Patent Application: US-99959509-A

Abstract:
the invention relates to methods for identifying compounds which modulate the interaction between stat3 an sp1 . a peptide is provided which is able to bind stat3 and interfere with the interaction of stat3 and sp1 . the invention provides methods for identifying compounds which are capable of binding to the peptide and thus release interference with the interaction between stat3 and sp1 , as well as methods for identifying inhibitors and enhancers of the stat3 sp1 interaction . compounds identified by the methods of the invention are useful in the repression or stimulation of appetite in a patient , useful for the treatment of leptin resistance , obesity and anorexia .

Description:
the details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention , by way of example . it will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details . dna constructs : the pomc promoter - luciferase construct ( pgl3 - pomc ) was a generous gift from dr . domenico accili ( columbia university , usa ), pcdna3 - flag - mfoxo1 from dr . fukamizu ( japan ), pn3 - sp1 fl - complete from dr . suske ( germany ). pxj40 - flag - stats was described previously ( 20 ). all the other dna constructs and primers used in this study , including truncation and mutation constructs based on pgl3 - pomc , are described in tables 1 and 2 . cell culture and luciferase assay : flp - inhek293 stable cell lines over - expressing obra ( 293 - obra ) or obrb ( 293 - obrb ) were described previously ( 21 ). cells were cultured in dulbecco &# 39 ; s minimal essential medium ( dmem , invitrogen ) containing 10 % fetal bovine serum ( fbs ) in a 37 ° c . incubator with 5 % co2 . one day after plating , cells were transfected with relevant dna constructs using fugene 6 ( roche ). 16 hr later , transfected cells were serum - starved for 5 hr before they were treated with recombinant leptin ( invitrogen ) or vehicle for 20 hr . cells were then washed with pbs and lysed in 200 μl of 1 × passive lysis buffer included in dual - luciferase reporter assay system ( promega ). luciferase activity was measured from cell extracts on aluminometer ( molecular devices ). the firefly luciferase activity was normalized against renilla luciferase activity . detection of obra and obrb in 293 stable cell lines : 293 - obra and 293 - obrb cells were harvested and lysed with lysis buffer , and incubated with leptin - coupled cnbr - activated sepharose beads ( sigma ) overnight . after repeated washing with lysis buffer , the beads with pulled down proteins were subjected to sds - page . leptin receptor expression was examined by using myc antibodies . leptin binding to stable hek293 cells : this was performed in six - well plates as previously described ( 21 ). briefly , 293 - obra or 293 - obrb cells were grown to ˜ 90 % confluence and washed with pbs . cells were incubated with approximately 60 , 000 cpm of murine recombinant 125i - leptin ( perkin - elmer ) alone , or 125i - leptin with excessive amount of unlabeled leptin ( 2 μg / well ) for 6 hr at 4 ° c . in a final volume of 1 ml pbs supplemented with 1 % ( w / v ) bsa ( fraction v , sigma ). at the end of incubation , unbound 125i leptin was removed by two pbs washes . 1 ml of 1n naoh was then added , and radioactivity in the lysate was measured using a wizard 1470 automatic gamma counter ( perkin - elmer ). nuclear extract preparation from 293 cells : cells after treatment with leptin or vehicle were washed twice and collected in cold pbs . the cell suspension was centrifuged at 1 , 300 rpm for 5 min . the resulting pellet was resuspended with hypotonic buffer ( 20 mm hepes ph 7 . 9 , 10 mm kcl , 1 mm edta , 1 mm na3vo4 , 10 % glycerol , 0 . 2 % np - 40 , 20 mm naf , 1 mm dtt and 1 × complete protease inhibitor ( roche )), and rocked at 4 ° c . for 10 min . the mixture was then centrifuged at 13 , 000 rpm for 30 sec , and high salt buffer ( 20 % glycerol , 420 mm nacl , 1 mm na3vo4 , 1 mm dtt and 1 × complete protease inhibitor in hypotonic buffer without np - 40 ) was added to resuspend the pellet . after 40 min rocking , the mixture was centrifuged at 13 , 000 rpm for 10 min at 4 ° c . the supernatant was collected as the nuclear extract . co - ip : 1 ) for stats - sp1 interaction , 293 - obrb cells were transfected with pxj40 - flag - mstat3 , and followed by leptin treatment . nuclear extracts were prepared from the cells and incubated with sp1 antibody for immunoprecipitation ( ip ). immunoblotting of the immunoprecipitation was performed using phospho - stat3 antibody ( cell signaling ). 