Growth Hormone-induced Diacylglycerol and Ceramide Formation via Gαi3 and Gβγ in GH4 Pituitary Cells
2002; Elsevier BV; Volume: 277; Issue: 50 Linguagem: Inglês
10.1074/jbc.m202130200
ISSN1083-351X
AutoresGele Liu, Liliane Robillard, Behzad Banihashemi, Paul R. Albert,
Tópico(s)Lipid metabolism and disorders
ResumoGrowth hormone (GH) secretion is regulated by indirect negative feedback mechanisms. To address whether GH has direct actions on pituitary cells, lipid signaling in GH4ZR7 somatomammotroph cells was examined. GH (EC50 = 5 nm) stimulated diacylglycerol (DAG) and ceramide formation in parallel by over 10-fold within 15 min and persisting for >3 h. GH-induced DAG/ceramide formation was blocked by pertussis toxin (PTX) implicating Gi/Goproteins and was potentiated 1.5-fold by activation of Gi/Go-coupled dopamine-D2S receptors, which had no effect alone. Following PTX pretreatment, only PTX-resistant Gαi3, not Gαo or Gαi2, rescued GH-induced DAG/ceramide signaling. GH-induced DAG/ceramide formation was also blocked in cells expressing Gβγ blocker GRK-ct. In GH4ZR7 cells, GH induced phosphorylation of JAK2 and STAT5, which was blocked by PTX and mimicked by ceramide analogue C2-ceramide or sphingomyelinase treatment to increase endogenous ceramide. We conclude that in GH4 pituitary cells, GH induces formation of DAG/ceramide via a novel Gαi3/Gβγ-dependent pathway. This novel pathway suggests a mechanism for autocrine feedback regulation by GH of pituitary function. Growth hormone (GH) secretion is regulated by indirect negative feedback mechanisms. To address whether GH has direct actions on pituitary cells, lipid signaling in GH4ZR7 somatomammotroph cells was examined. GH (EC50 = 5 nm) stimulated diacylglycerol (DAG) and ceramide formation in parallel by over 10-fold within 15 min and persisting for >3 h. GH-induced DAG/ceramide formation was blocked by pertussis toxin (PTX) implicating Gi/Goproteins and was potentiated 1.5-fold by activation of Gi/Go-coupled dopamine-D2S receptors, which had no effect alone. Following PTX pretreatment, only PTX-resistant Gαi3, not Gαo or Gαi2, rescued GH-induced DAG/ceramide signaling. GH-induced DAG/ceramide formation was also blocked in cells expressing Gβγ blocker GRK-ct. In GH4ZR7 cells, GH induced phosphorylation of JAK2 and STAT5, which was blocked by PTX and mimicked by ceramide analogue C2-ceramide or sphingomyelinase treatment to increase endogenous ceramide. We conclude that in GH4 pituitary cells, GH induces formation of DAG/ceramide via a novel Gαi3/Gβγ-dependent pathway. This novel pathway suggests a mechanism for autocrine feedback regulation by GH of pituitary function. Pituitary somatotrophs synthesize and secrete GH, 1The abbreviations used are: GH, growth hormone; IGF, insulin-like growth factor; DAG, diacylglycerol; PRL, prolactin; PTX, pertussis toxin; JAK, Janus kinase; STAT, signal transducing activator of transcription; SM, sphingomyelin; TLC, thin-layer chromatography; FBS, fetal bovine serum; PC-PLC, phosphatidyl choline phospholipase C. 1The abbreviations used are: GH, growth hormone; IGF, insulin-like growth factor; DAG, diacylglycerol; PRL, prolactin; PTX, pertussis toxin; JAK, Janus kinase; STAT, signal transducing activator of transcription; SM, sphingomyelin; TLC, thin-layer chromatography; FBS, fetal bovine serum; PC-PLC, phosphatidyl choline phospholipase C. which acts at the liver and other tissues to stimulate IGF formation, promoting somatic growth throughout the body (1Melmed S. Yamashita S. Yamasaki H. Fagin J. Namba H. Yamamoto H. Weber M. Morita S. Webster J. Prager D. Rec. Prog. Horm. Res. 1996; 51: 189-215Google Scholar, 2Giustina A. Veldhuis J.D. Endocr. Rev. 1998; 19: 717-797Google Scholar). Secretion of GH is stimulated by hypothalamic GH-releasing hormone and inhibited by the hypothalamic tetradecapeptide somatostatin and by IGF. In addition, somatostatin agonists (e.g. octreotide) and dopamine-D2 agonists (e.g. bromocryptine) are used clinically to treat acromegaly (a syndrome produced by hypersecretion of GH) and to inhibit somatomammotroph growth and GH production (3Colao A. Lombardi G. Lancet. 1998; 352: 1455-1461Google Scholar). Negative feedback via autocrine actions of GH at the pituitary has been postulated (4Asa S.L. Coschigano K.T. Bellush L. Kopchick J.J. Ezzat S. Am. J. Pathol. 2000; 156: 1009-1015Google Scholar) but is yet to be clearly demonstrated. The GH receptor is a member of the type I cytokine receptor superfamily, related to PRL and erythropoietin receptors that homodimerize to initiate signaling (5Gadina M. Hilton D. Johnston J.A. Morinobu A. Lighvani A. Zhou Y.J. Visconti R. O'Shea J.J. Curr. Opin. Immunol. 