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Leptin interferes with 3',5'-Cyclic Adenosine Monophosphate (cAMP) signaling to inhibit steroidogenesis… Lin, Qing; Poon, Ling S; Chen, Junling; Cheng, Linan; HoYuen, Basil; Leung, Peter C Oct 22, 2009

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ralReproductive Biology and ssBioMed CentEndocrinologyOpen AcceResearchLeptin interferes with 3',5'-Cyclic Adenosine Monophosphate (cAMP) signaling to inhibit steroidogenesis in human granulosa cellsQing Lin†1,2, Song Ling Poon†1, Junling Chen1, Linan Cheng3, Basil HoYuen1 and Peter CK Leung*1Address: 1Department of Obstetrics and Gynecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, V6H 3V, Canada, 2Department of Obstetrics and Gynecology, Beijing Friendship Hospital-Affiliate of Capital University of Medical Sciences, Beijing, PR China and 3School of Medicine, Shanghai Jiao Tong University, Shanghai, 200030, PR ChinaEmail: Qing Lin - qing.lin1008@gmail.com; Song Ling Poon - songlp@yahoo.com; Junling Chen - chen_junling@hotmail.com; Linan Cheng - linanc2@yahoo.com; Basil HoYuen - bhoyuen@interchange.ubc.ca; Peter CK Leung* - peleung@interchange.ubc.ca* Corresponding author    †Equal contributorsAbstractBackground: Obesity has been linked to an increased risk of female infertility. Leptin, anadipocytokine which is elevated during obesity, may influence gonadal function through modulatingsteroidogenesis in granulosa cells.Methods: The effect of leptin on progesterone production in simian virus 40 immortalizedgranulosa (SVOG) cells was examined by Enzyme linked immunosorbent assay (ELISA). The effectof leptin on the expression of the steroidogenic enzymes (StAR, P450scc, 3betaHSD) in SVOG cellswas examined by real-time PCR and Western blotting. The mRNA expression of leptin receptorisoforms in SVOG cells were examined by using PCR. SVOG cells were co-treated with leptin andspecific pharmacological inhibitors to identify the signaling pathways involved in leptin-reducedprogesterone production. Silencing RNA against leptin receptor was used to determine that theinhibition of leptin on cAMP-induced steroidogenesis acts in a leptin receptor-dependent manner.Results and Conclusion: In the present study, we investigated the cellular mechanismsunderlying leptin-regulated steroidogenesis in human granulosa cells. We show that leptin inhibits8-bromo cAMP-stimulated progesterone production in a concentration-dependent manner.Furthermore, we show that leptin inhibits expression of the cAMP-stimulated steroidogenic acuteregulatory (StAR) protein, the rate limiting de novo protein in progesterone synthesis. Leptininduces the activation of ERK1/2, p38 and JNK but only the ERK1/2 (PD98059) and p38 (SB203580)inhibitors attenuate the leptin-induced inhibition of cAMP-stimulated StAR protein expression andprogesterone production. These data suggest that the leptin-induced MAPK signal transductionpathway interferes with cAMP/PKA-stimulated steroidogenesis in human granulosa cells.Moreover, siRNA mediated knock-down of the endogenous leptin receptor attenuates the effectof leptin on cAMP-induced StAR protein expression and progesterone production, suggesting thatthe effect of leptin on steroidogenesis in granulosa cells is receptor dependent. In summary, leptinacts through the MAPK pathway to downregulate cAMP-induced StAR protein expression andprogesterone production in immortalized human granulosa cells. These results suggest a possiblePublished: 22 October 2009Reproductive Biology and Endocrinology 2009, 7:115 doi:10.1186/1477-7827-7-115Received: 12 May 2009Accepted: 22 October 2009This article is available from: http://www.rbej.com/content/7/1/115© 2009 Lin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 8(page number not for citation purposes)mechanism by which gonadal steroidogenesis could be suppressed in obese women.Reproductive Biology and Endocrinology 2009, 7:115 http://www.rbej.com/content/7/1/115BackgroundThe major function of the female gonad is to differentiateand release mature oocytes for fertilization and successfulpropagation