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Rapid Inhibition of Leptin Signaling by Glucocorticoids in Vitro and in Vivo

2004; Elsevier BV; Volume: 279; Issue: 19 Linguagem: Inglês

10.1074/jbc.m310864200

1083-351X

Ryoko Ishida-Takahashi, Shigeo Uotani, Takahiro Abe, Mikako Degawa‐Yamauchi, Tetsuya Fukushima, Naruhiro Fujita, Hiroyuki Sakamaki, Hironori Yamasaki, Yoshihiko Yamaguchi, Katsumi Eguchi,

Adipokines, Inflammation, and Metabolic Diseases

Elevated secretion of glucocorticoids (GCs) or hypersensitivity to GCs has a permissive effect on the development of obesity and leads to abnormalities of body fat distribution. Recent studies demonstrated GCs act as antagonists of leptin in rodents. However, little is known about the interaction between GCs and leptin signaling. In the present study, we investigated the effects of GCs on leptin action in vitro and in vivo. GCs rapidly inhibited the leptin-induced STAT3 phosphorylation in a dose- and time-dependent manner, as assayed by Western blotting using anti-phosphospecific-STAT3 in human hepatoma cell lines (Huh7) transiently expressing long form leptin receptor. GCs also inhibited the leptin-induced JAK2 tyrosine phosphorylation but unaltered the specific binding of 125I-leptin to the cells. Parallel experiments, however, demonstrated that the inhibitory effects of GCs were not observed in either IL-6- or LIF-induced STAT3 phosphorylation. Furthermore, we examined the feeding behavior and hypothalamic leptin signaling following intracerebroventricular (icv) infusion of GCs prior to icv leptin infusion in Sprague-Dawley rats. The food intake after 24 h of icv leptin injection increased 3-fold in GCs-treated animals. In addition, central infusion of GCs resulted in a marked reduction of hypothalamic STAT3 phosphorylation in response to icv infusion of leptin. To clarify the molecular mechanism by which GCs rapidly reduce leptin-induced JAK/STAT signaling, we examined the intracellular signal transduction pathway potentially mediated by GCs. PD98059, a specific MEK inhibitor, blocked the inhibitory effects of GCs on leptin-induced JAK/STAT activation in Huh7 cells. These results suggest GCs antagonize leptin action by a rapid inhibition of the leptin-induced JAK/STAT pathway partly via MAPK cascade. Elevated secretion of glucocorticoids (GCs) or hypersensitivity to GCs has a permissive effect on the development of obesity and leads to abnormalities of body fat distribution. Recent studies demonstrated GCs act as antagonists of leptin in rodents. However, little is known about the interaction between GCs and leptin signaling. In the present study, we investigated the effects of GCs on leptin action in vitro and in vivo. GCs rapidly inhibited the leptin-induced STAT3 phosphorylation in a dose- and time-dependent manner, as assayed by Western blotting using anti-phosphospecific-STAT3 in human hepatoma cell lines (Huh7) transiently expressing long form leptin receptor. GCs also inhibited the leptin-induced JAK2 tyrosine phosphorylation but unaltered the specific binding of 125I-leptin to the cells. Parallel experiments, however, demonstrated that the inhibitory effects of GCs were not observed in either IL-6- or LIF-induced STAT3 phosphorylation. Furthermore, we examined the feeding behavior and hypothalamic leptin signaling following intracerebroventricular (icv) infusion of GCs prior to icv leptin infusion in Sprague-Dawley rats. The food intake after 24 h of icv leptin injection increased 3-fold in GCs-treated animals. In addition, central infusion of GCs resulted in a marked reduction of hypothalamic STAT3 phosphorylation in response to icv infusion of leptin. To clarify the molecular mechanism by which GCs rapidly reduce leptin-induced JAK/STAT signaling, we examined the intracellular signal transduction pathway potentially mediated by GCs. PD98059, a specific MEK inhibitor, blocked the inhibitory effects of GCs on leptin-induced JAK/STAT activation in Huh7 cells. These results suggest GCs antagonize leptin action by a rapid inhibition of the leptin-induced JAK/STAT pathway partly via MAPK cascade. Leptin, a hormone secreted by adipocytes (1Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11753) Google Scholar), acts via a long form of the leptin receptor (OBRb) expressed in specific nuclei of the hypothalamus (2Schwartz M.W. Seeley R.J. Campfield L.A. Burn P. Baskin D.G. J. Clin. Investig. 