rottlerin and Hypoxia

Topics: Acetophenones; Adaptation, Physiological; Animals; Benzophenanthridines; Benzopyrans; Cell Hypoxia; Cytoprotection; Gene Expression Regulation; Genistein; Hypoxia; L-Lactate Dehydrogenase; Male; Myocytes, Cardiac; Oxygen; Phosphatidylinositol 3-Kinases; Primary Cell Culture; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Wortmannin

Diabetic retinopathy is one of the most common complications in patients with diabetes and affects ~75% of them within 15 years of the onset of the disease. Activation of protein kinase C (PKC) is a key feature of diabetes mellitus and may be involved in the pathogenesis of diabetic retinopathy. The present study aimed to examine the translocation of protein kinase C (PKC) isoforms, which are triggered by high an moderately high glucose levels as well as hypoxic conditions. The underlying cell mechanisms of PKC translocation in primary cultured human retinal endothelial cells (HRECs) were also investigated. The expression levels of PKC isoforms were assessed using western blot analysis. Cell proliferation was determined using the MTT assay and DNA synthesis was assessed by bromodeoxyuridine incorporation. Translocation of PKC isoforms was examined by western blot analysis and immunofluorescence. The expression of PKC α, βI, βII, δ and ε was detected, while PKC ζ was not detected in HRECs. The results of the present study were consistent with the findings of a previous study by our group, reporting that moderately high glucose levels and hypoxia, but not high glucose levels, significantly increased cell proliferation. It was demonstrated that the PKC δ isoform was translocated from the cytosol to the membrane only under moderately high glucose conditions, while PKC α and ε isoforms were translocated from the cytosol to the membrane at high glucose conditions. In addition, PKC βI was translocated under all three conditions. Translocation of PKC βII was comparable among all groups. Furthermore, rottlerin, an inhibitor of PKC δ, blocked cell proliferation, which was induced by moderately high glucose levels, but not by hypoxia. Ro32-0432, an inhibitor of PKC α, βI and ε, did not significantly affect proliferation of HRECs in all treatment groups. In conclusion, the present study suggested that PKC α, βI, βII, δ and ε were expressed in primary cultured HRECs, whereas PKC ζ was not. Cell proliferation induced by moderately high glucose concentrations was associated with translocation of the PKC δ isoform; however, hypoxic conditions did not induce translocation.

Topics: Acetophenones; Benzopyrans; Cell Proliferation; Cells, Cultured; Endothelial Cells; Gene Expression; Glucose; Humans; Hypoxia; Indoles; Primary Cell Culture; Protein Kinase C-delta; Protein Transport; Pyrroles; Retinal Pigment Epithelium

In the present study, to clarify the role of protein kinase C (PKC) in the regulation of monocarboxylate transporter 4 (MCT4) expression, we examined the regulation mechanism of MCT4 expression in human rhabdomyosarcoma (RD) cells, an in vitro skeletal muscle model. Exposure of RD cells to PMA, a PKC activator, for 24 h resulted in a two-fold increase in the amount of lactic acid in the growth medium. In parallel to an increase in lactic acid release from RD cells, the level of MCT4 mRNA and protein were also significantly increased in RD cells. A PKC inhibitory study indicated that PMA-induced stimulation of MCT4 expression can be mediated through a novel PKC isoform, especially PKCδ. Moreover, rottlerin, a selective PKCδ inhibitor, decreased PMA-induced MCT4 promoter activity. Deletion and mutational analysis suggested that the potential hypoxia-response elements (HREs) played a major role in the observed modulation of MCT4 expression by PMA. Furthermore, we found that small interfering RNA (siRNA)-mediated knockdown of hypoxia-inducible factor 1α (HIF-1α) significantly inhibited PMA-induced MCT4 promoter activity. Our results show that the effects of PMA on MCT4 expression are mediated through an indirect pathway partially involving PKCδ and HIF-1α transcription factor.

Topics: Acetophenones; Base Sequence; Benzopyrans; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lactic Acid; Molecular Sequence Data; Monocarboxylic Acid Transporters; Muscle Cells; Muscle Proteins; Muscle, Skeletal; Promoter Regions, Genetic; Protein Kinase C; Protein Kinase Inhibitors; Response Elements; Rhabdomyosarcoma; RNA, Messenger; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Hypoxia inducible factor-1 (HIF-1) is central to most adaptation responses of tumors to hypoxia, and consists of a hypoxia inducible HIF-1alpha or -2alpha subunit, and a constitutively expressed HIF-1beta subunit. Previously, mitochondrial uncouplers, rottlerin and FCCP, were shown to increase the rate of cellular O(2 )consumption. In this study, we determined that mitochondrial uncouplers, rottlerin and FCCP, significantly decreased hypoxic as well as normoxic HIF-1 transcriptional activity which was in part mediated by down-regulation of the oxygen labile HIF-1alpha and HIF-2alpha protein levels in PC-3 and DU-145 prostate cancer cells. Our results also revealed that mitochondrial uncouplers decreased the expression of HIF target genes, VEGF and VEGF receptor-2. Taken together, our results indicate that functional mitochondria are important in HIF-1alpha and HIF-2alpha protein stability and transcriptional activity during normoxia as well as in hypoxia, and that mitochondrial uncouplers may be useful in the inhibition of HIF pathway in tumors.

Topics: Acetophenones; Basic Helix-Loop-Helix Transcription Factors; Benzopyrans; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Line, Tumor; Cell Shape; Deferoxamine; Enzyme Inhibitors; Gene Expression Regulation; Genes, Reporter; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Ionophores; Mitochondria; Siderophores; Uncoupling Agents; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2