2001; 13: 363-373Google Scholar). The GH receptor signals through the JAK2 tyrosine kinase-signal transducer and activator of transcription 5 (STAT5) transcription factor pathway to induce gene expression (6Kelly P.A. Goujon L. Sotiropoulos A. Dinerstein H. Esposito N. Edery M. Finidori J. Postel-Vinay M.C. Horm. Res. 1994; 42: 133-139Google Scholar, 7Herrington J. Smit L.S. Schwartz J. Carter-Su C. Oncogene. 2000; 19: 2585-2597Google Scholar, 8Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell. Signal. 2001; 13: 599-616Google Scholar, 9Herrington J. Carter-Su C. Trends Endocrinol. Metab. 2001; 12: 252-257Google Scholar). Phosphorylation on residue Tyr-694 by JAK2 is obligatory for STAT5 activation (10Gouilleux F. Wakao H. Mundt M. Groner B. EMBO J. 1994; 13: 4361-4369Google Scholar). The two STAT5 variants, STAT5a and STAT5b, have 90% identical protein sequences and are independently regulated and activated in various cell types (11Park S.H. Liu X. Hennighausen L. Davey H.W. Waxman D.J. J. Biol. Chem. 1999; 274: 7421-7430Google Scholar). Studies using STAT5a or STAT5b knockout mice have demonstrated that STAT5b, but not STAT5a, is required for GH-induced regulation of IGF1 and sex-specific steroidogenic enzymes in liver (11Park S.H. Liu X. Hennighausen L. Davey H.W. Waxman D.J. J. Biol. Chem. 1999; 274: 7421-7430Google Scholar, 12Davey H.W. Xie T. McLachlan M.J. Wilkins R.J. Waxman D.J. Grattan D.R. Endocrinology. 2001; 142: 3836-3841Google Scholar, 13Davey H.W. Park S.H. Grattan D.R. McLachlan M.J. Waxman D.J. J. Biol. Chem. 1999; 274: 35331-35336Google Scholar). While STAT5 activation is implicated in many GH actions, other signaling pathways not involving STAT5 appear to be recruited for GH-induced stimulation of other pathways including MAPK phosphorylation and phosphatidyl inositol 3′-kinase or protein kinase C activation (14Moutoussamy S. Renaudie F. Lago F. Kelly P.A. Finidori J. J. Biol. Chem. 1998; 273: 15906-15912Google Scholar, 15Moutoussamy S. Kelly P.A. Finidori J. Eur. J. Biochem. 1998; 255: 1-11Google Scholar) in a cell type-dependent manner (16Love D.W. Whatmore A.J. Clayton P.E. Silva C.M. Endocrinology. 1998; 139: 1965-1971Google Scholar). Ceramide is a novel second messenger implicated in regulation of cell differentiation, proliferation, inflammation, and apoptosis (17Liu B. Obeid L.M. Hannun Y.A. Semin. Cell Dev. Biol. 1997; 8: 311-322Google Scholar, 18Basu S. Kolesnick R. Oncogene. 1998; 17: 3277-3285Google Scholar, 19Liu G. Kleine L. Hebert R.L. Crit. Rev. Clin. Lab. Sci. 1999; 36: 511-573Google Scholar). Ceramide plays an important role in signaling of a subgroup of cytokine receptors that includes tumor necrosis factor and interleukin-1 receptors (5Gadina M. Hilton D. Johnston J.A. Morinobu A. Lighvani A. Zhou Y.J. Visconti R. O'Shea J.J. Curr. Opin. Immunol. 2001; 13: 363-373Google Scholar, 15Moutoussamy S. Kelly P.A. Finidori J. Eur. J. Biochem. 1998; 255: 1-11Google Scholar, 20Raines M.A. Kolesnick R.N. Golde D.W. J. Biol. Chem. 1993; 268: 14572-14575Google Scholar, 21Waters M.J. Shang C.A. Behncken S.N. Tam S.P. Li H. Shen B. Lobie P.E. Clin. Exp. Pharmacol. Physiol. 1999; 26: 760-764Google Scholar). However, the coupling of the GH/PRL-related family of receptors to ceramide has not been reported. We therefore examined whether GH might influence ceramide formation in pituitary cells as part of an autocrine feedback pathway and whether dopamine-D2 agonists would influence GH action. Rat pituitary tumor GH4C1 cells synthesize and secrete PRL and GH and provide an excellent model of pituitary somatomammotrophs used for over 30 years (22Albert P.R. Vitam. Horm. 1994; 48: 59-109Google Scholar). In this report we have identified a novel induction of DAG and ceramide formation by GH that is blocked by PTX, implicating the involvement of Gi/Go proteins (23Birnbaumer L. Cell. 1992; 71: 1069-1072Google Scholar). The contribution of specific Gα subunits to GH autocrine signaling pathways was addressed using PTX-insensitive mutants of Gαi2, Gαi3, and Gαoindividually transfected into GH4ZR7 pituitary cells (GH4C1 cells transfected with the dopamine-D2S receptor (24Albert P.R. Neve K.A. Bunzow J.R. Civelli O. J. Biol. Chem. 1990; 265: 2098-2104Google Scholar, 25Banihashemi B. Albert P.R. Mol. Endocrinol. 