of the species. The follicular maturation, ovu-lation and corpus luteum function of mammalian ovariesis regulated by the interaction between gonadotropinreleasing hormone, gonadotropins, ovarian sex steroidsand a variety of peptide hormones [1]. Dysfunction in thebiosynthesis of sex steroid hormones could impair nor-mal ovarian function or even cause infertility.Leptin, a key hormone in energy homeostasis and neu-roendocrine function, has a permissive role in initiatingpuberty and is crucial in the pathogenesis of reproductivedysfunction in several disease states of energy imbalance[2]. In obesity, there is an expansion of the adipose tissuewith increased leptin production [3]. Accumulating evi-dence suggests that increased leptin levels contribute tothe pathogenesis of reproductive abnormalities includinginfertility, polycystic ovary syndrome (PCOS), anovula-tion, disruption of the menstrual cycle, hyperinsulinemiaand many other conditions [4-6].Recent studies that have identified expression of the leptinreceptor in several peripheral tissues (eg. ovary, testis andadrenal gland) strongly suggest that leptin may have adirect effect on downstream endocrine targets of thereproductive axis [7,8]. In vitro studies conducted on the-cal and granulosa cells have shown that leptin has nega-tive effects on ovarian steroid output in rodent, bovineand human models. In particular, it has been found that:1) Leptin antagonizes insulin action in human granulosacells and thereby inhibits their gonadotropin-stimulatedprogesterone production [9]; 2) Leptin stimulates therelease of proinflamatory cytokines and prostaglandins inhuman placenta [4]; 3) High serum and follicular fluidleptin may account for decreased fertilization, implanta-tion and pregnancy rates of IVF in PCOS women [10].Conditions with abnormally elevated leptin concentra-tions and impaired ovarian function may be due to dis-turbed steroidogenesis. However, the mechanisms bywhich leptin modulates steroidogenesis in the ovaryremain elusive.In the present study, we investigated the underlying mech-anisms through which leptin modulates steroidogenesisin human granulosa cells. We found that treatment ofimmortalized granulosa cells with increasing doses of lep-tin inhibited 8-bromo cAMP-induced steroidogenesiswith downregulation of the de novo produced steroidog-enic acute regulatory (StAR) protein. In addition, we dem-onstrated that cAMP-regulated steroidogenic enzymes andprogesterone production could be inhibited by leptin viaMethodsCell culture and chemicalsStudies of human ovarian granulosa cells have been lim-ited by the small numbers and short life span in culture ofcells currently obtained from clinical material. UsingSV40 large T antigen, we have reproducibly immortalizedfreshly explanted human granulosa cells obtainedthrough an in vitro fertilization program. The use ofimmortalized granulosa cells for this study was approvedby the University of British Columbia Research EthicsBoard. This cell line was shown to posses the same ster-oidogenic capabilities as primary human granulosa cells[11]. Cells are maintained in growth medium(M199:MCDB 105 (Sigma-Aldrich Corp., St. Louis, MO)containing 10% (v/v) fetal bovine serum (FBS; HycloneLaborataries Inc., Logan, UT), 100 units/mL penicillin,100 g/mL streptomycin (Sigma) under a humidifiedatmosphere of 5% CO2 at 37°C and changed every 3 days.For each experiment, 2 × 105 immortalized granulosa cellswere incubated in serum free medium for 4 h prior totreatment. Human recombinant leptin, 8-bromo cAMP,and SB203580 were purchased from Sigma. PD98059 andSP600125 were purchased from Calbiochem (San Diego,CA).RNA extraction and semiquantitave PCRTotal RNA was extracted with Trizol reagent (InvitrogenInc., Burlington, ON, Canada) according to the manufac-turer's instructions. Reverse transcription was performedin a mixture containing 5 M random hexamer, 200 MdNTP, 2 U/l MMLV trasnscriptase (Promega, Madison,WI) and 1 g RNA with the corresponding buffer at 42°Cfor 90 min followed by 85°C for 10 min. The cDNA wasfurther amplified by specific primer pairs, including