1996; 98: 1101-1106Crossref PubMed Scopus (1372) Google Scholar) and regulates food intake and energy expenditure (3Halaas J.L. Gajiwalak K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Science. 1995; 269: 543-546Crossref PubMed Scopus (4230) Google Scholar, 4Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Science. 1995; 269: 540-543Crossref PubMed Scopus (3869) Google Scholar, 5Campfield L.A. Smith F.J. Guisez Y. Devos R. Burn P. Science. 1995; 269: 546-549Crossref PubMed Scopus (3067) Google Scholar). 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Chem. 2000; 275: 14563-14572Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar) on the receptor, similar to STATs proteins (8Baumann H. Morella K.K. White D.W. Dembski M. Bailon P.S. Kim H. Lai C.F. Tartaglia L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8374-8378Crossref PubMed Scopus (763) Google Scholar, 9Bjorbaek C. Uotani S. da Silva B. Flier J.S. J. Biol. Chem. 1997; 272: 32686-32695Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar, 10Banks A.S. Davis S.M. Bates S.H. Myers Jr., M.G. J. Biol. Chem. 2000; 275: 14563-14572Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar). Although leptin can activate STAT1, STAT3, STAT5, and STAT6 (11Wang Y. Kuropatwinski K.K. White D.W. Hawley T.S. Hawley R.G. Tartaglia L.A. Baumann H. J. Biol. Chem. 1997; 272: 16216-16223Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 12Ghilardi N. Ziegler S. Wiestner A. Stoffel R. Heim M.H. Skoda R.C. Proc. Natl. Acad. Sci. U. S. 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Cell. 1998; 92: 573-585Abstract Full Text Full Text PDF PubMed Scopus (3149) Google Scholar, 23Shimada M. Tritos N.A. Lowell B.B. Flier J.S. Maratos-Flier E. Nature. 1998; 396: 670-674Crossref PubMed Scopus (990) Google Scholar). Hypercortisolism resulting from exogenous administration or endogenous overproduction of glucocorticoids (GCs) is known to cause central obesity associated with hyperinsulinemia and also hyperleptinemia (24Leal-Cerro A. Considine R.V. Peino R. Venegas E. Astorga R. Casanueva F.F. Dieguez C. Horm. Metab. Res. 1996; 28: 711-713Crossref PubMed Scopus (98) Google Scholar). GCs-mediated regulation of cytokine production and action has been extensively investigated. GCs inhibit the transcription of most proinflammatory cytokines such as IL-1, IL-2, IL-6, IL-8, interferon-γ, and tumor necrosis factor-α (25Brattsand R. Linden M. Aliment. Pharmacol. Ther. 1996; 10: 81-90Crossref PubMed Scopus (289) Google Scholar). 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However, the molecular basis for the cross-talk between the OBRb and GCs pathway has remained unclear. In the present study, we report the acute effects of DEX on the leptin-induced JAK/STAT pathway in vitro and in vivo. We also further investigated the intracellular signal transduction mechanism of GCs-mediated modulation of leptin signaling. Materials—Recombinant mouse leptin was obtained from R&D Systems, Inc. (Minneapolis, MN). All reagents for cell culture were obtained from Invitrogen. Anti-JAK2 and -STAT3 antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-STAT5 and -STAT6 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphospecific JAK2 was from BioSource (Camarillo, CA). Anti-STAT1, -phosphospecific STAT1, STAT3, STAT5, and STAT6 were from Cell Signaling Technology (Beverly, MA). DEX, IL-6, LIF, CelLytic-M mammalian cell lysis/extraction reagent, and CelLytic-MT mammalian tissue lysis/extraction reagent were purchased from Sigma. Nitrocellulose membranes were from