Peroxynitrite modulates MnSOD gene expression in lung epithelial cells

Peroxynitrite (ONOO−) is a strong oxidant derived from nitric oxide (√NO) and superoxide (O2•−), reactive nitrogen (RNS) and oxygen species (ROS) present in inflamed tissue. Other oxidant stresses, e.g., TNF-α and hyperoxia, induce mitochondrial, manganese-containing superoxide dismutase (MnSOD) gene expression. These experiments tested whether ONOO− regulated MnSOD gene expression in human lung epithelial (A549) cells. 3-morpholinosydnonimine HCl (SIN-1) (10 or 1000 μM) increased MnSOD mRNA, but did not change hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA. Authentic peroxynitrite (ONOO−) (100–500 μM) also increased MnSOD mRNA but did not change constitutive HPRT mRNA expression. ONOO− stimulated luciferase gene expression driven by a 2.5 kb fragment of the rat MnSOD gene 5′ promoter region. MnSOD gene induction due to ONOO− was inhibited effectively by L-cysteine (10 mM) and partially inhibited by N-acetyl cysteine (50 mM) or pyrrole dithiocarbamate (10 mM). √NO from 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA NONOate) (100 or 1000 μM) did not change MnSOD or HPRT mRNA. Neither H2O2 nor NO2−, breakdown products of SIN-1 and ONOO−, had any effect on MnSOD mRNA expression; however, ONOO− and SIN-1 did not increase MnSOD protein content detectable by western blots, nor did they increase MnSOD enzymatic activity. Increased steady state [O2•−] in the presence of √NO yields ONOO−, and ONOO− has direct, stimulatory effects on MnSOD transcript expression.