pretreatment. On the other hand, in leptin-pretreated cells. fetal calf serum
retained
the ability to induce SOCS-3 mRNA, suggesting that leptin pretreatment of
CHO-OBRI cells causes leptin-resistant signaling at a step upstream of the
sots-3
gene.
Because induction of sots genes by cytokines has been reported to require
STAT activation, STAT DNA-binding activities by leptin in CHO-OBRI cells was
measured using an electrophoretic-mobility-shift-assay (EMSA) specific for
STAT1
and STAT3 using methods well known in the art. Leptin rapidly induced
activation
of STAT DNA-binding activities with maximal levels detected after ~5 minutes
of
leptin treatment. However, as demonstrated by EMSA. leptin was unable to
activate
STAT for up to 24 hours after leptin pretreatment. Yet in the same leptin-
pretreated
cells, TNF-a retained a full ability to activate STAT, suggesting that leptin
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pretreatment of CHO cells causes blockade of leptin signaling at a step
upstream of
STAT activation.
Because proximal leptin signaling involves tyrosine phosphorylation by JAK
kinases of the leptin receptor, leptin pretreatment of CHO-OBRI cells was
tested for
the ability to inhibit subsequent stimulation of leptin receptor
phosphorylation.
Pretreatment with 3 or 100 rul~I leptin for 1 hour blocked the ability of
fresh leptin to
induce receptor phosphorylation. Binding of tracer leptin was not
significantly
affected by prior leptin treatment as measured 1.5-24 hours after leptin
pretreatment;
therefore, the reduced level of leptin receptor phosphorylation was not due to
10 downregulation of the leptin receptor itself. Collectively, these data
demonstrate
that leptin pretreatment of CHO-OBRI cells results in blockade of proximal
leptin
signaling without affecting surface leptin receptor expression.
EQUIVALENTS
While this invention has been particularly shown and described with references
15 to preferred embodiments thereof, it will be understood by those skilled in
the art
that various changes in form and details may be made therein without departing
from the spirit and scope of the invention as defined by the appended claims.
Those
skilled in the art will recognize or be able to ascertain using no more than
routine
experimentation, many ;.quivalents to the specific embodiments of the
invention
20 described specifically herein. Such equivalents are intended to be
encompassed in
the scope of the claims.