Schleicher & Schuell. Anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase were purchased from Amersham Biosciences. Cell Culture and Transient Transfection—Huh7 cells were grown in RPMI supplemented with 5% fetal calf serum, 50 units/ml penicillin G, and 50 μg/ml streptomycin at 37 °C under 5% CO2. The cDNA encoding mouse leptin receptor long form (OBRb) was generated as described previously (9Bjorbaek C. Uotani S. da Silva B. Flier J.S. J. Biol. Chem. 1997; 272: 32686-32695Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar). For binding assays, Huh7 cells were grown in 24-well plates and transfected using 0.5 μl of LipofectAMINE (Invitrogen) and 700 ng of DNA per well according to the manufacturer's protocol. For Western blotting experiments, cells were grown in 6-well plates and transfected using 7 μl of LipofectAMINE and 2 μg of DNA per well. At 48 h post-transfection including 15 h of serum deprivation, cells were incubated for 5 or 15 min with 1 nm leptin. Cells were then washed three times with ice-cold phosphate-buffered saline (phosphate-buffered saline), dissolved in lysis buffer, CelLytic-M, and centrifuged at 12,000 rpm at 4 °C for 20 min. Binding Assays—Huh7 cells expressing OBRb were incubated with 100,000 cpm of 125I-leptin (PerkinElmer Life Sciences) in RPMI medium containing 1 mg/ml bovine serum albumin (Sigma) at 4 °C for 2 h in the presence or absence of 100 nm unlabeled mouse recombinant leptin. The cells were then washed three times with ice-cold phosphate-buffered saline and dissolved in lysis buffer consisting of 1% Nonidet P-40, 0.5% Triton X-100, and 1 n NaOH. The radioactivity in the lysates was measured in a gamma counter. Nonspecific binding was determined as the radioactivity bound in the presence of 100 nm unlabeled leptin. Animals—Adult male Sprague-Dawley rats obtained from Shizuoka Laboratory Animal Center (Shizuoka, Japan) were housed at 22 °C in a 12–12-h light-dark cycle environment (lights on at 7:00 and off at 19:00) and provided with standard laboratory diet (Oriental Yeast, Tokyo) and tap water ad libitum. The experimental protocol was approved by the Animal Care and Use Committee at Nagasaki University. Placement of icv Cannulae—At 9 weeks of age and weighing 320–360 g, a 23-gauge stainless steel guide cannula was stereotaxically implanted into the third cerebral ventricle (6 mm anterior to the interaural line, 7.7 mm in depth from the brain surface) by using the SR-6N Stereotaxic Instrument (Narishige Scientific Instrument Laboratory, Tokyo, Japan) under pentobarbital anesthesia (40 mg/kg, intraperitoneal administration). A stainless steel wire stylet (29 gauge) was inserted into the guide cannula and was tightly fixed on the skull with dental cement to prevent leakage of the cerebrospinal fluid. icv Infusion of Dexamethasone—After a 7-day recovery period, the rats were divided into three groups as follows: the control group, the leptin group, and the DEX/leptin group. The rats were injected icv with a bolus of vehicle (6.0 μl of phosphate-buffered saline with 0.1% of bovine serum albumin) for the control group, leptin (7.5 μg dissolved in 6.0 μl of vehicle) for the leptin group, and DEX (5.0 μg dissolved in 6.0 μl of vehicle) and leptin (7.5 μg dissolved in 6.0 μl of vehicle) for the DEX/leptin group via the cannula (1.0 μl/min). All treatments were performed as close to 19:00 as possible, when the dark phase began. To measure the food intake of each group, the food was weighed 24 h after the icv administration of DEX, leptin, or vehicle. Tissue Excision and Preparation—At 12 h after the injection, leptin (7.5 μg dissolved in 6.0 μl of vehicle) or the same volume of vehicle was administered via the icv cannula (1.0 μl/min) to the leptin group and the DEX/leptin group. At 30 min after the infusion, the rats were decapitated, and the whole hypothalamus was dissected in ice-cold saline on the ice and immediately frozen in liquid nitrogen until the assays. The hypothalamus was homogenized