for-ward long-form leptin receptor (OBRb) (5'-CCA GAAACG TTT GAG CAT CT-3') and reverse OBRb (5'-CAAAAG CAC ACC ACT CTC TC-3'), forward short-form lep-tin receptor (OBRa) (5'-GAA GGA GTG GGA AAA CCAAAG-3') and reverse OBRa (5'-CCA CCA TAT GTT AACTCT CAG-3'), forward GAPDH (5'-TCC CAT CAC CATCTT CCA-3') and reverse GAPDH (5'-CAT CAC GCC ACAGTT TCC-3').Real-time PCRThe primers used for SYBR Green real-time RT-PCR weredesigned using the Primer Express Software v2.0 (AppliedBiosystems, Foster City, CA). The specific primer pairsused are forward StAR (5'-AAACTTACGTGGCTACTCAG-CATC-3') and reverse StAR (5'-GACCTGGTTGATGAT-GCTCTTG-3'), forward P450scc (5'-CAGGAGGGGTGGACACGAC-3') and reverse P450scc(5'-AGGTTGCGTGCCATCTCATAC-3'), forward 3-HSD(5'-GCCTTCAGACCAGAATTGAGAGA-3') and reverse 3-Page 2 of 8(page number not for citation purposes)the MAPK pathway. HSD (5'-TCCTTCAAGTACAGTCAGCTTGGT-3'), forwardGAPDH (5'-ATGGAAATCCCATCACCATCTT-3') andReproductive Biology and Endocrinology 2009, 7:115 http://www.rbej.com/content/7/1/115reverse GAPDH (5'-CGCCCCACTTGATTTTGG-3'). Real-time PCR was performed using the ABI prism 7000Sequence 10 Detection System (Applied Biosystems)equipped with a 96-well optical reaction plate. The reac-tions were set up with 16.5 l SYBRR Green PCR MasterMix (Applied Biosystems). All real-time experiments wererun in triplicate and a mean value was used for the deter-mination of mRNA levels. Negative controls, containingwater instead of sample cDNA, were used in each experi-ment. Relative quantification of the mRNA levels for StAR,P450scc and 3-HSD2 in ovarian cancer cells was per-formed using the comparative CT method with GAPDH asan internal standard and with the formula 2-Ct.Enzyme-Linked Immunosorbent Assay (ELISA)Immunoplates were pre-coated with IgG (Calbiochem)and nonspecific binding sites were blocked with 0.1% (w/v) BSA buffer. IgG binds to the Fc fragment of the proges-terone antibody while the Fab fragment is competitivelybound by both biotinylated progesterone (EastCoast Bio,Inc. North Berwick, ME) and progesterone in the samples.The biotinylated progesterone interacts with streptavidin-horseradish peroxidase (SA-HRP) (EastCoast Bio), whichcatalyzes the substrate solution composed of 3,3',5,5'-tetramethylbenzidine (TMB) (Dojindo Inc., Gaithersburg,MD), to produce a blue-colored solution. The enzyme-substrate reaction is stopped by the addition of sulphuricacid (H2SO4) and the solution turns yellow. The intensityof the yellow color is directly proportional to the amountof biotinylated peptide-SA-HRP complex but inverselyproportional to the amount of the progesterone in thesamples. A standard curve of progesterone with knownconcentrations was established accordingly, and the pro-gesterone concentrations in the samples were determinedby extrapolation to this standard curve. The intra- andinter-assay CV of the progesterone assay were 5.6 and10.2% respectively.Western blottingThe cells were washed twice with ice-cold PBS and lysed inRIPA buffer (150 mM NaCl, 50 mM Tris-base (pH 7.5),1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate,and 0.1% (v/v) SDS). Twenty micrograms of total proteinwere run on 12% SDS-PAGE gels and electrotransferred toa nitrocellulose membrane (Biorad laboratories, Her-cules, CA). The membrane was immunoblotted using spe-cific primary antibodies (StAR, P450scc, 3-HSD and -actin: Santa Cruz Biotechnology Inc., Santa Cruz, CA;ERK, phosphorylated ERK, JNK, phosphorylated JNK andp38: Cell Signaling Technology Inc., Danvers, MA; phos-phorylated p38: Biosource, Invitrogen) at 4°C overnight.After washing, the membranes were incubated with horse-radish peroxidase-conjugated secondary antibody for 1 h,Statistical analysisELISA and real-time PCR normalized data are representedas mean ± S.E.M. of three independent experiments. West-ern blot normalized data are represented as mean ± S.D.of three independent experiments. Statistically significantdifferences between treatments and controls were deter-mined by either two-way ANOVA followed by Bonferronitest (Figure 1 and Figure 2) or one-way ANOVA followedby