Cellular stress protection and increased cell proliferation induced by the protein p8 in INS-1 beta cells via activation of the phosphatidylinositol-3'-kinase and MAP kinase pathways

Overexpression of the stress protein p8 is known to increase glucose dependent cell proliferation in insulin producing cells. In this study we investigated p8 associated intracellular signal transduction in the glucose dependent beta cell line INS-1. INS-1 beta cells stably overexpressing p8 (p8-INS-1) were used. These cells displayed a 6fold increase in p8-mRNA levels and a 2fold increase in cell proliferation as compared to wild type INS-1 beta cells. Cell proliferation was measured using [3H]-thymidine incorporation. Activation of mitogenic signalling proteins was assessed using co-immunoprecipitation and immunoblot analysis. p8 protein is ubiquitinated and degraded by the proteasome pathway. Inhibition of proteasome dependent protein degradation by lactacystin resulted in increased cell proliferation in the presence of low glucose concentrations (0–6 mM) whereas at higher glucose concentrations no such effect was observed in INS-1 beta cells. p8 dependent cell proliferation seems to be mediated via the phosphatidylinositol-3'-kinase (PI3K) pathway and protein kinase C (PKC), because coimmunoprecipitation assays demonstrated protein binding of both PI3K and PKCzeta to p8. Inhibition of PKCzeta resulted in complete inhibition of cell proliferation in INS-1 beta cells. In contrast, inhibition of PI3K lead only to partial reduction in cell proliferation suggesting PI3K independent activation of PKCzeta in p8 overexpressing cells. Further, protein association of p8 to MAPK (erk1/2) was observed, whereas no association of p8 to p38MAPK could be detected. These results suggest that p8 is an important protein involved in cell proliferation and cell protection during stress in insulin producing cells. p8 effects are dependent on ubiquitination and proteasomal degradation of the protein and are mediated via PI3K, PKCzeta and MAPK dependent signalling pathways. These results yield insight into the regulation of nutrient dependent cell proliferation and protection from cell stress in pancreatic beta cells.