2002; 16: 2393-2404Google Scholar)). In PTX-insensitive G protein mutants, the carboxyl-terminal ribosyl-acceptor cysteine was changed to a nonacceptor serine. The Cys-to-Ser mutation is a structurally conservative change, and the mutant G proteins remain functional following PTX pretreatment (26Chuprun J.K. Raymond J.R. Blackshear P.J. J. Biol. Chem. 1997; 272: 773-781Google Scholar, 27Ghahremani M.H. Cheng P. Lembo P.M. Albert P.R. J. Biol. Chem. 1999; 274: 9238-9245Google Scholar, 28Ghahremani M.H. Forget C. Albert P.R. Mol. Cell. Biol. 2000; 20: 1497-1506Google Scholar). The role of Gβγ subunits was evaluated by using the carboxyl-terminal domain of G protein-coupled receptor kinase (GRK-ct), a selective Gβγ scavenger (29Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Google Scholar). In GH4ZR7 cells, dopamine-D2S receptor activation potentiated GH-induced DAG and ceramide formation. We have identified Gαi3 and Gβγ as crucial for both GH-induced ceramide formation and dopamine-D2-induced potentiation of the GH response. Apomorphine, dopamine, Staphylococcus aureus SMase, PTX, 1,2-dioleoyl-rac-glycerol (C18:1[cis]-9), DAG, puromycin, and all other drugs, standards, and salts were purchased from Sigma. Human GH (iodination grade) and Escherichia coli DAG kinase (13 units/mg protein) were from Calbiochem (San Diego, CA). Sera, media, and Geneticin (G418) were obtained from Invitrogen, Inc. [γ-32P]ATP and [α-32P]dCTP (>3000 Ci/mmol) were from AmershamBiosciences. Thin-layer chromatography (TLC) plates (0.25 mm thick) were purchased from Whatman. Solvents were supplied by BDH. Plasmids pY3 and pCMV-LacZ II were obtained from the American Type Culture Collection (Manassas, VA). The cDNAs encoding wild-type rat Gαo, Gαi1, Gαi2, and Gαi3 were generously provided by Dr. Randall Reed, Johns Hopkins University, Baltimore, MD. Phospho-STAT5 (Tyr-694) antibody, phosphoplus® STAT3 (Tyr-705) and phosphoplus® p44/42 MAPK antibody kits were purchased from New England Biolabs (Mississauga, Ontario, Canada). As previously described (27Ghahremani M.H. Cheng P. Lembo P.M. Albert P.R. J. Biol. Chem. 1999; 274: 9238-9245Google Scholar), PTX-insensitive Gαi/o mutants were generated by point mutation of rat cDNAs (30Jones D.T. Reed R.R. J. Biol. Chem. 1987; 262: 14241-14249Google Scholar) encoding Gαi2 and Gαi3 subunits at cysteine 351 (352 for Gαi2). The TGT (cysteine codon) was mutated to TCT (serine) and confirmed by Sanger dideoxynucleotide sequencing. The mutant Gαi2 and Gαi3 cDNAs were FLAG-tagged at the initiator ATG codon, and the cDNAs were subcloned in KpnI-EcoRI-cut pcDNA3 (Invitrogen) to generate Gαi2-PTX, Gαi3-PTX, and Gαo-PTX. The carboxyl-terminal domain of OK-GRK2 cDNA (31Lembo P.M. Ghahremani M.H. Albert P.R. Mol. Endocrinol. 1999; 13: 138-147Google Scholar), beginning from Thr-493, was tagged at the amino-terminal with RGS-His6, and the His-GRK-ct fragment was cloned into pcDNA3 to produce the GRK-ct construct. GH4ZR7 cells and derivative clones were maintained in Ham's F10 medium with 8% fetal bovine serum (FBS) at 37 °C, 5% CO2. Gαi2-PTX, Gαi3-PTX, and Gαo-PTX (20 μg) were cotransfected individually with pGK-puro (2 μg) into GH4ZR7 cells using calcium phosphate co-precipitation. The transfected cells were cultured in F10 + 8% FBS containing puromycin (20 μg/ml) for 3–4 weeks. Antibiotic-resistant clones were picked (24 clones/transfection) and tested for expression of the corresponding Gαi/oproteins by Western blot analysis. The following Gαi2-PTX, Gαi3-PTX, Gαo-PTX and GRK-ct clones were selected for analysis, respectively: Gi2Z 24, Gi3Z 15, Gαo 15, and GRKZ 17. These cells express about 2- to 3-fold times the endogenous level of total Gα protein in GH4ZR7 cells, suggesting that the ratio of PTX-insensitive/endogenous Gα proteins in the clones was 1–2-fold (25Banihashemi B. Albert P.R. Mol. Endocrinol. 2002; 16: 2393-2404Google Scholar). Equivalent numbers of cells were cultured in ten 10-cm plates with Ham's F10 medium plus 8% FBS in a humidified atmosphere of 5% CO2, 95% air at 37 °C, grown to 80–90% confluence, and placed in serum-free F10 medium for 16 h. For PTX treatment, the cells were treated with 10 ng/ml PTX for 16 h prior to experimentation. Cells were rinsed with serum-free F10 medium and treated with experimental compounds at 37 °C as indicated. Following incubations, cells were twice rinsed with ice-cold PBS and lipids extracted (32Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). After centrifugation at 500 ×g for 1 min at 4 °C, the supernatants were aspirated and the cells were lyzed with 0.5 ml of chloroform/methanol/HCl (20:40:1, v/v/v), and sonicated in 5-s intervals × 6 on ice. Cells were rinsed with 1 ml of chloroform and 0.3 ml of 1 m NaCl and spun at 14,000 × g for 15 min at 4 °C. The upper aqueous layer was discarded, and the lower lipid-containing layer was transferred to a 1-ml glass Chrompack