Redox activation of DUSP4 by N-acetylcysteine protects endothelial cells from Cd2+-induced apoptosis

Abstract

Redox imbalance is a primary cause for endothelial dysfunction (ED). Under oxidant stress, many critical proteins regulating endothelial function undergo oxidative modifications that lead to ED. Cellular levels of GSH, the primary reducing source in cells, can significantly regulate cell function via reversible protein thiol modification. N-Acetyl cysteine (NAC), a precursor for GSH biosynthesis, is beneficial for many vascular diseases; however, the detailed mechanism of these benefits is still not clear. From HPLC analysis, NAC significantly increases both cellular GSH and BH4 levels. Immunoblotting of eNOS and DUSP4, a dual-specificity phosphatase with a cysteine as its active residue, revealed that both enzymes are up-regulated by NAC. EPR spin-trapping further demonstrated that NAC enhances NO generation from cells. Long-term exposure to Cd2+ contributes to DUSP4 degradation and the uncontrolled activation of p38 and ERK1/2, leading to apoptosis. Treatment with NAC prevents DUSP4 degradation and protects cells against Cd2+-induced apoptosis. Moreover, the increased DUSP4 expression can redox regulate p38 and ERK1/2 pathways from hyper-activation, providing a survival mechanism against the toxicity of Cd2+. DUSP4 gene knockdown further supports the hypothesis that DUSP4 is an antioxidant gene, critical in the modulation of eNOS translation, and thus protects against Cd2+-induced stress. Depletion of intracellular GSH by BSO makes cells more susceptible to Cd2+-induced apoptosis. Pre-treatment with NAC prevents p38 over-activation and thus protects the endothelium from this oxidative stress. Therefore, the identification of DUSP4 activation by NAC provides a novel target for future drug design.

N-acetylcysteine stimulates protein synthesis in enterocytes independently of glutathione synthesis

“…Data are mean ± SEM, n = 8. *P < 0.05 (Barajas-Espinosa et al 2014;Kim et al 2001). In recent years, we found that dietary supplementation with a small amount of NAC relative to dietary provision of cysteine enhanced small-intestinal mucosal mass in LPS-challenged young pigs (Hou et al 2012, suggesting that NAC may have hitherto unrecognized effects other than simply serving as a precursor of cysteine for GSH synthesis in cells.…”

“…Hence, it has generally been assumed that the efficacy of NAC in treating clinical diseases is due to its antioxidant/radical scavenger properties (Kim et al 2001). However, through modulating levels of various target genes and/or proteins, NAC has also been reported to regulate cell cycle and apoptosis, including the inhibition of proliferation of normal human cells and transformed cells (Samuni et al 2013), prevention of apoptosis of neuronal cells and fibroblasts (Cossarizza et al 1995), and enhancement of survival of PC12 and endothelial cells (Barajas-Espinosa et al 2014). Apparently, there are discrepancies among these studies with regard to effects of NAC on cell growth and apoptosis because the mechanisms responsible for the biological function of NAC are incompletely understood.…”

“…According to published reports, NAC has long been thought to exert positive effects in intestinal cells mainly through GSH synthesis and antioxidative reactions (Barajas-Espinosa et al 2014;Kim et al 2001). However, alternative mechanisms for the biological function of NAC are possible but largely unknown.…”

Loss of Multidrug Resistance–Associated Protein 1 Potentiates Chronic Doxorubicin-Induced Cardiac Dysfunction in Mice

“…The protein concentrations of heart ventricle homogenates were determined with the bicinchoninic acid protein assay. The measurement of GSH and GSSG by high-performance liquid chromatography was performed exactly as described (Deng et al, 2015) and used by others (Yamazaki et al, 2008(Yamazaki et al, , 2012Yamazaki and Villarreal, 2011;Kennedy et al, 2013;Barajas-Espinosa et al, 2014). The use of authentic GSH and GSSG to develop the standard curves ensured quantitation of the correct entities by high-performance liquid chromatography, thus validating the assay.…”

Elevated Glutathione Is Not Sufficient to Protect against Doxorubicin-Induced Nuclear Damage in Heart in Multidrug Resistance–Associated Protein 1 (Mrp1/Abcc1) Null Mice

“…The HPLC assay for GSH and GSSG is based on that developed by Senft et al (2000), and is used widely (Yamazaki et al, 2008(Yamazaki et al, , 2012Yamazaki and Villarreal, 2011;Kennedy et al, 2013;Barajas-Espinosa et al, 2014). Mouse heart homogenate was diluted with 0.5 volume of buffer A containing 15% metaphosphoric acid and mixed thoroughly, followed by centrifugation at 12,000 rpm for 15 minutes at 4°C.…”