Tukey test (Figure 3, 4, 5). Statistical significance wasset at P < 0.05.ResultsLeptin attenuates the 8-bromo cAMP-stimulated progesterone production in human granulosa cellsThe gonadotropin-stimulated progesterone production ingranulosa and luteal cell is critical for normal uterinefunction, establishment and maintenance of pregnancy,and mammary gland development [1,12]. To gain insightinto the impact of leptin on gonadotropin-stimulatedsteroidogenesis, we co-treated 8-bromo cAMP withincreasing concentration of leptin (10 ng/ml, 100 ng/ml,1000 ng/ml) for 24 h in immortalized human granulosacells. Results showed that leptin inhibited the 8-bromocAMP-stimulated progesterone production in a concen-tration-dependant manner, suggesting that leptin inter-feres with gonadotropin-stimulated progesteroneproduction in these cells (Figure 1).Leptin inhibited 8-bromo cAMP-stimulated progesterone production n human granulosa cellsFigure 1Leptin inhibited 8-bromo cAMP-stimulated proges-terone production in human granulosa cells. Cells were treated with increasing concentrations of leptin (10 ng/ml - 1000 ng/ml) in the presence or absence of 1 mM 8-bromo cAMP for 24 h. Media were collected and assayed for progesterone production by ELISA. Each data point in the fig-ure represents the mean ± SEM of three independent exper-iments. * above the column indicates that those groups differ significantly from untreated control (P < 0.05); (ctrl = con-Page 3 of 8(page number not for citation purposes)and visualized using the ECL system (GE Healthcare Bio-Science, Piscataway NJ).trol).Reproductive Biology and Endocrinology 2009, 7:115 http://www.rbej.com/content/7/1/115Leptin inhibits the 8-bromo cAMP-induced StAR mRNA and protein induction in granulosa cellsThe regulation of StAR, P450scc enzyme and 3-HSD iscrucial for regulating steroid hormone production in ster-oidogenic cells [13,14]. Using real-time PCR, we moni-tored the effect of leptin on these steroidogenic enzymes.Results showed that leptin inhibited the transcriptionalmRNA level of StAR (Figure 2A), but not P450scc (Figure2B) or 3-HSD (Figure 2C), in a temporally-defined man-ner. In concordance with the mRNA regulation, the pro-tein level of StAR induced by 8-bromo cAMP isActivation of ERK1/2 and p38 are involved in the leptin regulated 8-bromo cAMP-stimulated StAR protein expression and progesterone productionAs we have previously shown, immortalized human gran-ulosa cells only expressed the short form leptin receptor(Ob-Ra) (Figure 3A) [15]. It is documented that the MAPKpathway is the dominant signaling pathway downstreamof Ob-Ra [16,17]. Thus, leptin treatment of human gran-ulosa cells over different time intervals was used to moni-tor the activation of the MAPK pathway. As shown in ourresults, leptin induced the phosphorylation of ERK1/2Leptin inhibited 8-bromo cAMP-stimulated StAR mRNA and protein expressionFigure 2Leptin inhibited 8-bromo cAMP-stimulated StAR mRNA and protein expression. Cells were treated with leptin (1000 ng/ml) in the presence or absence of 8-bromo cAMP (1 mM) for different time scale (1, 3, 6, 12, 24 h) and total RNA was analyzed with real time RT-PCR to measure the temporal change of StAR mRNA (A), P450scc mRNA (B) and 3-HSD mRNA (C) levels with GAPDH as internal standard. Cell lysates from cotreatment of leptin with 8-bromo cAMP for 24 h was sub-jected to Western blot analysis to monitor the protein levels of StAR, P450scc and 3-HSD (D). The blots are a Western blot-ting representative data from three independent experiments. Each data point in the figure represents the mean ± SEM of three independent experiments. * above the bars in "A" indicate that cotreatment of leptin with cAMP group significantly differ from cAMP treated group in the particular time scale (P < 0.05). (ctrl = control).Page 4 of 8(page number not for citation purposes)significantly inhibited by the administration of leptinafter 24 h of treatment (Figure 2D).