in lysis buffer, CelLytic-MT, and centrifuged at 14,000 rpm at 4 °C for 10 min. The protein concentration of the supernatant was assayed using a BCA Protein Assay Reagent kit (Pierce). Western Blotting—In the in vitro study, 40 μl of the cell lysates was mixed with SDS sample buffer, and in the in vivo study, it was a 200-μg protein of the supernatant. The mixture was boiled for 5 min before the samples were applied to SDS-PAGE. After electrophoresis, proteins were transferred to a nitrocellulose membrane and blocked by incubation for 1 h at room temperature with 5% non-fat dry milk in Tris-buffered saline, 0.1% Tween 20 (TBST). The membranes were incubated with anti-phospho-STAT1, STAT3, STAT5, STAT6, or JAK2 antibody (1:1,000) in 5% milk in TBST overnight at 4 °C. After washing three times at room temperature, the membranes were reacted with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (1:2,000) in 1% milk in TBST for 60 min at room temperature, and again washed six times with TBST. The targeted proteins were detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce) following the instructions of the manufacturer. The membranes were stripped in Restore Western Blot Stripping Buffer (Pierce) according to the manufacturer's instructions. The membranes were then reprobed with anti-STAT1, STAT3 (1:1,000), STAT5, STAT6 (1:200), or JAK2 (1:1,000) antibodies. Statistical Analysis—Differences between two groups were determined by two-tailed Student's t test. p value < 0.05 was considered to be statistically significant. All values are expressed as means ± S.E. Effects of DEX on STAT3 Phosphorylation in Vitro —It is well known that administration of GCs to rodents and humans results in obesity associated with hyperleptinemia in addition to hyperinsulinemia. In this study, we examined the effects of DEX on leptin-induced JAK/STAT signaling pathway using human hepatoma cell lines (Huh7). Due to a lack of endogenous leptin receptor, we transiently transfected OBRb cDNA to Huh7 cells (33Chen J. Kunos G. Gao B. FEBS Lett. 1999; 20: 162-168Crossref Scopus (63) Google Scholar). Leptin has been shown to activate STAT1, STAT3, STAT5, and STAT6 in other cell lines (8Baumann H. Morella K.K. White D.W. Dembski M. Bailon P.S. Kim H. Lai C.F. Tartaglia L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8374-8378Crossref PubMed Scopus (763) Google Scholar, 12Ghilardi N. Ziegler S. Wiestner A. Stoffel R. Heim M.H. Skoda R.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6231-6235Crossref PubMed Scopus (734) Google Scholar). We tested which STAT proteins might be involved in leptin signaling in Huh7 cells. Huh7 cells transiently expressing long form leptin receptor (OBRb) were stimulated with 1 nm of leptin at room temperature for 15 min. The cell lysates were subsequently immunoblotted with antibodies for phospho-STAT1, STAT3, STAT5, or STAT6. The bands could only be detected with the antibody for phospho-STAT3. When these membranes were stripped and reprobed with each respective anti-STAT antibody, STAT1, STAT3, STAT5, and STAT6 proteins were all detected in the Huh7 cells (Fig. 1A). We thus tested whether DEX affects the leptin-induced STAT3 phosphorylation in Huh7 cells. The Huh7 cells transiently expressing OBRb were pretreated with physiologically relevant concentrations of DEX sequentially stimulated with leptin. The cell lysates were immunoblotted with anti-phospho-STAT3 antibodies. To investigate the dose response of inhibitory effects of DEX on leptin-induced STAT3 phosphorylation, the cells expressing OBRb were pretreated with 0–10 nm DEX for 1 h at 37 °C before 1 nm leptin stimulation for 15 min at room temperature. A 1-h DEX pretreatment inhibited the leptin-induced STAT3 phosphorylation in a dose-dependent manner. A significant inhibitory effect of DEX was detected from 0.1 nm DEX pretreatment (Fig. 1B). The leptin-induced STAT3 phosphorylation was completely inhibited by 10 nm DEX pretreatment. Furthermore, to determine the