vial, dried under a stream of O2-free N2 gas, and redissolved in 200 μl of chloroform. The samples were stored at −80 °C until analysis. The particulate protein interface was air-dried, dissolved in 0.5 ml of 2m NaOH, and assayed for protein according to Lowry's method. DAG and ceramide were quantified using the DAG kinase method (33Preiss J. Loomis C.R. Bishop W.R. Stein R. Niedel J.E. Bell R.M. J. Biol. Chem. 1986; 261: 8597-8600Google Scholar, 34Wright T.M. Rangan L.A. Shin H.S. Raben D.M. J. Biol. Chem. 1988; 263: 9374-9380Google Scholar). A blank tube and a standard ceramide/DAG tube were included as controls. For each sample, 10 μl of DAG kinase (20 milliunits), 50 μl of reaction buffer (100 mm imidazole, pH 6.6, 25 mm MgCl2and 2 mm EGTA), 10 μl of 20 mmdithiothreitol, and 10 μl of [γ-32P]ATP (2.5 × 105 dpm/nmol) were added and incubated at 25 °C for 30 min, and the reactions terminated by addition of 0.5-ml ice-cold chloroform/methanol (1:2 v/v). The lipids were separated and extracted by addition of 0.5 ml of chloroform and 0.5 ml of 1 m NaCl spun at 14,000 × g for 3 min, and the upper aqueous phase was discarded. The organic phase was sequentially washed with 0.5 ml of 1% perchloric acid, 0.3 ml of chloroform/methanol (1:2 v/v), 0.2 ml of chloroform, and 0.2 ml of water, dried under N2, and reconstituted in 25 μl of chloroform/methanol (95:5, v/v). The samples were spotted onto a Silica Gel 60 TLC plate, heat-activated, and developed in a solvent mixture of chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1, v/v/v/v/v). Since DAG kinase can use ceramide or DAG as substrate, [32P]ceramide-phosphate represented ceramide production and [32P]phosphatidic acid represented DAG production. The TLC plates were exposed to phosphor screens for 18 h, and [32P]ceramide-phosphate and [32P]phosphatidic acid were quantified using the Molecular Dynamics System ImageQuaNT computer software. Results are expressed as percentage of control. The binding assay was performed using 50 μg of protein and 15,000 cpm/sample of125I-hGH (2150 Ci/mmol, PerkinElmer Life Sciences) in a final volume of 300 μl (0.021 nm final concentration) of TME buffer (75 mm Tris, pH 7.4, 12.5 mmMgCl2, 1 mm EDTA) containing 0.1% BSA (35Klempt M. Bingham B. Breier B.H. Baumbach W.R. Gluckman P.D. Endocrinology. 1993; 132: 1071-1077Google Scholar). To assess nonspecific binding 1 nm unlabeled hGH was added to the reaction. Incubation at room temperature was stopped after 30 min by the addition of 500 μl of cold 100 mm Tris, pH 7.4. The reaction was then filtered through GF/C filters and washed three times with 5 ml of cold 100 mm Tris, pH 7.4. Triplicate measurements were performed for all samples. Cells were treated as described above. Cell pellets were frozen on dry ice/ethanol and stored at –80 °C. Samples were sonicated 10–15 s, heated at 95 °C for 5 min, and centrifuged, and 40 μl/sample loaded onto SDS-PAGE gel and electrotransferred to polyvinylidene difluoride membrane. The membrane was blocked (1 h, room temperature), probed with primary antibody (1:1000, overnight, 4 °C), washed in TBST (10 mmTris-HCl, pH 8, 150 mm NaCl, and 0.05% Tween 20) and incubated with horseradish peroxidase-conjugated secondary antibody (1:2000) and horseradish peroxidase-conjugated anti-biotin antibody (1:1000) to detect biotinylated protein markers (2 h at room temperature). The blot was then washed, incubated with LumiGLO (1 min), and exposed to x-ray film. Exposures in the linear range (gray scale) were scanned and quantified using the UnScanIt program (Silk Scientific Inc., Orem, Utah). The data were analyzed by repeated measure using analysis of variance for each set of experiments. Differences of p < 0.05 were considered statistically significant. The acute action of GH on endogenous DAG and ceramide levels in GH4ZR7pituitary cells was assessed by the DAG kinase assay. The cells were washed to remove extracellular (secreted) GH and assayed in serum-free medium. GH induced a 10-fold increase in both DAG and ceramide production in a concentration-dependent manner from 10−10 to 10−6m at 20 min with an EC50 of ∼5 nm (Fig. 1). Addition of exogenous SMase (0.1 units/ml) was included as a positive control to demonstrate the hydrolysis of endogenous SM to form ceramide. The phosphorylated DAG and ceramide species co-migrated with the respective standards, confirming the identity of the products. GH (10−7m) robustly increased both DAG and ceramide production in parallel, which was maximal within 15 min and declined but remained significantly elevated at 3 h (Fig. 2). Low levels of GH are secreted by GH4C1 