(Figure 3B), p38 (Figure 3C) and JNK (Figure 3D) in atime-dependent manner. To further elucidate the signal-Reproductive Biology and Endocrinology 2009, 7:115 http://www.rbej.com/content/7/1/115Page 5 of 8(page number not for citation purposes)Leptin induced MAPK pathway in human granulosa cellsFigure 3Leptin induced MAPK pathway in human granulosa cells. (A) The expression of leptin receptor in human immortalized granulosa cells were monitored by semi-quanti-tative RT-PCR. Upper bands showed the expression of short form leptin receptor (Ob-Ra) in 4 different passages of immortalized granulosa cells. Lower band is to detect the expression of long form leptin receptor (Ob-Rb) in immor-talized granulosa cells. GAPDH was used as internal stand-ard. Cells were treated with leptin (1000 ng/ml) for different time scale (5, 15, 30, 60 min) and cell lysates were collected and subjected to Western blot analysis to monitor the expression of phosphorylated ERK1/2 (B), phosphorylated p38 (C) and phosphorylated JNK (D). Total ERK1/2, p38, JNK and -actin were detected as internal standard. Upper bands are Western blotting representative datas from three independent experiments. Lower bars were integrated opti-cal density (IOD) of target phosphorylated protein expres-sion with target total protein normalization. Each data point in the figure represents the mean ± SD of three independent experiments. * above the bars indicates that those groups differ significantly from untreated control (P < 0.05). (ctrl = control).Figure 4Leptin acted through ERK1/2 and p-38 pathways tomodulate steroidogenesis in human granulosa cells.Cells were treated with leptin (1000 ng/ml) in thepresence or absence of ERK1/2 inhibitor (PD98059,25 M), p38 inhibitor (SB203580, 10 M) and JNKinhibitor (SP600125, 25 M) for 24 h. Cell lysateswere collected and subjected to Western blot analy-sis to monitor the expression of StAR protein (A)and media were collected and assayed for progester-one production by ELISA (B). Upper bands in (A) areWestern blotting representative data from threeindependent experiments. Lower bars were inte-grated optical density (IOD) of StAR protein expres-sion with -actin normalization. Each data point in(A) represents the mean ± SD of three independentexperiments. Each data point in (B) represents themean ± SEM of three independent experiments. *above the bars indicate that those groups differ sig-nificantly from untreated control (P < 0.05).Reproductive Biology and Endocrinology 2009, 7:115 http://www.rbej.com/content/7/1/115ing pathways involved in the leptin-inhibited steroido-genesis in human granulosa cells, we usedpharmacological inhibitors to individually block theERK1/2, p38 and JNK pathways. The effectiveness of eachinhibitor was tested prior to use in the current experi-ments (data not shown). Results showed that pretreat-ment for 30 minutes with the ERK1/2 inhibitor PD98059or the p38 inhibitor SB203580 prior to co-treatment with8-bromo cAMP and leptin reversed the inhibition of lep-tin on 8-bromo cAMP-induced StAR protein expression(Figure 4A) and progesterone production (Figure 4B) ingranulosa cells. Interestingly, pretreatment with the JNKinhibitor SP600125 did not inhibit the leptin effect. Theseresults suggest that the ERK1/2 and p38 MAPK pathwaysare necessary for leptin-mediated interference of cAMP-stimulated steroidogenesis in human granulosa cells.Leptin modulates steroidogenesis in human granulosa cells through the short form leptin receptor (OB-Ra)To further confirm the role of the leptin receptor in leptin-modulated steroidogenesis in human granulosa cells, weused siRNA technology to knockdown the endogenousexpression of the leptin receptor in these cells. The effec-tiveness of the siRNA was confirmed by Western blot anal-ysis (Figure 5A). Knockdown of the leptin receptor ingranulosa cells impaired the ability of leptin to inhibit 8-bromo cAMP-stimulated StAR protein expression (Figure5B) and progesterone production (Figure 5C). Theseresults confirm that leptin acts through its receptor onimmortalized human granulosa cells to initiate the MAPKpathway and downregulate the cAMP-induced steroido-genesis.DiscussionCompelling evidence demonstrates a direct inhibitoryaction of leptin