Activation of Sphingosine Kinase-1 Mediates Inhibition of Vascular Smooth Muscle Cell Apoptosis by Hyperglycemia

Abstract

Vascular smooth muscle cell (VSMC) apoptosis plays an essential role in vascular development and atherosclerosis. Hyperglycemia inhibits VSMC apoptosis, which may contribute to the development of diabetic vasculopathy. In the present study, we analyzed the mechanism of high-glucoseinduced anti-apoptotic effect in cultured human aortic smooth muscle cells (HASMCs). Compared with normoglycemia, exposure of HASMCs to hyperglycemia but not mannitol significantly increased sphingosine kinase 1 (SK1) activity but not SK2 activity. This increase was inhibited by protein kinase C (PKC) inhibitor GF109203X, the antioxidant N-acetylcysteine, and the reduced form of glutathione. The mechanism of SK1 activation by high glucose involves plasma membrane translocation. In addition, hyperglycemia markedly inhibited serum withdrawalinduced apoptosis in HASMCs. Importantly, inhibition of SK1 by either a competitive inhibitor N,N-dimethylsphingosine or expression of dominant-negative mutant of SK1(G82D) or specific small interference RNA knockdown substantially attenuated hyperglycemia-induced anti-apoptotic effect and anti-apoptotic protein Bcl-2 expression in HASMCs. Moreover, SK1-mediated anti-apoptotic effect requires the intracellular effects of sphingosine-1-phosphate. We conclude that hyperglycemia stimulates SK1 activity via PKC-and oxidative stress-dependent pathways, leading to decreased apoptosis in HASMCs. Taken together, these observations have important implications for understanding the roles of the SK1 signaling pathway in the pathogenesis of diabetic vasculopathy. Diabetes

Down-regulation of Mammalian Sterile 20–Like Kinase 1 by Heat Shock Protein 70 Mediates Cisplatin Resistance in Prostate Cancer Cells

“…Histone-associated DNA fragments were quantitated by the Cell Death Detection ELISA (Roche; ref. 17). Briefly, the cytoplasmic fractions were added to the 96-well ELISA plates precoated with the antihistone monoclonal antibody and incubated for 90 min at room temperature.…”

The Orphan Nuclear Receptor Nur77 Suppresses Endothelial Cell Activation Through Induction of IκBα Expression

“…15 Briefly, pBHGlox⌬E1,3Cre, including the ⌬E1 adenoviral genome, was cotransfected with the pDC shuttle vector containing the gene of interest into Ad293 cells using FuGene 6 Transfection Reagent (Roche, Indianapolis, IN). The viruses were propagated in Ad293 cells and purified using CsCl 2 banding, followed by dialysis against 20 mmol/L Tris-buffered saline with 10% glycerol.…”

Arterial smooth muscle cells dysfunction in hyperglycaemia and hyperglycaemia associated with hyperlipidaemia: from causes to effects

“…Although the mechanism responsible for the accelerated SMCs proliferation in diabetes is still under debate, recent reports underline the involvement of anti-apoptotic protein Bcl-2 that is over-expressed in high glucose conditions. The up-regulation of Bcl-2 perturbs the balance between SMCs apoptosis and proliferation, reduces apoptosis, and contribute finally to enhanced SMCs proliferation, as demonstrated in both human diabetic arteries (Ruiz et al, 2006) and cultured human aortic SMCs (You et al, 2007). In the latter, decreased apoptosis was directly linked to the high glucose effects; thus, excess glucose stimulates sphingosine kinase 1 activity via PKC and oxidative stress-dependent pathways, leading to reduced apoptosis.…”

“…The inner mitochondrial membrane is responsible for the generation of ROS implicated in SMCs dysfunction. Recent evidence indicates that in human aortic SMCs oxidative stress-dependent pathways and PKC conduct to stimulation of sphingosine kinase-1 activity, an enzyme that decreases cells apoptosis (You et al, 2007). Glycated albumin and insulin act synergistic triggering potent increase in proliferation of aortic SMCs (Rong et al, 2007).…”