title: Inhibition of Raf-1 alters multiple downstream pathways to induce pancreatic beta-cell apoptosis.
abstract: The serine threonine kinase Raf-1 plays a protective role in many cell types, but its function in pancreatic beta-cells has not been elucidated. In the present study, we examined whether primary beta-cells possess Raf-1 and tested the hypothesis that Raf-1 is critical for beta-cell survival. Using reverse transcriptase-PCR, Western blot, and immunofluorescence, we identified Raf-1 in human islets, mouse islets, and in the MIN6 beta-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 beta-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 beta-cell survival, via both Erk-dependent and Bad-dependent mechanisms. Conversely, acutely inhibiting phosphatidylinositol 3-kinase Akt had more modest effects on beta-cell death. These studies identify Raf-1 as a critical anti-apoptotic kinase in pancreatic beta-cells and contribute to our understanding of survival signaling in this cell type.
authors.affiliation: Laboratory of Molecular Signaling in Diabetes, Diabetes Research Group, Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Emilyn U
authors.initials: EU
authors.lastname: Alejandro
authors.forename: James D
authors.initials: JD
authors.lastname: Johnson
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000201:enzymology*; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C494819:BAD protein, human; C494826:BAX protein, human; C494820:Bad protein, mouse; C494827:Bax protein, mouse; D047428:Protein Kinase Inhibitors; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases; C505409:Casp3 protein, mouse; D053148:Caspase 3