on steroid hormone secretion. Such effectshave been independently reported by different groups inthe three major steroidogenic tissues, namely the adrenalgland, the ovary and the testis [18-22]. However, themechanisms for such an inhibitory action are only par-tially characterized, and little attention has been paid tothe molecular events involved in leptin-induced inhibi-tion of progesterone secretion in granulosa cells. To gaininsight into the mechanisms whereby leptin suppressesprogesterone secretion in vitro, we correlated the hormo-nal secretory responses to mRNA expression levels ofStAR, P450scc and 3-HSD in cAMP-stimulated immor-talized human granulosa cells after exposure to high dos-age of recombinant human leptin. In the present study wedemonstrate that the physiological induction of StAR pro-tein by cAMP, a rate-limiting step in steroidogenesis insteroidogenic cells, is significantly reduced by leptin treat-ment. These results confirm the predominantly inhibitoryonstration that the leptin short form receptor wasinvolved in leptin-mediated inhibition of cAMP-stimu-lated steroidogenesis via the activation of the MAPK sign-aling pathway.Leptin was shown to be distributed in the intact ovary andhave distinct changes in its localization during folliculo-genesis [23]. Study suggested that leptin may be producedlocally and act in autocrine or paracrine way to affect ster-oidogenesis in the human ovary [23]. During obesity,both serum and follicular fluid levels of leptin may be ashigh as 100 ng/ml [24,25]and our study provides evi-dence that this high concentration of leptin suppressedthe production of progesterone in granulosa cells. Moreimportantly, treatment with a 1000 ng/ml of high dosageleptin also strikingly inhibited the cAMP-induced ster-oidogenesis in granulosa cells. This is in agreement withfindings in PCOS demonstrating conspicuous occurrenceof leptin in the wall of polycystic follicles, higher localconcentration of leptin independent of the serum levelwith hormonal dysregulation in granulosa cells [26]which may account for the decreased pregnancy rate inthese PCOS women [10].Several reports have now demonstrated expression of bio-logically active isoforms of the leptin receptor in the endo-crine pancreas [27], the ovary [8] and the placenta [28].Consistent with this widespread expression, leptin candirectly modulate the activity of these glands. Leptin hasbeen shown to inhibit insulin secretion from pancreatic -cells [29]. Leptin decreases the production of estradioland progesterone from ovarian granulosa cells [30,31], atleast partly via inhibition of the electron transport proteinadrenodoxin [31]. Finally, leptin can modulate the releaseof hCG from human trophoblast cells in culture [32]. Inour cell model, it was shown that only the leptin shortform (Ob-Ra) receptor is expressed in the culture system.Interestingly, using siRNA to knockdown the endogenousleptin receptor reversed the inhibition of leptin on cAMP-induced StAR protein expression and progesterone pro-duction. These results suggest that Ob-Ra is functional inour culture system and that this leptin receptor isoformplays a role in the regulation of steroidogenesis in humangranulosa cells.Leptin receptors are structurally similar to the class Icytokine receptor family [33]. In humans, the leptinreceptor (OB-R) is produced in several alternativelyspliced forms, designated OB-Ra, OB-Rb, OB-Rc, OB-Re[34]. Each of these isoforms has an extracellular domainand a transmembrane domain in common, with a varia-ble intracellular domain that is characteristic for each ofthe isoforms. Based on the variable intracellular domainPage 6 of 8(page number not for citation purposes)effects that leptin exerts on progesterone production inhuman granulosa cells. In addition, it was the first dem-the isoforms are classified as short (OB-Ra), long (OB-Rb)and secreted (OB-Re) leptin receptor. Other than the clas-Reproductive Biology and Endocrinology 2009, 7:115 http://www.rbej.com/content/7/1/115sical JAK/STAT signaling pathways [35], leptin may actthrough OB-Ra or OB-Rb to trigger the