(taken from the application) The focus of this proposal is to assess the role(s) of the PHAS-I/mTOR signaling pathway in growth factor and nutrient-mediated Beta-cell growth. An increase in translation of mRNA into protein is required for the mitogenic response of cells. PHAS-I, a recently identified binding protein of the initiation factor, eIF-4E, exerts a key role in this process. Phosphorylation of PHAS-I results in dissociation of the PHAS-I.eIF-4E complex, allowing eIF-4 to interact with other factors and initiate translation, cell cycle progression and proliferation. Recent studies with rapamycin, an immunosuppressant and anti-proliferative agent, have implicated the mammalian target of rapamycin (mTOR) as an upstream regulator of PHAS-I phosphorylation. Our findings indicate that in the Beta-cell, glucose mediates the phosphorylation of PHAS-I by stimulating insulin secretion which interacts in an autocrine manner with its own insulin receptor. Furthermore, rapamycin inhibits both glucose stimulated protein synthesis by islets and serum-induced cell proliferation by the Beta-cell line, RINm5F, suggesting a role for the PHAS-I/mTOR pathway in Beta-cell growth. The objective of specific aim 1 of this proposal is to determine if insulin and other growth factors utilize the same PHAS-I/mTOR signaling pathway to up-regulate protein translation, cell cycle progression and Beta-cell proliferation. Our recent studies with pancreatic islets and Beta-cell lines have indicated that amino acids are absolutely required for insulin and growth factors to activate the PHAS- I/mTOR pathway. Furthermore, amino acids alone dose-dependently stimulate the phosphorylation of PHAS-I which is further enhanced by insulin and growth factors. The objective of specific aim 2 is to define the cellular mechanism whereby amino acids alone and in synergy with insulin and other growth factors may utilize the PHAS-I/mTOR pathway. Studies will be performed to identify which amino acids are capable of mediating these effects, and to what extent the ability of growth factors to promoter Beta-cell growth are associated with enhanced transport and/or metabolism. of these active amino acids. In studies designed to identify endogenous growth factors that may promote Beta-cell growth via the PHAS-I/mTOR PATHWAY, we discovered that keratinocyte growth factor (KGF), a novel member of the FGF family, and KGF receptor (KGFR) are expressed endogenously by islets and the Beta-cell line, RINm5F. The objective of specific aim 3 is to further characterize the regulation of KGF and KGFR expression by pancreatic islets and Beta-cell lines at the level of mRNA, protein and activity, and also evaluate if KGF mediates its effects in a paracrine manner on ductal cells and/or in an autocrine manner on Beta- cells via the PHAS-I/mTOR signaling pathway. An understanding of the PHAS- I/mTOR signaling pathway to generate mitogenic mediators will provide important new insights to enhance the ability of growth factors and nutrients to stimulate Beta-cell growth.