cells at a rate of 0.2 ng/ml/min or 10−11 mol/liter/min (36Albert P.R. Tashjian Jr., A.H. J. Biol. Chem. 1984; 259: 15350-15363Google Scholar), sufficient to reach a threshold concentration (10−9m) for DAG/ceramide formation in 1.5 h following initiation of treatments (see “Materials and Methods”). However, GH is also metabolized, hence the actual GH concentration under culture conditions may be lower and did not appear to interfere with actions of exogenous GH.Figure 2Sustained GH-induced increases in DAG and ceramide production in GH4ZR7cells. GH4ZR7 cells treated with 10−7m GH for 15 min and 3 h. Lipids were extracted from cells and separated as described under “Material and Methods”, and a representative image is shown. Below, the quantified data from three independent experiments are expressed as mean ± S.E. *, p < 0.03, and **,p < 0.01. B, blank; C, control;Std, standard.View Large Image Figure ViewerDownload (PPT) We recently showed that in Balb/c-3T3 fibroblasts, activation of the D2S receptor induces DAG and ceramide formation that is blocked by PTX, which inactivates Gi/Goproteins. 2Liu, G., Robillard, L., Banihashemi, B., and Albert, P. R., in press. Cells were pretreated with or without 10 ng/ml PTX for 16 h, a concentration that blocks Gi/Go-mediated signaling in these cells (22Albert P.R. Vitam. Horm. 1994; 48: 59-109Google Scholar). PTX treatment blocked GH-induced DAG and ceramide formation, thus implicating Gi/Go proteins (Fig. 3). By contrast, PTX or dopamine-D2 agonist apomorphine (10−6m) alone did not alter DAG or ceramide formation. Importantly, PTX treatment did not change the level of specific 125I-GH binding sites measured in crude membranes from GH4ZR7 cells. Specific 125I-GH binding was 118 ± 45 fmol/mg in GH4ZR7cells (mean ± S.E., n = 3), and binding in PTX-treated cells was 104 ± 8% of control binding, indicating that blockade of GH-induced ceramide by PTX was not due to loss of receptor sites. To examine further whether activation of the D2S receptor modulates DAG or ceramide formation, GH4ZR7 cells were incubated with GH, apomorphine (a D2 receptor agonist) or both GH and apomorphine (Fig. 4). Although dopamine-D2S receptor activation alone did not influence DAG or ceramide formation, apomorphine potentiated by 1.5- to 2-fold times the GH-induced formation of DAG and ceramide. In parental GH4C1 cells, which lack dopamine receptors, GH induced both DAG and ceramide formation but this effect was not enhanced by apomorphine (data not shown), indicating that apomorphine-induced potentiation is mediated via activation of dopamine-D2S receptors present on GH4ZR7 cells. Pretreatment with PTX blocked GH-induced ceramide production in GH4ZR7 and GH4C1 cells and also completely blocked DAG/ceramide production by apomorphine/GH (Fig. 5), indicating that D2S-induced potentiation of GH action involves Gi/Goproteins.Figure 5Both GH - and apomorphine/GH-induced DAG and ceramide formation is blocked by PTX pretreatment.GH4ZR7 cells were treated as described in previous figures, and representative image of labeled DAG and ceramide products is shown above, and below averages of three experiments (mean ± S.E.), *, p < 0.03 and **,p < 0.01, compared with control. A: apomorphine; B: blank; C: control; G: GH; P: PTX; S: sphingomyelinase; Std: standard; or as indicated.View Large Image Figure ViewerDownload (PPT) We examined which subunit(s) of G proteins mediate DAG or ceramide signaling induced by GH or apomorphine/GH in combination using GH4ZR7cells stably transfected with PTX-insensitive Gα mutants (Gαi2-PTX and Gαi3-PTX cells) (25Banihashemi B. Albert P.R. Mol. Endocrinol. 2002; 16: 2393-2404Google Scholar). As observed in wild-type GH4ZR7 cells and in Gαi2-PTX and Gαi3-PTX clones, the level of ceramide production induced by combination of apomorphine and GH was greater than for GH alone (Fig. 6 and data not shown). To examine the importance of Gαi2-PTX and Gαi3-PTX, cells were pretreated with PTX to block endogenous Gi/oproteins and challenged with GH or apomorphine/GH in combination. PTX blocked completely DAG and ceramide production stimulated by GH or apomorphine/GH in Gαi2-PTX cells (Fig. 6). However in Gαi3-PTX cells, both DAG and ceramide production were at least 50% resistant to PTX pretreatment (Fig. 7). Since ∼50% of the total Gαi3 was PTX-sensitive endogenous protein (25Banihashemi B. Albert P.R. Mol. Endocrinol. 