Growth Hormone-induced Diacylglycerol and Ceramide Formation via Gαi3 and Gβγ in GH4 Pituitary Cells

Abstract

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. 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 B. Albert P.R. Mol. Endocrinol. 2002; 16: 2393-2404Google Scholar). of cells were cultured in plates with Ham's F10 medium 8% FBS in a of 5% at 37 °C, to and in F10 medium for h. PTX the cells were with PTX for to were with F10 medium and with at 37 as Following cells were with and W.J. J. Biochem. Physiol. Scholar). at for min at °C, the were and the cells were with of and in on were with of and of and at for 15 min at The was and the was to a a of and in of The were at analysis. The protein was in of and for protein to DAG and ceramide were using the DAG kinase J. R. J. Biol. Chem. Scholar, J. Biol. Chem. Scholar). and a were as of DAG kinase (20 of mm mm mm of and of [γ-32P]ATP were and at for 30 and the by of The were and by of of and of at for and the was The was with of of of and of and in of The were a and in a of DAG kinase ceramide or DAG as ceramide production and DAG The plates were to for and and were using the are as of The was using of protein and in a of of mm mm containing M. B. Endocrinology. 1993; Scholar). To was to the at was 30 min by the of of mm The was through and times with 5 of mm were for all were as described Cell were on and at were at for 5 and and and to The was blocked with antibody in mm and and with antibody and antibody to protein (2 at The blot was with and to in the were and using the The were by using of for of of were The of GH on endogenous DAG and ceramide in cells was by the DAG kinase The cells were to GH and in GH induced a 10-fold increase in both DAG and ceramide production in a manner from to at min with an of of was as a to the of endogenous to ceramide. The DAG and ceramide with the standards, the of the GH both DAG and ceramide production in which was within 15 min and but at of GH are by GH4C1 cells at a of or P.R. J. Biol. Chem. Scholar), to a for DAG/ceramide formation in following of and However, GH is also the GH be and not appear to with actions of GH-induced in DAG and ceramide production in GH4ZR7 cells with GH for 15 min and h. were from cells and as described and and a is the from are as and We that in activation of the receptor induces DAG and ceramide formation that is blocked by PTX, which and P. in were with or PTX for a that signaling in cells (22Albert P.R. Vitam. Horm. 1994; 48: 59-109Google Scholar). PTX treatment blocked GH-induced DAG and ceramide formation, implicating Gi/Go proteins PTX or dopamine-D2 not DAG or ceramide formation. PTX treatment not the level of specific in from GH4ZR7 was in = and in cells was 8% of that of GH-induced ceramide by PTX was not to of receptor To whether activation of the receptor DAG or ceramide formation, GH4ZR7 cells were with GH, (a receptor or both GH and dopamine-D2S receptor activation not influence DAG or ceramide formation, potentiated by to times the GH-induced formation of DAG and ceramide. In GH4C1 cells, which receptors, GH induced both DAG and ceramide formation but this effect was not by not that potentiation is via activation of dopamine-D2S receptors on GH4ZR7 with PTX blocked GH-induced ceramide production in GH4ZR7 and GH4C1 cells and also blocked DAG/ceramide production by that potentiation of GH GH and DAG and ceramide formation is blocked by PTX cells were as described in and of DAG and ceramide is and of and with or as We examined which of G proteins DAG or ceramide signaling induced by GH or in using transfected with PTX-insensitive Gα mutants and B. Albert P.R. Mol. Endocrinol. 2002; 16: 2393-2404Google Scholar). As in wild-type GH4ZR7 cells and in and the level of ceramide production induced by of and GH was for GH and not To the of and Gαi3-PTX, cells were with PTX to endogenous and with GH or in PTX blocked DAG and ceramide production stimulated by GH or in cells in cells, both DAG and ceramide production were at to PTX pretreatment of the total Gαi3 was endogenous protein B. Albert P.R. Mol. Endocrinol. 2002; 16: 2393-2404Google Scholar), a of of the would be from the of PTX-insensitive Gαi3, but not Gαi2, plays a crucial role in both and DAG and ceramide formation. To the role of Gαo subunits in GH-induced lipid GH4ZR7 cells were transfected with Gαo-PTX. In cells, PTX blocked DAG and ceramide formation induced by the of and GH that Gαi2-PTX, Gαo-PTX not GH-induced lipid DAG and ceramide formation is rescued by GH4ZR7 cells expressing PTX-insensitive Gαi3 cDNA were for min with or and GH or with PTX pretreatment are as in is a of DAG and ceramide and are as and to DAG and ceramide signaling induced by of and expressing PTX-insensitive Gαo were for min with or and GH, or with PTX pretreatment of DAG and ceramide is are as in 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 was used to 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 B. Albert P.R. Mol. Endocrinol. 