time course of DEX inhibitory effects on leptin-induced STAT3 phosphorylation, the cells were pretreated in 10 nm DEX for various times. The inhibition of leptin-induced STAT3 phosphorylation was detected after a 5-min pretreatment with 10 nm DEX and inhibited completely after 1 h (Fig. 1C). All membranes were stripped and reprobed with anti-STAT3 antibodies. The total amount of STAT3 protein was not affected by DEX. OBRb is a member of the class I cytokine receptor superfamily, which includes receptors for IL-6, LIF, prolactin, erythropoietin, and growth hormone (GH). We performed parallel experiments with Huh7 cells expressing endogenous IL-6 and LIF receptor. DEX showed no effect on IL-6- or LIF-induced STAT3 phosphorylation in either a dose- or time-dependent manner (Fig. 1, D and E). Effects of DEX on JAK2 Phosphorylation in Vitro —JAK2 is presumed to be STAT3 tyrosine kinases involved in signaling by the cytokine subfamily to which OBRb belongs. We therefore examined whether DEX affects leptin-induced JAK2 tyrosine phosphorylation by Western blot analysis with anti-phospho-JAK2 antibody. Because Huh7 cells did not have enough endogenous JAK2 protein to be detected by using Western blotting, both JAK2 and OBRb were transiently co-transfected into the cells. When Huh7 cells co-transfected with JAK2 and OBRb were stimulated with 1 nm of leptin for 5 min, JAK2 tyrosine phosphorylation was increased to a greater extent compared with that in cells co-transfected with empty vector and OBRb. The degree of JAK2 tyrosine phosphorylation in response to leptin appears to be correlated with the relative amount of JAK2 protein. However, leptin-induced STAT3 phosphorylation in cells co-transfected with JAK2 and OBRb was almost equal to that in cells co-transfected with empty vector and OBRb (Fig. 2A). In parallel experiments, we examined IL-6- or LIF-induced JAK2 tyrosine phosphorylation in Huh7 cells transfected with empty vector or JAK2 cDNA. We could not detect the tyrosine phosphorylation of endogenous JAK2 in Huh7 cells transfected with empty vector after stimulation by IL-6 or LIF. Each ligand markedly stimulated the tyrosine phosphorylation of JAK2 in cells transfected with JAK2 (Fig. 2B). Similar to the results obtained for STAT3 phosphorylation mediated via leptin, the amount of JAK2 protein did not affect STAT3 tyrosine phosphorylation in response to either ligand. Next, we examined the effects of DEX on leptin-induced JAK2 phosphorylation using Huh7 cells co-transfected with JAK2 and OBRb. The cells expressing both JAK2 and OBRb were incubated with or without 10 nm DEX for 1 h and stimulated with 1 nm leptin for 5 min. DEX completely inhibited the leptin-induced JAK2 tyrosine phosphorylation in cells co-expressing JAK2 and OBRb, as assayed by Western blotting using anti-phosphospecific JAK2 (Fig. 2C). The membrane was stripped and reprobed with anti-JAK2 antibodies. The total amount of JAK2 protein was not affected by DEX. We did not determine the tyrosine kinase activity of JAK2 in this study. In general, the catalytic activity of JAK2 was stimulated by phosphorylated tyrosine at position 1007 (34Ihle J.N. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1144) Google Scholar). We used anti-phosphospecific JAK2 antibody that recognizes this critical site for JAK2 kinase activity. Therefore, phosphorylation of JAK2 should be correlated with the activity of JAK2 in the present study. We next examined the effects of DEX on leptin-induced STAT3 phosphorylation in Huh7 cells co-transfected with JAK2 and OBRb. Similar to previous results (Fig. 1, B and C), DEX attenuated the phosphorylation of STAT3 in cells co-expressing JAK2 and OBRb (Fig. 2C). Effects of Dexamethasone on 125I-Leptin Binding—To test whether DEX decreases the cell surface expression of leptin receptor in Huh7 cells transiently expressing OBRb, we studied the effects of DEX on 125I-leptin binding to the cells. Cells were incubated with 0–100 nm of DEX for 1 h and then