5 % of cell lysate used in each colp sample was loaded as input . 2 ) for stat3 - foxo1 interaction , 293 - obrb cells transfected with expression vectors of pxj40 - flag - mstat3 and pcdna3 - myc - mfoxo1 were serum - starved and treated with leptin ( 50 nm ) for 30 min , and then lysed in lysis buffer ( 20 mm tris - cl , ph 7 . 5 , 150 mm nacl , 1 % triton - x - 100 , 10 mm naf , 1 mm edta , 1 mm na3vo4 , 1 mm pmsf , supplemented with protease inhibitors ). ˜ 500 μg cell lysate was incubated for 2 hr with 1 μg flag ( sigma ), myc ( santa cruz biotechnology ) antibodies , or control igg , respectively , followed by ip with protein a + g sepharose beads ( sigma ) for 1 hr . the immunoprecipitates were washed 4times in lysis buffer and subjected to sds - page and immunoblotting with antibodies against flagor myc . 5 % of cell lysate used in each colp sample was loaded as input . 3 ) for foxo1 effects on stat3 - sp1 interaction , pxj40 - flag - mstat3 and increasing amount of pcdna3 - myc - mfoxo1 were transfected into 293 - obrb cells . cells were harvested for nuclear fractionation after leptin treatment . binding of stat3 to sp1 in nuclear extracts was examined by ip with flag antibody and ib with myc ( for stat3 ) and sp1 antibodies . immunoblotting : cells were lysed in 1 × cell lysis buffer ( cell signaling ) containing 1 mm pmsf . lysate was incubated on ice for 20 min with gentle rocking and centrifuged at 20 , 000 × g for 10 min at 4 ° c . equivalent amount of samples were analyzed by sds - page and immunoblotting using antibodies against phospho - stat3 ( cell signaling technology ); pan - stat3 , foxo1 , sp1 and myc ( santa cruz biotechnology ); flag ( sigma ); and myc ( polyclonal , upstate ). emsa : two pairs of oligonucleotides : wild type ( gag gcc cgc cgc ccc cct and gaa gggggg cgg cgg gc ) and sp1 binding site mutant sequence ( gag gct tgt tgc ccc cct and gaa ggg gaa caa cgg gc ) were annealed , and about 100 ng of the probes were labelled with 50 μci of 32p dctp by klenowexo -( neb ). after labelling , the probes were purified by using g - 50column , and radioactivity was measured with ls6500 multi - purpose scintillation counter ( beckmam coulter ). 5 μg of nuclear protein was incubated with the probe with 20 , 000 cpm in dna - protein loading buffers ( 50 mm nacl , 10 mm triscl ph 7 . 5 , 0 . 5 mm edta , 1 mm mgcl2 , 4 % ficoll , 0 . 5 mm dtt and 1 × complete protease inhibitor ) in a total volume of 12 μl at room temperature for 15 min . the mixture was resolved by 4 % page gel in 0 . 5 × tbe , and the gel dried at 80 ° c . by using a gel dryer ( bio - rad ) for 2 hr . super sensitive x - ray film ( kodak ) was exposed for 48 hr at − 80 ° c . and then developed . immunocytochemistry : 293 - obrb cells were transfected with relevant plasmids one day after they were plated on poly - lysine coated coverslips . after leptin or mock treatment , the cells were washed with pbs , fixed in pbs containing 4 % paraformaldehyde for 10 min , permeabilized in pbs containing 0 . 5 % triton x - 100 for 10 min , and blocked in icc buffer ( 3 % bsa , 3 % goat serum , and 0 . 15 % triton x - 100 in pbs ) for 1 hr at room temperature . the cells were then probed by using stat3 and foxo1 antibodies , and fluorescence conjugated secondary antibodies ( invitrogen ). coverslips were mounted on slides and sealed for observation by confocal microscopy . statistical analysis : the data were presented as means ± s . e . m . comparisons of data were made using two - tailed student &# 39 ; s t - test for independent data . the significance limit was set at p & lt ; 0 . 05 . to understand how stat3 signaling may be inhibited downstream of its activation , a cell - based system was established to investigate how stat3 mediates leptin regulation of gene expression . the cell - based system includes stable expression of obrb , and transient expression of firefly luciferase under the pomc promoter . pomc promoter was chosen to study stat3 - mediated leptin regulation because : 1 . pomc is a key anorexigenic neuropeptide that is regulated by leptin and stat3 ( 19 ), 2 . pomc expression is reduced in leptin - resistant dio mice ( 18 ). leptin regulates energy homeostasis mainly through its central action by binding and activating the long form leptin receptor obrb , but not the other forms ( 5 , 6 ). hek 293 cell lines with stable expression of obrb ( 293 - obrb ) were established as an in vitro system to study leptin