2002; 16: 2393-2404Google Scholar), a recovery of 50% of the response would be expected from the remaining fraction of PTX-insensitive Gi3 proteins. Thus Gαi3, but not Gαi2, plays a crucial role in both GH- and apomorphine/GH-induced DAG and ceramide formation. To examine the role of Gαo subunits in GH-induced lipid signaling, GH4ZR7 cells were stably transfected with Gαo-PTX. In these cells, PTX completely blocked DAG and ceramide formation induced by the combination of apomorphine and GH (Fig. 8), indicating that like Gαi2-PTX, Gαo-PTX does not rescue GH-induced lipid signaling.Figure 7Apomorphine/GH-induced DAG and ceramide formation is rescued by Gαi3-PTX. GH4ZR7 cells expressing PTX-insensitive Gαi3 cDNA were treated for 20 min with 10−6m apomorphine or apomorphine and 10−7m GH without or with PTX pretreatment (10 ng/ml, 16 h). Abbreviations are as in previous figures.Above is a representative image of labeled DAG and ceramide products, and below averaged data are expressed as mean ± S.E. *, p < 0.03 and **, p < 0.01.View Large Image Figure ViewerDownload (PPT)Figure 8Gαo-PTX subunit fails to rescue DAG and ceramide signaling induced by combination of apomorphine and GH. GH4ZR7cells expressing PTX-insensitive Gαo were treated for 20 min with 10−6m apomorphine or apomorphine and 10−7m GH, without or with PTX pretreatment (10 ng/ml, 16 h). A representative image of labeled DAG and ceramide products is shown. Abbreviations are as in previous figures.View Large Image Figure ViewerDownload (PPT) As a selective Gβγ scavenger (29Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Google Scholar), the carboxyl-terminal domain of G protein-coupled receptor kinase (GRK-ct) was used to examine the role of Gβγ subunits in signaling to ceramide formation. We have transfected GRK-ct into GH4ZR7 cells and identified expression of GRK-ct by Western blot (25Banihashemi B. Albert P.R. Mol. Endocrinol. 2002; 16: 2393-2404Google Scholar). Neither apomorphine nor apomorphine/GH induced DAG or ceramide formation in GRK-ct cells (Fig. 9). This suggests that Gβγ subunits are necessary for ceramide formation induced by the combination of apomorphine and GH. Based on the results above, we examined the influence of GH, apomorphine, PTX, and ceramide on well known and potential downstream pathways of the GH receptor including phosphorylation of JAK2, STAT5 (Fig. 10), STAT3 or MAPK. In GH4ZR7 cells, GH alone increased phosphorylation of JAK2 (100% increase over basal) and STAT5 (40% increase), which was more strongly enhanced with both apomorphine and GH (160% increase over basal for phospho-JAK2, 90% increase for phospho-STAT5). Treatment with a ceramide analogue (C2-ceramide) or SMase (to increase endogenous ceramide) also increased JAK2 phosphorylation by 90 and 150%, and STAT5 phosphorylation by 60 and 90%, respectively. Interestingly, PTX-blocked apomorphine/GH-induced STAT5 phosphorylation by 50%, further supporting a role for the PTX-sensitive ceramide pathway in GH-induced STAT5 phosphorylation in these cells. By contrast, these compounds elicited no changes in STAT3 or MAPK phosphorylation (data not shown). Our results indicate that GH induces a G protein-dependent increase in lipid metabolism to generate DAG and ceramide in GH4 cells. Previous studies in pre-adipocyte Ob1771 cells (37Doglio A. Dani C. Grimaldi P. Ailhaud G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1148-1152Google Scholar, 38Catalioto R.M. Ailhaud G. Negrel R. Biochem. Biophys. Res. Commun. 1990; 173: 840-848Google Scholar) and in pancreatic β-cells (39Sjoholm A. Zhang Q. Welsh N. Hansson A. Larsson O. Tally M. Berggren P.O. J. Biol. Chem. 2000; 275: 21033-21040Google Scholar) have shown that GH induces DAG formation via activation of PC-PLC. By analogy, GH may activate PLC in GH4 cells to induce DAG formation. Both DAG and ceramide formation were induced in parallel, suggesting interconversion between these lipids possibly via SM synthase, which can convert DAG into ceramide, leading to depletion of SM (40van Helvoort A. van't Hof W. Ritsema T. Sandra A. van Meer G. J. Biol. Chem. 1994; 269: 1763-1769Google Scholar, 41Luberto C. Hannun Y.A. J. Biol. Chem. 1998; 273: 14550-14559Google Scholar). Alternately, DAG can activate acidic SMase to generate ceramide (42Schutze S. Machleidt T. Kronke M. J. Leukoc. Biol. 1994; 56: 533-541Google Scholar, 43Schutze S. Wiegmann K. Machleidt T. Kronke M. Immunobiology. 