Role of microRNA in nitrogen mustard mediated skin pathogenesis

Alkylating mustard vesicants have been employed as chemical warfare agents. Their potential use against civilian populations poses a significant threat to public health. Dermal exposure to the war gas sulfur mustard and related vesicants results in inflammation, edema, blistering and impaired wound healing. We investigated the mechanisms mediating these responses using PAM212 mouse keratinocytes and the model vesicant nitrogen mustard, mechlorethamine (HN2) and examining changes in the expression pattern of genes mediating inflammation and blistering. Exposure to HN2 resulted concentration (1 to 30 µM) and time (0 to 24 h) related increases in mRNA levels encoding the pro-inflammatory proteins iNOS, COX2, IL-1â, and TNFa and the oxidative stress markers, heme oxygenases, as well as alterations in metalloproteinase levels, molecules important in blistering and wound healing. Messenger RNA levels were evaluated using quantitative real-time PCR (QPCR)and semi-quantitative reverse transcriptase PCR. Changes to multiple cellular processes prompted us to speculate that HN2 may affect microRNA (miR), small endogenous noncoding RNAs (miR)that that broadly modulate gene expression. Cellular levels of regulatory miR were evaluated using polymerase chain reaction (PCR) arrays and QPCR. We found that levels of miR 183; a regulator of gene expression important in cell migration and matrix-epithelial interactions were enhanced up to 35 fold by treatment of cells with 3 µM HN2. Likewise, levels of miR 21, a central regulator of diverse genes important in oxidative stress and inflammation, were enhanced 6-10 fold by HN2. Increases in miR levels were limited by the immunosuppressant, rapamycin, and by inhibitors of inflammatory responses including phosphoinositide-3-kinase (LY294002), and the p38-MAP kinase inhibitor (SB203580). Taken together these data indicate that HN2 alters expression of genes central to inflammation, matrix-stromal interactions and oxidative stress. We speculate that noncoding RNAs such as miR 21 and miR 183 have a role to play in HN2 mediated pathobiology.