2002; 16: 2393-2404Google Scholar). induced DAG or ceramide formation in GRK-ct cells This suggests that Gβγ subunits are for ceramide formation induced by the of and on the we examined the influence of GH, PTX, and ceramide on and pathways of the GH receptor including phosphorylation of STAT5 STAT3 or In GH4ZR7 cells, GH phosphorylation of JAK2 increase over and STAT5 which was with both and GH increase over for 90% increase for with a ceramide analogue or increase endogenous also JAK2 phosphorylation by and and STAT5 phosphorylation by and STAT5 phosphorylation by a role for the ceramide pathway in GH-induced STAT5 phosphorylation in no in STAT3 or MAPK phosphorylation not that GH induces a G increase in lipid to generate DAG and ceramide in GH4 in cells A. C. P. G. Sci. S. A. Scholar, G. R. Biochem. Res. 1990; Scholar) and in A. N. A. O. M. J. Biol. Chem. 2000; Scholar) have that GH induces DAG formation via activation of GH in GH4 cells to induce DAG formation. DAG and ceramide formation were induced in suggesting via which DAG into to of A. T. A. G. J. Biol. Chem. 1994; 269: Scholar, C. Hannun Y.A. J. Biol. Chem. 1998; 273: Scholar). DAG to generate ceramide S. T. M. J. Biol. 1994; Scholar, S. T. M. Scholar). of DAG to ceramide would for the identical Gαi3 and Gβγ of lipid formation. The actions of GH in GH4 cells were to PTX pretreatment, a role for GH receptors with and a family of negative the of cytokine proteins J. Vitam. Horm. 2000; Scholar, P.A. Waxman D.J. J. Biol. Chem. 1999; 274: Scholar). of the GH receptor to G proteins is and of GH receptors or proteins as proteins with G proteins remain is that receptors with G In cells, proteins by were to the PRL receptor using a but not with with a direct Biochem. Res. 1990; Scholar). In addition, GH-induced have been reported. GH-induced activation in A. C. P. G. Sci. S. A. G. R. Biochem. Res. 1990; Scholar) and DAG formation and in A. N. A. O. M. J. Biol. Chem. 2000; Scholar) are activation of the PRL receptor in cells PTX of proteins and induces Endocrinology. Scholar, P.R. Endocrinology. Scholar, J. Biol. Chem. 1992; Scholar). are with coupling of the GH receptor to proteins to DAG, which be to ceramide. to Gi/Go GH from the Gi/Go-coupled receptor to induce DAG and ceramide formation had no was an GH and signaling potentiated GH-induced lipid signaling and and DAG and ceramide formation were both rescued by and blocked by suggesting a crucial role for for both The dopamine-D2 receptor to activation of in pituitary cells P.M. J. J.D. 1992; 8: Scholar) via of Gβγ to the Rev. Pharmacol. 1997; Scholar), and is to to in GH4 pituitary The mechanism by which GH receptors to to be but GH receptors appear to with proteins from receptors as or In GH coupling of of and activation of and blocked P. R.J. Sci. S. A. Scholar, J. Endocrinol. 1998; Scholar). GH induce of coupling to J. Endocrinol. 1998; Scholar, Endocrinology. 1999; Scholar), and coupling to DAG/ceramide formation. of ceramide Y.A. Obeid L.M. J. Biol. Chem. 2002; Scholar), in the of and coupling might for to induce ceramide formation in GH4 pituitary that C2-ceramide and sphingomyelinase induce activation in GH4 cells, suggesting a GH-induced in DAG/ceramide and the pathway. with sphingomyelinase was to increase ceramide and was to JAK2 and in cultured C. M.A. 2001; Scholar). as for ceramide formation, GH-induced activation was by and was blocked by PTX, suggesting that both G and pathways to activation in ceramide signaling GH-induced In to GH-induced ceramide formation other signaling G. Kleine L. Hebert R.L. Crit. Rev. Clin. Lab. Sci. 1999; 36: 511-573Google Scholar). GH (16Love D.W. Whatmore A.J. Clayton P.E. Silva C.M. Endocrinology. 1998; 139: 1965-1971Google Scholar, J. Biol. Chem. 1999; 274: Scholar) and ceramide 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 to the MAPK in other cell but we no induction of by GH or ceramide in GH4 Ceramide other pathways including the and but the of pathways in GH4 cells is not negative feedback pathways GH secretion at the level of the and the level of the GH GH-releasing hormone and somatostatin in GH secretion at the pituitary Park S. Coschigano K.T. Kopchick J.J. Endocrinology. 2001; 142: Scholar, J. Endocrinology. 1998; 139: Scholar, H. A. M.H. G. Mol. Endocrinol. 1997; Scholar). GH-induced IGF formation is to be the negative feedback pathway to inhibit GH 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). is to that GH for a 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, C.M. R.L. M. J. Clin. Endocrinol. Metab. Scholar, R.J. F. A. R. L. Clin. Endocrinol. 1987; 26: Scholar). The GH receptor is in rat and pituitary and and GH, suggesting a role for GH to secretion from the pituitary C. T. M.J. G. J. Clin. Endocrinol. Metab. Scholar, M.J. R. F. G. 1994; Scholar, S. H. Endocrinology. 1993; Scholar, S. J. Endocrinol. Scholar). However, the signaling of the GH receptor in pituitary cells has not been of a novel G of GH to induce DAG/ceramide as as activation in GH4 cells suggests a role for GH in regulation of pituitary function. GH4 cells are a pituitary cell that has provided an important model of somatotrophs that synthesize and secrete of GH that are to autocrine GH-induced actions (22Albert P.R. Vitam. Horm. 1994; 48: 59-109Google Scholar). C2-ceramide has been to inhibit GH secretion from rat pituitary cells T. Endocrinology. 1999; Scholar), suggesting that GH-induced ceramide formation negative feedback of GH direct actions of GH on DAG and ceramide provide a to address direct actions of GH in regulation of in