MAPK cascade intwo different ways, i.e. via tyrosine phosphorylation ofJAK2 receptor-associated activation or independently ofreceptor phosphorylaton [17,36]. Although the moleculesthat are involved in transmitting the leptin signal have notexpression of specific target genes, such as c-fos and egr-1,that participate in cell proliferation and differentiation[37]. The other two members of the MAP kinase family,p38 MAP kinase and NH2-terminal c-Jun kinase/stress-activated protein kinase (JNK) have also been shown to beactivated by leptin [38,39].Many reports have demonstrated the involvement of theMAPK pathway in steroidogenesis in granulosa cells. Acti-vation of the MAPK cascade by gonadotropins mediatesdown-regulation of steroidogenesis via attenuation ofStAR expression [40]. EGF (epidermal growth factor)stimulates steroidogenesis of granulosa cells through acti-vation of the ERK-related MAP kinase pathway [41].PGF2 (prostaglandin F2)-induced MAPK activation isassociated with modulation of progesterone production[42]. In the present study, we demonstrated that leptin iscapable of inducing the phosphorylation of ERK, phos-phorylation of p38 and phosphorylation of JNK. Interest-ingly, only the ERK and p38 inhibitors were able toreverse leptin mediated inhibition of StAR protein expres-sion and progesterone production in granulosa cells. Thissuggests that these two MAPK cascades are involved in lep-tin mediated inhibition of steroidogenesis in granulosacells.ConclusionIn conclusion, we demonstrate that the StAR protein par-ticipates in the physiological inhibition of granulosa cellsfunction by leptin. This leptin-dependent fine tuning ofovarian function could be of clinical relevance in obesityand related disorders as well as in the pathogenesis ofinfertility.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsQL and SLP designed the study and performed the exper-iments and participated in discussion of the results anddrafted the manuscript. BHY, LC and PCKL were respon-sible for supervision of this work. PCKL was responsiblefor the conception, design, discussion of the results, draft-ing and critical revision of the manuscript. All authorsread and approved the final manuscript.AcknowledgementsThis work was supported by an operating grant from the Canadian Insti-tutes of Health Research (to P.C.K.L.). P.C.K.L. is the recipient of Distin-guished Scientist award of the Child & Family Research Institute. S.L.P. was recipient of Studentship Award from the Interdisciplinary Women's Repro-ductive Health Research Training Program.ReferencesLeptin acted through short form leptin receptor to downreg-ulate the 8-bromo cAMP-stimulat d steroidogenesis in human granul sa cellsFigure 5Leptin acted through short form leptin receptor to downregulate the 8-bromo cAMP-stimulated ster-oidogenesis in human granulosa cells. Cells were trans-fected with leptin receptor siRNA (si-OBR) for 24 h to reduce the endogenous expression of leptin receptor in human granulosa cells. Western blot analysis was used to monitor the efficiency of the siRNA (A). Transfected cells were then treated with leptin (1000 ng/ml) in the presence or absence of 8-bromo cAMP (1 mM) for 24 h and cell lysates were collected to detect the expressions of StAR protein (B) and media were collected and assayed for pro-gesterone production (C). Blots in (B) are Western blotting representative data from three independent experiments. -actin was detected as internal standard. Each data point in (C) represents the mean ± SEM of three independent exper-iments. * above the bars indicate that those groups differ sig-nificantly from untreated control in either vehicle or si-OBR group respectively (P < 0.05).Page 7 of 8(page number not for citation purposes)been completely elucidated, activated MEKs (MAPK/ERKkinases) phosphorylate ERKs, leading finally to the1. McGee EA, Hsueh AJ: Initial and cyclic recruitment of ovarianfollicles.  Endocr Rev 2000, 21(2):200-214.Reproductive Biology and Endocrinology 2009, 7:115 http://www.rbej.com/content/7/1/1152. 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