ABSTRACT
Insulin secretion from pancreatic beta cells is stimulated by glucose metabolism. However, the relative importance of metabolizing glucose via mitochondrial oxidative phosphorylation versus glycolysis for insulin secretion remains unclear. von Hippel-Lindau (VHL) tumor suppressor protein, pVHL, negatively regulates hypoxia-inducible factor HIF1alpha, a transcription factor implicated in promoting a glycolytic form of metabolism. Here we report a central role for the pVHL-HIF1alpha pathway in the control of beta-cell glucose utilization, insulin secretion, and glucose homeostasis. Conditional inactivation of Vhlh in beta cells promoted a diversion of glucose away from mitochondria into lactate production, causing cells to produce high levels of glycolytically derived ATP and to secrete elevated levels of insulin at low glucose concentrations. Vhlh-deficient mice exhibited diminished glucose-stimulated changes in cytoplasmic Ca(2+) concentration, electrical activity, and insulin secretion, which culminate in impaired systemic glucose tolerance. Importantly, combined deletion of Vhlh and Hif1alpha rescued these phenotypes, implying that they are the result of HIF1alpha activation. Together, these results identify pVHL and HIF1alpha as key regulators of insulin secretion from pancreatic beta cells. They further suggest that changes in the metabolic strategy of glucose metabolism in beta cells have profound effects on whole-body glucose homeostasis.
Subject(s)
Carbohydrate Metabolism/genetics , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/physiology , Animals , Cells, Cultured , Electrophysiology , Fluorometry , Glucose Tolerance Test , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Immunoblotting , Immunohistochemistry , Insulin Secretion , Mice , Mice, Mutant Strains , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Von Hippel-Lindau Tumor Suppressor Protein/geneticsABSTRACT
We have previously shown that PKC inhibitors interfere with the Ets1/Smad3-dependent regulation of parathyroid hormone-related protein (PTHrP) P3 promoter activity by TGFbeta in invasive MDA-MB-231 breast cancer cells. By examining PKC expression in a variety of breast cancer cell lines, the protein level of PKCalpha was found to be much higher in Ets1-expressing MDA-MB-231 and MDA-MB-435 breast cancer cells than in Ets1-deficient MCF-7 and SK-BR3 cells. No correlation of Ets1 expression with the expression of other PKC subtypes (PKCbeta1, PKCbeta2, PKCdelta or PKCepsilon) could be observed. In contrast to MDA-MB-231 cells, PKCalpha-deficient MCF-7 cells do not support Ets1-induced activation of the PTHrP P3 promoter suggesting that PKCalpha may be important for Ets1 activity. A constitutively active form of PKCalpha was found to potentiate the P3 promoter activation by Ets1 alone and in synergy with Smad3. PKCalpha, but not PKCepsilon, also induced phosphorylation of the Ets1 protein. Both PKCalpha effects on Ets1 depended on the exon VII domain of Ets1. Using verapamil and ionomycin, we could show that PKCalpha induces Ets1 phosphorylation independent of calcium mobilization. Collectively, our data suggest that PKCalpha may regulate Ets1 activity in invasive breast cancer cells.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Protein Kinase C/physiology , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Blotting, Western , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Cell Line , Cell Line, Tumor , Cell Nucleus/metabolism , Chloramphenicol O-Acetyltransferase/metabolism , Cytosol/metabolism , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Humans , Ionomycin/pharmacology , Ionophores/pharmacology , Luciferases/metabolism , Phosphorylation , Plasmids/metabolism , Protein Kinase C/metabolism , Protein Kinase C-alpha , Protein Kinase C-epsilon , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins c-ets , Smad3 Protein , Trans-Activators/metabolism , Transfection , Verapamil/pharmacologyABSTRACT
Transcription factor Ets1 is expressed in invasive breast cancer cells. In T-cells, the splicing variant DeltaVII-Ets1 is naturally produced along with full length Ets1 (fl-Ets1). Though its function is unknown, the lack of important inhibitory domains predicts a regulatory role of DeltaVII-Ets1 in fl-Ets1-controlled transcription. Examining the expression status of DeltaVII-Ets1 in invasive Ets1-producing MDA-MB-231 breast cancer cells, we found that the DeltaVII-Ets1 protein could only be detected when nuclear proteins from these cells were fractionated by ionic exchange chromatography. When overexpressed, DeltaVII-Ets1 was found to be partially degraded in breast cancer cells, but not in Jurkat T-cells or SK-Mel melanoma cells. In contrast, no proteolytic products resulted from ectopic expression of fl-Ets1 suggesting that breast cancer cells are able to specifically cleave DeltaVII-Ets1. Overexpression of DeltaVII-Ets1 reduced survival of MDA-MB-231 cells, but not of MCF-7 cells. A mutant version of DeltaVII-Ets1, lacking first 129 N-terminal amino acids, had no effect. These data suggest that Ets1-producing invasive breast cancer cells specifically downregulate DeltaVII-Ets1, as it may be able to adversely affect the survival of these cells.
Subject(s)
Alternative Splicing , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Down-Regulation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acids/chemistry , Blotting, Western , Cell Line , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Survival , Chromatography, Ion Exchange , Green Fluorescent Proteins , Humans , Jurkat Cells , Luminescent Proteins/metabolism , Mutation , Neoplasm Invasiveness , Plasmids/metabolism , Protein Structure, Tertiary , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins c-ets , Subcellular Fractions , Time Factors , Transcription, Genetic , TransfectionABSTRACT
Von Hippel-Lindau (VHL) tumour suppressor gene inactivation is linked to the development of haemangioblastomas in the central nervous system and retina, often in association with other tumours, such as clear-cell carcinomas of the kidney and phaeochromocytomas. Here we show that the VHL protein (pVHL) is a microtubule-associated protein that can protect microtubules from depolymerization in vivo. Both the microtubule binding and stabilization functions of pVHL depend on amino acids 95-123 of pVHL, a mutational 'hot-spot' in VHL disease. From analysis of naturally occurring pVHL mutants, it seems that only point mutations such as pVHL(Y98H) and pVHL(Y112H) (that predispose to haemangioblastoma and phaeochromocytoma, but not to renal cell carcinoma) disrupt pVHL's microtubule-stabilizing function. Our data identify a role for pVHL in the regulation of microtubule dynamics and potentially provide a link between this function of pVHL and the pathogenesis of haemangioblastoma and phaeochromocytoma in the context of VHL disease.