1995; 193: 193-203Google Scholar). Interconversion of DAG to ceramide would account for the identical Gαi3 and Gβγ dependencies of GH-mediated lipid formation. The actions of GH in GH4 cells were sensitive to PTX pretreatment, indicating a role for Gi/Goproteins. Upon activation, GH receptors dimerize, associate with JAK2, and recruit a family of negative regulators, the SOCS (suppressors of cytokine signaling) proteins (44Finidori J. Vitam. Horm. 2000; 59: 71-97Google Scholar, 45Ram P.A. Waxman D.J. J. Biol. Chem. 1999; 274: 35553-35561Google Scholar). Coupling of the GH receptor to PTX-sensitive G proteins is relatively unexplored, and potential interactions of GH receptors or associated proteins such as SOCS proteins with G proteins remain elusive. There is some evidence that GH-like receptors interact with G proteins. In Nb2 cells, Gαi proteins labeled by PTX-mediated ADP ribosylation were cross-linked to the PRL receptor using a 16-Å cross-linking agent, but not cross-linkers with shorter molecular lengths, consistent with a direct physical interaction (46Too C.K. Shiu R.P. Friesen H.G. Biochem. Biophys. Res. Commun. 1990; 173: 48-52Google Scholar). In addition, some PTX-sensitive GH-induced responses have been reported. For example, GH-induced PC-PLC activation in Ob1771 preadipocytes (37Doglio A. Dani C. Grimaldi P. Ailhaud G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1148-1152Google Scholar,38Catalioto R.M. Ailhaud G. Negrel R. Biochem. Biophys. Res. Commun. 1990; 173: 840-848Google Scholar) and GH-mediated DAG formation and mitogenesis in pancreatic β-cells (39Sjoholm A. Zhang Q. Welsh N. Hansson A. Larsson O. Tally M. Berggren P.O. J. Biol. Chem. 2000; 275: 21033-21040Google Scholar) are PTX-sensitive actions. Similarly, activation of the homologous PRL receptor in Nb2 lymphoma cells enhances PTX labeling of Gi proteins (suggesting activation) and induces PTX-sensitive mitogenesis (47Larsen J.L. Dufau M.L. Endocrinology. 1988; 123: 438-444Google Scholar, 48Too C.K. Murphy P.R. Friesen H.G. Endocrinology. 1989; 124: 2185-2192Google Scholar, 49Larsen J.L. J. Biol. Chem. 1992; 267: 10583-10587Google Scholar). Taken together, these results are consistent with coupling of the GH receptor to PTX-sensitive Gi proteins to activate PLC thereby generating DAG, which can be converted to ceramide. Although coupled to Gi/Go proteins, GH signaled differently from the Gi/Go-coupled dopamine D2 receptor to induce PTX-sensitive DAG and ceramide formation since apomorphine alone had no effect. Nevertheless there was an interaction between GH and D2 signaling since apomorphine potentiated GH-induced lipid signaling and JAK2/STAT5 activation. Furthermore, GH- and apomorphine/GH-induced DAG and ceramide formation were both rescued by Gαi3-PTX and blocked by GRK-ct, suggesting a crucial role for Gαi3/Gβγ for both receptors. The dopamine-D2 receptor utilizes Gi3 to mediate activation of potassium channels in pituitary cells (50Lledo P.M. Homburger V. Bockaert J. Vincent J.D. Neuron. 1992; 8: 455-463Google Scholar) via binding of Gβγ to the GIRK potassium channel (51Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Google Scholar), and is likely to couple to Gαi3/Gβγ in GH4 pituitary cells. The mechanism by which GH receptors couple to Gi3 remains to be elucidated, but GH receptors appear to interact with Gi proteins differently from Gi-coupled heptahelical receptors (such as adenosine or D2S receptors). In adipocytes, GH prevented coupling of adenosine receptor-mediated inhibition of cAMP and activation of phosphatidylinositol-specific-PLC and blocked PTX-induced ADP-ribosylation (52Roupas P. Chou S.Y. Towns R.J. Kostyo J.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1691-1695Google Scholar, 53Doris R.A. Kilgour E. Houslay M.D. Vernon R.G. J. Endocrinol. 1998; 158: 295-303Google Scholar). GH may induce relocalization of Gαi subunits, prevent their coupling to adenylyl cyclase (53Doris R.A. Kilgour E. Houslay M.D. Vernon R.G. J. Endocrinol. 1998; 158: 295-303Google Scholar, 54Yip R.G. Goodman H.M. Endocrinology. 1999; 140: 1219-1227Google Scholar), and allow efficient coupling to DAG/ceramide formation. Since sites of ceramide synthesis display discrete subcellular localization (55Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 25847-25850Google Scholar), differences in the localization of D2S- and GH-receptor coupling might account for their differing effectiveness to induce ceramide formation in GH4 pituitary cells. Our data show that C2-ceramide and sphingomyelinase induce JAK2/STAT5 activation in GH4 cells, suggesting a link between GH-induced changes in DAG/ceramide and the classical GH-receptor-mediated JAK/STAT pathway. Consistent with our results, sphingomyelinase was shown to increase ceramide levels and was found to activate JAK2 and STAT1/3 in cultured human fibroblasts (56Maziere C. Conte M.A. Maziere J.C. FEBS Lett. 2001; 507: 163-168Google Scholar). Importantly, as observed for GH-mediated ceramide formation, GH-induced JAK2/STAT5 activation was enhanced by apomorphine and was partially blocked by PTX, suggesting that both G protein-dependent and -independent pathways lead to JAK2/STAT5 activation in these cells. Thus Gi-mediated ceramide signaling regulates GH-induced JAK2/STAT5 activation. In addition to regulating JAK2/STAT5, GH-induced ceramide formation may activate other signaling cascades (19Liu G. Kleine L. Hebert R.L. Crit. Rev. Clin. Lab. Sci. 1999; 36: 511-573Google Scholar). Both GH (16Love D.W. Whatmore A.J. Clayton P.E. Silva C.M. Endocrinology. 