Inhibition of Raf-1 alters multiple downstream pathways to induce pancreatic β-cell apoptosis

The serine threonine kinase Raf-1 plays a protective role in many cell types, but its function in pancreatic β-cells has not been elucidated. In the present study, we examined whether primary β-cells possess Raf-1 and tested the hypothesis that Raf-1 is critical for β-cell survival. Using reverse transcriptase-PCR, Western blot, and immunofluorescence, we identified Raf-1 in human islets, mouse islets, and in the MIN6β-cell line. Blocking Raf-1 activity using a specific Raf-1 inhibitor or dominant-negative Raf-1 mutants led to a time- and dose-dependent increase in cell death, assessed by real-time imaging of propidium iodide incorporation, TUNEL, PCR-enhanced DNA laddering, and Caspase-3 cleavage. Although the rapid increase in apoptotic cell death was associated with decreased Erk phosphorylation, studies with two Mek inhibitors suggested that the classical Erk-dependent pathway could explain only part of the cell death observed after inhibition of Raf-1. An alternative Erk-independent pathway downstream of Raf-1 kinase involving the pro-apoptotic protein Bad has recently been characterized in other tissues. Inhibiting Raf-1 in β-cells led to a striking loss of Bad phosphorylation at serine 112 and an increase in the protein levels of both Bad and Bax. Together, our data strongly suggest that Raf-1 signaling plays an important role regulating β-cell survival, via both Erk-dependent and Bad-dependent mechanisms. Conversely, acutely inhibiting phosphatidylinositol 3-kinase Akt had more modest effects on β-cell death. These studies identify Raf-1 as a critical anti-apoptotic kinase in pancreatic β-cells and contribute to our understanding of survival signaling in this cell type.