subjected to 125I-leptin binding assay. DEX pretreatment had no significant effects on the specific binding of 125I-leptin to the cells expressing OBRb (Fig. 3). The Effects of icv Infusion of DEX on Leptin Action in Vivo —To test whether DEX antagonizes the inhibitory effect of leptin in vivo, we measured the cumulative food intake 24 h after the infusion in Sprague-Dawley rats. As expected, a single icv infusion of leptin significantly suppressed food intake compared with the vehicle-infused control rats. Cumulative food intake 24 h after combined icv infusion of leptin and DEX increased 3-fold compared with icv leptin infusion alone (Fig. 4A). Next we examined the effect of DEX on the leptin signaling in the hypothalamus. Western blotting was carried out using anti-phospho-STAT3 antibody as a primary antibody in the hypothalamus at 30 min after icv infusion of leptin. Pretreatment of icv DEX infusion for 12 h prior to infusion of leptin decreased the leptin-induced STAT3 phosphorylation in the hypothalamus without affecting the total amount of STAT3 protein (Fig. 4B). The Mechanism of the DEX Effects on JAK/STAT Signaling Pathway—The mechanism by which DEX inhibited leptin-induced JAK/STAT signaling was further examined. DEX rapidly inhibited the leptin-induced STAT3 phosphorylation, suggesting that these effects did not require de novo protein synthesis. DEX has been shown to modulate the activity of several protein kinases, including protein kinase C (35John C. Cover P. Solito E. Morris J. Christian H. Flower R. Buckingham J. Endocrinology. 2002; 143: 3060-3070Crossref PubMed Scopus (46) Google Scholar) and mitogen-activated protein kinase (MAPK) (36Qiu J. Wang P. Jing Q. Zhang W. Li X. Zhong Y. Sun G. Pei G. Chen Y. Biochem. Biophys. Res. Commun. 2001; 287: 1017-1024Crossref PubMed Scopus (48) Google Scholar). To determine whether the inhibitory effects of DEX on leptin-induced STAT3 phosphorylation are mediated through these kinases, we investigated the inhibitory effects of DEX by using the specific inhibitors of protein kinase C and MAPK. Rottlerin, an inhibitor of protein kinase C, and SB202190, a p38 MAPK inhibitor, had no effect on the DEX inhibition of leptin-induced STAT3 activation (Fig. 5A). In contrast, when 1 μm of PD98059, a MEK inhibitor, was added to the cells prior to treatment with DEX, the inhibitory effect of DEX on leptin-induced STAT3 phosphorylation was blocked. PD98059 by itself had no effect on the STAT3 phosphorylation (Fig. 5B). We also determined whether PD98059 affects the inhibition of signaling events upstream of STAT3 phosphorylation. We thus investigated the effect of PD98059 on leptin-induced JAK2 phosphorylation that is inhibited by DEX. Pretreatment of PD98059 prevented DEX-mediated inhibition of JAK2 phosphorylation. PD98059 itself did not alter the total amount of JAK2 protein or tyrosine phosphorylation of JAK2 (Fig. 5C). The current data demonstrate that DEX inhibits the leptin-induced STAT3 signaling pathway in a cultured cell line and also in the rat hypothalamus. GCs cause an increase in the level of ob gene expression and leptin secretion in isolated adipose tissue from rodents and humans (29Slieker L.J. Sloop K.W. Surface P.L. Kriauciunas A. LaQuier F. Manetta J. Bue-Valleskey J. Stephens T.W. J. Biol. Chem. 1996; 271: 5301-5304Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 35John C. Cover P. Solito E. Morris J. Christian H. Flower R. Buckingham J. Endocrinology. 2002; 143: 3060-3070Crossref PubMed Scopus (46) Google Scholar). On the other hand, GCs decrease gene expression for most cytokines including IL-6 and LIF (25Brattsand R. Linden M. Aliment. Pharmacol. Ther. 1996; 10: 81-90Crossref PubMed Scopus (289) Google Scholar). Furthermore, several studies suggested that GCs have a major ability to modify the actions of leptin in vivo (31Zakrzewska K.E. Cusin I. Sainsbury A. Rohner-Jeanrenaud F. Jeanrenaud B. Diabetes. 