1998; 139: 1965-1971Google Scholar, 57Yarwood S.J. Sale E.M. Sale G.J. Houslay M.D. Kilgour E. Anderson N.G. J. Biol. Chem. 1999; 274: 8662-8668Google Scholar) and ceramide (19Liu G. Kleine L. Hebert R.L. Crit. Rev. Clin. Lab. Sci. 1999; 36: 511-573Google Scholar, 20Raines M.A. Kolesnick R.N. Golde D.W. J. Biol. Chem. 1993; 268: 14572-14575Google Scholar) have been shown to activate the MAPK cascade in other cell types, but we observed no induction of p42/44-MAPK by either GH or ceramide in GH4 cells. Ceramide regulates other pathways including the SAPK/JNK cascade, and several proapoptotic pathways, but the roles of these pathways in GH4 cells is not known. Multiple negative feedback pathways regulate GH secretion at the level of the hypothalamus and pituitary. At the level of the hypothalamus, GH inhibits GH-releasing hormone synthesis and enhances somatostatin release, resulting in decreased GH secretion at the pituitary (58Peng X.D. Park S. Gadelha M.R. Coschigano K.T. Kopchick J.J. Frohman L.A. Kineman R.D. Endocrinology. 2001; 142: 1117-1123Google Scholar, 59Kamegai J. Unterman T.G. Frohman L.A. Kineman R.D. Endocrinology. 1998; 139: 3554-3560Google Scholar, 60Zheng H. Bailey A. Jiang M.H. Honda K. Chen H.Y. Trumbauer M.E. Van der Ploeg L.H. Schaeffer J.M. Leng G. Smith R.G. Mol. Endocrinol. 1997; 11: 1709-1717Google Scholar). GH-induced IGF formation is believed to be the primary negative feedback pathway to inhibit GH synthesis in somatotrophs (1Melmed S. Yamashita S. Yamasaki H. Fagin J. Namba H. Yamamoto H. Weber M. Morita S. Webster J. Prager D. Rec. Prog. Horm. Res. 1996; 51: 189-215Google Scholar, 2Giustina A. Veldhuis J.D. Endocr. Rev. 1998; 19: 717-797Google Scholar). In addition, Gi/Go-coupled dopamine-D2 and somatostatin receptors also inhibit GH secretion and somatomammotroph growth (3Colao A. Lombardi G. Lancet. 1998; 352: 1455-1461Google Scholar). It is tempting to speculate that GH may negatively regulate its own secretion; however, evidence for a non-IGF-mediated autocrine pituitary feedback by GH is indirect (4Asa S.L. Coschigano K.T. Bellush L. Kopchick J.J. Ezzat S. Am. J. Pathol. 2000; 156: 1009-1015Google Scholar, 61Nakamoto J.M. Gertner J.M. Press C.M. Hintz R.L. Rosenfeld R.G. Genel M. J. Clin. Endocrinol. Metab. 1986; 62: 822-826Google Scholar, 62Ross R.J. Borges F. Grossman A. Smith R. Ngahfoong L. Rees L.H. Savage M.O. Besser G.M. Clin. Endocrinol. 1987; 26: 117-123Google Scholar). The GH receptor is expressed in rat and human anterior pituitary and binds and internalizes radiolabeled GH, suggesting a role for GH to regulate its secretion from the pituitary (63Mertani H.C. Pechoux C. Garcia-Caballero T. Waters M.J. Morel G. J. Clin. Endocrinol. Metab. 1995; 80: 3361-3367Google Scholar, 64Mertani H.C. Waters M.J. Jambou R. Gossard F. Morel G. Neuroendocrinology. 1994; 59: 483-494Google Scholar, 65Harvey S. Baumbach W.R. Sadeghi H. Sanders E.J. Endocrinology. 1993; 133: 1125-1130Google Scholar, 66Fraser R.A. Siminoski K. Harvey S. J. Endocrinol. 1991; 128: R9-R11Google Scholar). However, the signaling of the GH receptor in pituitary cells has not been investigated. Our finding of a novel G protein-mediated action of GH to induce DAG/ceramide as well as JAK2/STAT5 activation in GH4 cells suggests a role for GH in regulation of pituitary function. GH4 cells are a pituitary cell strain that has provided an important model of somatotrophs that synthesize and secrete levels of GH that are sufficient to mediate autocrine GH-induced actions (22Albert P.R. Vitam. Horm. 1994; 48: 59-109Google Scholar). Interestingly, C2-ceramide has been shown to inhibit GH secretion from rat anterior pituitary cells (67Negishi T. Chik C.L. Ho A.K. Endocrinology. 1999; 140: 5691-5697Google Scholar), suggesting that GH-induced ceramide formation could mediate negative feedback inhibition of GH secretion. Previously unexplored direct actions of GH on DAG and ceramide may provide a more sensitive method to address direct actions of GH in regulation of somatotroph function in vivo.
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