regulation of pomc promoter activity . hek 293 cells over - expressing obra ( 293 - obra ) was used as a negative control . in these cell lines , only a single copy of the gene construct with c - terminal myc tagging ( fig1 a , upper panel ) was integrated into the genome to ensure consistent expression level of respective receptors . since expression level of the receptors in the stable cell lines was not abundant enough for direct detection from cell lysate by western blotting , the proteins were concentrated by using leptin - coupled beads . obra or obrb could be detected only in respective stable cell lines , but not the control ( fig1 a , lower panel ). to further confirm the expression of obra or obrb , and to validate their proper localization and orientation on the cell surface in these cell lines , cells were incubated with 125i - labeled leptin in the presence or absence of excessive unlabeled leptin . 125i - labeled leptin could bind both 293 - obra and 293 - obrb to a similar extent ( fig1 b ). radioactivity of 125i - labeled leptin , indicative of leptin binding , was not detectable in control cells or in the presence of excessive unlabeled leptin ( fig1 b ). a plasmid containing the luciferase gene driven by pomc promoter was introduced into 293 - obrb and the control 293 - obra by transient transfection to test whether 293 - obrb cells could be used as an in vitro system to study leptin regulation of promoter activity . we used the pomc promoter containing − 646 to + 65 of the pomc gene , as full promoter activity requires no more than 480 by dna fragment upstream of transcription initiation site ( 13 , 22 ). leptin treatment induced stat3 phosphorylation only in 293 - obrb cells ( fig1 c ). similarly , leptin stimulated luciferase activity was only observed in 293 - obrb , but not in 293 - obra cells ( fig1 d ), consistent with previous findings that only obrb is capable of leptin signal transduction ( 6 ). taken together , 293 - obrb was a suitable system in studying pomc promoter activity regulation by stat3 - mediated leptin signaling . in early stages of leptin resistance , levels of phospho - stat3 are comparable in mice on high fat diet with those on normal chow diet , indicating that impairment of leptin signalling lies downstream of stat3 activation ( 10 ). to mimic the early stages of leptin resistance , in which stat3 phosphorylation was not reduced , 293 - obrb cells were transfected with the amount of stat3 that resulted in maximal level of leptin induced pomc promoter activation ( data not shown ). an increasing amount of foxo1 cdna was introduced on the background of constant stat3 level ( fig2 a ) to test whether foxo1 could interfere with leptin - induced pomc promoter activity . foxo1 expression levels increased proportionately with increasing amounts of cdna used for transfection ( fig2 a ). although leptin - induced stat3 phosphorylation was not affected by increasing foxo1 expression , leptin - regulation of pomc promoter activity , as indicated by luciferase activity , was abolished at high expression levels of foxo1 ( fig2 b ). leptin - regulation of pomc promoter activity was not affected when increasing amount of a similar - sized control protein was introduced ( data not shown ). these data demonstrate that high levels of foxo1 could interfere with leptin signalling , and suggest foxo1 acted at a step downstream of stat3 activation . to further delineate at which step increasing foxo1 affected leptin signalling , we tested whether foxo1 suppressed stat3 translocation into nucleus after leptin activation . 293 - obrb cells were transfected with increasing amount of foxo1 cdna on the background of constant stat3 level , and nuclear and cytoplasmic components were separated by fractionation . as expected , foxo1 protein levels increased in the nuclear fraction ( fig3 a , second panel ) with increasing amount of foxo1 cdna ; while phosphorylated stat3 in the nucleus remained at the same level regardless of foxo1 expression levels ( fig3 a , first panel ). to directly visualize the effects of foxo1 on leptin - induced stat3 activation and translocation into the nucleus , we performed immunocytochemistry and confocal microscopy were performed on 293 - obrb cells expressing stat3 alone or stat3 plus foxo1 . stat3 signals were mostly cytoplasmic without leptin stimulation ( fig3 b , panel a & amp ; b ), but concentrated in the nucleus in leptin - treated samples ( fig3 b , panel c & amp ; d ). the extent of stat3 translocation into the