1997; 46: 717-719Crossref PubMed Google Scholar, 32Zakrzewska K.E. Cusin I. Stricker-Krongrad A. Boss O. Ricquier D. Jeanrenaud B. Rohner-Jeanrenaud F. Diabetes. 1999; 48: 365-370Crossref PubMed Scopus (172) Google Scholar). However, the molecular mechanisms responsible for this antagonism remain unclear. In the present study, a rapid inhibitory effect of DEX on the leptin-induced STAT3 phosphorylation was observed from 0.1 nm of DEX pretreatment in cultured cells expressing OBRb. This concentration in blood level can be considered within the physiologically relevant range (37Halleux C.M. Servais I. Reul B.A. Detry R. Brichard S.M. J. Clin. Endocrinol. Metab. 1998; 83: 902-910Crossref PubMed Scopus (115) Google Scholar). Little is known about the pharmacokinetics of DEX in the central nervous system of humans. One study reported the concentration of DEX in plasma and central nervous system after intravenous administration of DEX to rabbits (38Lamiable D. Vistelle R. Nguyen-Khac M. Millart H. J. Chromatogr. 1988; 434: 315-319Crossref PubMed Scopus (10) Google Scholar). Comparing these two concentrations, central nervous system:plasma ratio is around 10% after 6 h from DEX administration. This inhibitory effect of DEX was not observed in other cytokine receptors such as IL-6 and LIF receptor. We are unable to explain the difference between leptin and the IL-6 or LIF system with regard to the effects of DEX. Although OBR is a member of the class I cytokine receptor family, it has been demonstrated that OBR transduces the signal via its homooligomerization without gp130, the signal transducing component for IL-6 and LIF receptor (39Nakashima K. Narazaki M. Taga T. FEBS Lett. 1997; 403: 79-82Crossref PubMed Scopus (75) Google Scholar). Furthermore, the structural analysis revealed that the extracellular domain of OBR contains two repeating cytokine receptor domain/fibronectin type 3 domains, in contrast to other members of the class I family (40Fong T.M. Huang R.C. Tota M.R. Mao C. Smith T. Varnerin J. Karritskiy V.V. Krause J.E. Van der Ploeg L.H.T. Mol. Pharmacol. 1998; 53: 234-240Crossref PubMed Scopus (134) Google Scholar). Hypersecretion of corticosterone has been observed in genetically obese rodents (41Ahima R.S. Prabakaran D. Flier J.S. J. Clin. Investig. 1998; 101: 1020-1027Crossref PubMed Scopus (549) Google Scholar) and obese humans (42Strain G.W. Zumoff B. Strain J.J. Levin J. Fukushima D.K. Metabolism. 1980; 29: 980-985Abstract Full Text PDF PubMed Scopus (127) Google Scholar). Continuous central administration of GCs to normal rats resulted in obesity associated with hyperleptinemia, in keeping with the increase in hypothalamic neuropeptide Y levels (32Zakrzewska K.E. Cusin I. Stricker-Krongrad A. Boss O. Ricquier D. Jeanrenaud B. Rohner-Jeanrenaud F. Diabetes. 1999; 48: 365-370Crossref PubMed Scopus (172) Google Scholar). A central bolus injection of low dose leptin showed no significant effects in normal rats, but significantly decreased food intake and body weight in adrenalectomized rats (31Zakrzewska K.E. Cusin I. Sainsbury A. Rohner-Jeanrenaud F. Jeanrenaud B. Diabetes. 1997; 46: 717-719Crossref PubMed Google Scholar). In the present study, an icv bolus administration of GCs (DEX) to normal rats attenuated the inhibitory effects of exogenous leptin on food intake. In agreement with those in vitro findings, GCs markedly inhibit leptin-induced STAT3 phosphorylation in the rat hypothalamus. These results suggest the short term effects of GCs on food intake may be partly mediated via an inhibition of central leptin signaling. The findings in the present study demonstrated that the inhibitory effect of GCs occurs at JAK2 upstream of STAT3 in Huh7 cells. DEX has been reported to cause a rapid decrease of the cell surface number in GH receptors, and subsequently to inhibit the tyrosine phosphorylation of JAK2 in fibroblasts (43King A.P.J. Tseng M. Logsdon C.D. Billestrup N. Carter-Su C. J. Biol. Chem. 1996; 271: 18088-18094Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). GH receptor is a member of the cytokine receptor superfamily that includes receptors for IL-6, LIF, G-CSF, prolactin, and leptin. We have shown previously that in contrast to GH receptor, no significant effects on leptin binding were observed in DEX-pretreated Chinese hamster ovary cells stably expressing OBRa or OBRb (44Uotani S. Bjorbaek C. Tornoe J. Flier J.S. Diabetes. 