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 GH 4 ZR 7 somatomammotroph cells was examined. GH (EC 50 ‫؍‬ 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 G i /G o proteins and was potentiated 1.5-fold by activation of G i /G o -coupled dopamine-D2S receptors, which had no effect alone. Following PTX pretreatment, only PTX-resistant G␣ i 3, not G␣ o or G␣ i 2, rescued GHinduced DAG/ceramide signaling. GH-induced DAG/ceramide formation was also blocked in cells expressing G␤␥ blocker GRK-ct. In GH 4 ZR 7 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 GH 4 pituitary cells, GH induces formation of DAG/ceramide via a novel G␣ i 3/ G␤␥-dependent pathway. This novel pathway suggests a mechanism for autocrine feedback regulation by GH of pituitary function.Pituitary somatotrophs synthesize and secrete GH, 1 which acts at the liver and other tissues to stimulate IGF formation, promoting somatic growth throughout the body (1, 2). 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 (3). Negative feedback via autocrine actions of GH at the pituitary has been postulated (4) 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 (5). The GH receptor signals through the JAK2 tyrosine kinase-signal transducer and activator of transcription 5 (STAT5) transcription factor pathway to induce gene expression (6 -9). Phosphorylation on residue Tyr-694 by JAK2 is obligatory for STAT5 activation (10). The two STAT5 variants, STAT5a and STAT5b, have 90% identical protein sequences and are independently regulated and activated in various cell types (11). 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 (11-13). 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 3Ј-kinase or protein kinase C activation (14, 15) in a cell type-dependent manner (16). Ceramide is a novel second messenger implicated in regulation of cell differentiation,...

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