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In previous studies we demonstrated that vasoactive intestinal peptide (VIP) mediation, and interactions between mitogen-activated protein kinase (MAPK) and cAMP/protein kinase A (PKA) signaling pathways are implicated in insulin-like growth factor I (IGF-I)- and VIP-induced lactotroph proliferation. These facts led us to investigate the intracellular mechanisms involved in IGF-I- and VIP-induced lactotroph proliferation. Exposure of cultured male rat pituitary cells to IGF-I (10⁻⁷ M) or VIP (10⁻⁷ M) stimulated the MAPK cascade. Studies in GH4C1 cells, with an expression vector for Rap1 GTPase-activating protein (Rap1 GAP1), demonstrated reduced VIP-induced MAPK activation, indicating that VIP-dependent activation of the extracellular signal-regulated kinase (ERK) pathway requires PKA-Rap1 signaling. IGF-I induced cAMP-response element (CRE)-binding protein (CREB) phosphorylation through the Ras-MAPK pathway, whereas VIP phosphorylated CREB directly via PKA. The mechanisms that regulate IGF-I-and VIP-CREB-dependent gene transcription were examined using GH4C1 cells transiently transfected with a CRE reporter gene. IGF-I and VIP stimulation of CRE-mediated transcription required activation of both Ras-MAPK and cAMP/PKA signaling. This activation was blocked in the presence of Rap1 GAP1. In summary, we showed that IGF-I and VIP stimulated MAPK activity and the phosphorylation of CREB in pituitary cells. Furthermore, VIP-dependent activation of PKA-Rap1-ERK pathways mediated VIP and IGF-I effects on CREB-dependent transcription in GH4C1 cells. Thus, it is possible that VIP- and IGF-I-induced lactotroph proliferation may involve Rap1.

In previous studies we demonstrated that IGF-I induces proliferation of pituitary lactotrophs. In addition to its mitotrophic actions, IGF-I is known to prevent apoptosis induced by diverse stimuli in several cell types. In this study, we investigated the action of IGF-I on pituitary cell survival and the intracellular signaling transduction pathway implicated in this effect. Treatment of cultured male rat pituitary cells with IGF-I (10(-7) M) for 24 h prevented pituitary cell death induced by serum deprivation. The protective effect of IGF-I was blocked by phosphoinositide 3-kinase (PI3-kinase) inhibitor, LY294002, but was unaffected by PD98059, which inhibits MAP/ERK kinase (MEK1). IGF-I activation of PI3-kinase induced the phosphorylation and activation of the serine/threonine kinase Akt. Moreover, IGF-I increased the phosphorylation of the pro-apoptotic factor Bad and the levels of the anti-apoptotic protein Bcl-2 through the PI3-kinase pathway in primary pituitary cells.