Proteome Analysis and Functional Expression Identify Mortalin as an Antiapoptotic Gene Induced by Elevation of [Na ⁺ ] _i /[K ⁺ ] _i Ratio in Cultured Vascular Smooth Muscle Cells

Abstract

Abstract-Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in remodeling of vessel walls, one of the major determinants of long-term blood pressure elevation and an independent risk factor for cardiovascular morbidity and mortality. Recently, we have found that apoptosis in cultured VSMCs can be inhibited by inversion of the intracellular [Na ϩ ]/[K ϩ ] ratio after the sustained blockage of the Na ϩ ,K ϩ -ATPase by ouabain. To understand the mechanism of ouabain action, we analyzed subsets of hydrophilic and hydrophobic VSMC proteins from control and ouabain-treated cells by 2-dimensional electrophoresis. Ouabain treatment led to overexpression of numerous soluble and hydrophobic cellular proteins. Among proteins that showed the highest level of ouabain-induced expression, we identified mortalin (also known as GRP75 or PBP-74), a member of the heat shock protein 70 (HSP70) superfamily and a marker for cellular mortal and immortal phenotypes. Northern and Western blotting and immunocytochemistry all have confirmed that treatment of VSMCs with ouabain results in potent induction of mortalin expression. Transient transfection of cells with mortalin cDNA led to at least a 6-hour delay in the development of apoptosis after serum deprivation. The expression of tumor suppressor gene, p53, in mortalin-transfected cells was delayed to the same extent, and the expressed protein showed abnormal perinuclear distribution, suggesting that p53 is retained and inactivated by mortalin. Our studies therefore define a new [Na Key Words: apoptosis Ⅲ vascular smooth muscle Ⅲ proteome Ⅲ ion transport Ⅲ ouabain R emodeling of the blood vessel plays an important role in a variety of human vascular disorders, including hypertension, 1-3 atherosclerosis, 4 arterial injury, and restenosis after angioplasty. 5-7 Apoptosis (programmed cell death) of vascular smooth muscle cells (VSMCs) has recently been identified as the main factor contributing to the regulation of their number during remodeling, 8 -12 which inspired numerous studies of the mechanisms of the induction and progression of VSMC apoptosis. The execution phase of apoptosis in VSMCs is triggered similarly to that in the other cell types by activation of the caspase cascade, cleavage of intracellular proteins, and final disintegration of the cell. In contrast, the induction phase is specific for different subtypes of remodeling and involves the integration of multiple pro-and antiapoptotic signals, including the expression of death receptors, protooncogenes, and tumor suppressor genes. [13][14][15][16][17][18][19] Our recent studies showed that inhibition of the VSMC Na ϩ ,K ϩ pump with ouabain, or in K ϩ -free medium, rescues cells from apoptosis triggered by a number of factors including serum deprivation. 20 Equimolar substitution of extracellular Na ϩ with K ϩ completely abolished the effect of ouabain 20 showing that antiapoptotic action was indeed caused by the inversion of [NaThe development of cell death was blocked upstream of caspase-3 activati...

Na + /K + pump and endothelial cell survival: [Na + ] i /[K + ] i -independent necrosis triggered by ouabain, and protection against apoptosis mediated by elevation of [Na + ] i

“…What is the nature of the intracellular Na + sensor triggering antiapoptotic signals? Is expression of early response genes and mortalin detected in VSMC under [Na + ] i elevation [37,38] involved in the inhibition of PAEC apoptosis as shown in Fig. 7?…”

On the brotherhood of the mitochondrial chaperones mortalin and heat shock protein 60

“…In a proteome analysis of oubain-treated vascular smooth muscle cells (VSMCs), mortalin/mtHsp70 was identified as one of the important antiapoptotic genes (Taurin et al 2002). Its overexpression suppresses apoptosis from various stressors, eg, arsenite in rat lung epithelial cells (Lau et al 2004), differentiation agent 1,25-dihydroxyvitamin D3 in rat gliomas (Baudet et al 1998), and glucose starvation and ischemia reperfusion in Chinese hamster lung (CHL) cells (Gao et al 2003).…”

Overexpression of Mitochondrial Hsp70/Hsp75 Protects Astrocytes against Ischemic Injury in vitro

“…Studies have indicated that Hsp75 upregulation suppresses apoptosis induced by arsenite in lung epithelial cells (Lau et al, 2004), mercury in renal cells (Stacchiotti et al, 2006), and glucose starvation and ischemia reperfusion in lung cells (Gao et al, 2003). Hsp75 overexpression has been shown to delay apoptotic response to serum deprivation in muscle cells (Taurin et al, 2002), and decrease oxidative stress during GD in a neuronal cell line (Liu et al, 2005). Hsp75 is an important molecular chaperone in mitochondria, which is a crucial part of the mitochondrial protein import machinery (Krimmer et al, 2000).…”