1999; 48: 279-286Crossref PubMed Scopus (207) Google Scholar). Consistent with previous findings, the present observations showed DEX had no effects on the cell surface number of OBRb in Huh7 cells transiently expressing OBRb. It was reported previously that GCs inhibit IL-2-induced JAK/STAT signaling that suppresses the expression of IL-2 receptor and JAK3 in primary human T cells (27Bianchi M. Meng C. Ivashkiv L.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9573-9578Crossref PubMed Scopus (114) Google Scholar). A more recent study demonstrated that inhibition of STAT1 expression as a mechanism by which GCs inhibit INF-γ signaling (28Hu X. Li W. Meng C. Ivashkiv L.B. J. Immunol. 2003; 170: 4833-4839Crossref PubMed Scopus (127) Google Scholar). The inhibitory effects on the expression of these proteins involved in JAK/STAT signaling were mediated by a genomic mechanism of GCs action. Apart from the classic genomic action of GCs, it has been well documented that GCs also have rapid intracellular signal transduction pathway action (45Buttgereit F. Scheffold A. Steroids. 2002; 67: 529-534Crossref PubMed Scopus (243) Google Scholar). Because the inhibitory effect of DEX on leptin signaling occurred so rapidly, it is suggested that the mechanism does not require de novo protein synthesis. We showed that PD98059, an inhibitor of MEK, significantly blocked the DEX effect on leptin-induced JAK2/STAT3 phosphorylation in Huh7 cells. The present results suggest that the upstream signaling protein MEK might be involved in the DEX-mediated inhibition of leptin signaling. There have been several reports that the MEK-ERK pathway participates in the regulation of cytokine-induced JAK/STAT activation. In PC12 cells, it was reported that GCs rapidly activate ERK1/2 MAPK in a MEK-dependent manner (36Qiu J. Wang P. Jing Q. Zhang W. Li X. Zhong Y. Sun G. Pei G. Chen Y. Biochem. Biophys. Res. Commun. 2001; 287: 1017-1024Crossref PubMed Scopus (48) Google Scholar). Furthermore, MEK-ERK cascade can mediate the inhibition of IL-6- but not INF-α-induced STAT3 activation in Chinese hamster ovary cells (46Sengupta T.K. Talbot E.S. Scherle P.A. Ivashkiv L.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11107-11112Crossref PubMed Scopus (204) Google Scholar). Recently, we reported ethanol inhibits leptin-induced STAT3 phosphorylation partly via p38 MAPK, activated by inflammatory cytokines and environmental stresses in Huh7 cells (47Degawa-Yamauchi M. Uotani S. Yamaguchi Y. Takahashi R. Abe T. Kuwahara H. Yamasaki H. Eguchi K. FEBS Lett. 2002; 525: 116-120Crossref PubMed Scopus (16) Google Scholar). A more recent study (48Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2634) Google Scholar) demonstrated that the targeted disruption of c-Jun amino-terminal kinase 1 (JNK1), also called stress-activated protein kinase, shows reduced adiposity and increased insulin sensitivity associated with enhanced insulin receptor signaling in both genetically and diet-induced obese mice. These findings suggest that MAPKs may play an important role in the modification of insulin and leptin action. In conclusion, the present results provide evidence for a direct cross-talk between the leptin and GCs signaling pathways in vitro and in vivo. In Huh7 cells, GCs rapidly inhibit the leptin-induced JAK/STAT pathway partly mediated via MAPK cascades. In vivo GCs attenuate hypothalamic leptin signal transduction pathway and reduce the sensitivity in response to central administration of leptin. We thank Dr. Seiichi Chiba, Dr. Hironobu Yoshimatsu, and Dr. Toshiie Sakata for helpful advice on animal studies.