nucleus as indicated by the stat3 signal in the nucleus , was indistinguishable between cells with and those without foxo1 ( fig3 b , panel c & amp ; d ). these data showed that foxo1 affected neither leptin - induced stat3 phosphorylation nor the subsequent stat3 translocation into the nucleus , and indicated that foxo1 - mediated inhibition of leptin - regulation of pomc promoter activity happened downstream of stat3 translocation into the nucleus , i . e ., high level of foxo1 prevents stat3 from activating the pomc promoter in the nucleus . to understand how foxo1 inhibits stat3 - mediated pomc promoter activation , the mode of interaction between stat3 and pomc promoter was investigated . a series of mutants with deletion in the promoter region of pomc ( mutants # 1 - 11 , fig4 a ) on the background of pgl3 - pomc ( wt , fig4 a ) were made to determine the essential sequence for stat3 - mediated leptin activation of pomc promoter activity . mutant constructs , along with pgl3 - pomc , were separately introduced into 293 - obrb cells , and luciferase activity of various pomc promoter constructs with or without leptin treatment was determined . deletion mutants without dna fragment between − 138 and − 88 (# 2 , 6 and 8 ) resulted in the loss of leptin regulation of pomc promoter activity , whereas all the mutants containing this fragment retained leptin regulation , including mutant # 11 containing only this dna fragment (− 138 to − 88 ) fused directly upstream of pomc promoter tata box ( fig4 b ), indicating that a dna binding element critical to leptin - enhanced pomc promoter activity lies between − 138 and − 88 by upstream from the transcription initiation site . to identify the dna fragment of pomc promoter responsible for normal leptin response the structure of pomc promoter was investigated . sequence analysis revealed that the dna element between − 138 and − 88 contained a consensus binding sequence to sp1 ( fig5 a ), a constitutive transcription factor present in most cell types ( 23 ). to verify whether the putative sp1 binding site interacts with sp1 , probe 1 , corresponding to the original sequence , and probe 2 , containing mutations in the putative sp1 binding site ( fig5 a ) were synthesised , and emsa was performed with nuclear extracts from 293 - obrb cells . a nuclear protein bound specifically to probe 1 , but not to probe 2 , and the binding to probe 1 was specifically inhibited by a sp1 antibody ( fig5 b ), but not by stat3 or foxo1 antibodies ( data not shown ). these data indicated that the bound nuclear protein was sp1 , and sp1 and probe 1 formed a specific complex . to examine the potential function of sp1 binding site in pomc promoter activity , mutant # 12 and 13 , were generated which contained point mutations within sp1 binding site and adjacent sequence ( mutant # 12 ) or within sp1 binding site only ( mutant # 13 ) ( fig5 c ). functional analysis of these mutants in 293 - obrb cells revealed the promoter activity of both mutants as well as their regulation by leptin were abolished ( fig5 d ), indicating that leptin - mediated transcriptional activation of pomc promoter was dependent on sp1 . leptin - mediated pomc promoter activity requires direct interaction of stat3 and sp1 the lack of stat3 binding consensus sequence in the dna element between − 138 and − 88 suggested that stat3 regulation of pomc promoter activity was through a way other than direct stat3 - dna interaction , i . e . stat3 acted through an intermediate protein to mediate leptin action . as leptin - induced pomc promoter activation was dependent on sp1 , we hypothesized that stat3 regulated pomc promoter through its interaction with sp1 . co - ip using sp1 antibody resulted in abundant phospho - stat3 signal in samples from leptin - treated , but not control 293 - obrb cells , whereas the control antibody did not pull down phospho - stat3 from either leptin - treated or control cells ( fig6 a ). these data indicated that sp1 could bind to phospho - stat3 specifically , and further suggested that stat3 could act through sp1 to mediate leptin regulation of pomc promoter activity . previous studies linked two putative stat3 binding sites (− 361 to − 353 , and − 76 to − 68 ) and one foxo1 binding site (− 375 to − 370 ) to pomc expression ( 13 , 22 ). in this study , however , deletion of these stat3 binding sites ( mutant 1 , 4 , and 9 , fig4 ), or foxo1 binding site ( mutant 4 , fig4 ) had little effect on leptin regulation of pomc promoter activity . furthermore , sp1 , but not stat3 or foxo1 , was able to complex with the 51 bp dna fragment essential for leptin regulation , suggesting that phosphorylated stat3 enhance pomc promoter activity through a mechanism that requires sp1 - pomc promoter complex , instead of a direct stat3 - pomc promoter interaction . sp1 is a constitutive transcription factor , and has been reported to serve as an intermediate in stat3 regulation of gene expression ( 30 - 32 ), e . g . stat3 mediates il - 6 induced vegf promoter activity by interacting with sp1 - dna complex ( 30 ). together with this study , these studies suggest an alternative mechanism to the established direct stat3 - dna interaction in hormone / cytokine signaling , ie . stat3 may regulate gene expression through its interaction with sp1 - dna complex ( fig7 a ). inhibition of stat3 - mediated leptin regulation of pomc promoter activity by foxo1 occurred at a step downstream of stat3 translocation into the nucleus ( fig3 ) and leptin action required a direct interaction of stat3 and sp1 . whether foxo1 could interfere with stat3 - sp1 complex formation , and thus prevent stat3 from acting on the pomc promoter was tested . to test whether foxo1 could bind to stat3 , co - ip was performed on samples from 293 - obrb cells . foxo1 was specifically coimmunoprecipitated in samples treated with antibody ( anti - flag ) against flag - tagged stat3 , but not the control antibody ( fig6 b ). conversely , stat3 was pulled down in samples treated with antibody ( anti - myc ) against myc tagged foxo1 , but not the control antibody ( fig6 c ). thus , the two - way co - ip experiments confirmed foxo1 - stat3 binding . whether increasing amount of foxo1 could reduce , and even abolish sp1 binding to stat3 was tested by co - ip of 293 - obrb cells that were transfected with increasing amount of foxo1 cdna . the ability of stat3 antibody to pull down sp1 was inhibited by foxo1 , and stat3 - sp1 binding was undetectable at high foxo1 expression levels ( fig6 d ). together , these data demonstrated that foxo1 could prevent stat3 - sp1 complex formation by binding to stat3 . foxo1 is a 652 amino acid protein . to identify the foxo1 sequences essential for stat3 interaction , a series of c - terminal deletion constructs were made and tested their interaction with stat3 by coimmunoprecipitation : foxo1 ( 1 - 167 ) and other longer foxo1 mutants were able to bind to stat3 , while foxo1 ( 1 - 123 ) failed to bind to stat3 , suggesting that the region between amino acid residues 123 - 167 is important for stat3 interaction . a deletion construct , foxo1 ( 1 - 123 )-( 168 - 652 ) was made which does not contain the region identified in the previous c - terminal deletion constructs . as a control , a foxo1 mutant was made that does not contain the region between 168 - 241 , foxo1 ( 1 - 167 )-( 242 - 652 ) . co - ip experiments using the above two deletion constructs confirmed the c - terminal deletion results that the region between 124 - 167 amino acids is necessary for stat3 interaction because the foxo1 ( 1 - 123 )-( 168 - 654 ) peptide was not able to bind stat3 , but foxo1 ( 1 - 167 )-( 242 - 652 ) did bind stat3 . in summary , these data demonstrate that 1 ) phospho - stat3 activates pomc promoter in response to leptin signaling through a mechanism that requires the sp1 binding site in the pomc promoter ; 2 ) leptin action can be inhibited by foxo1 at a step downstream of stat3 phosphorylation and translocation into the nucleus ; 3 ) foxo1 binds to stat3 and prevents stat3 from interacting with the sp1 - pomc promoter complex and consequently inhibits stat3 - mediated leptin action 4 ) foxo1 binding to stat3 requires residues within the 124 - 167 region . these data provide a potential mechanism for leptin resistance , in which an increased foxo1 antagonizes stat3 - mediated leptin signaling by interfering with stat3 sp1 - target gene promoter complex formation . in light of these data , the inventors have proposed a model of a potential mechanism of how foxo1 inhibits leptin regulation of pomc promoter : with increasing amounts of foxo1 expression , foxo1 binds to phosphorylated stat3 in the nucleus ( via amino acid residues in the 124 - 167 region ), and prevents stat3 from interacting with the sp1 - pomc promoter complex and consequently inhibits stat3 mediated leptin activation of pomc promoter ( fig7 b ). 1 . zhang , y ., proenca , r ., maffei , m ., barone , m ., leopold , l ., and friedman , j . m . 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