Identification of copper/zinc superoxide dismutase as a nitric oxide-regulated gene in human (HaCaT) keratinocytes: implications for keratinocyte proliferation

Recent studies have demonstrated an induction of expression of inducible ni tric oxide synthase that is associated with several inflammatory diseases o f the skin. To define the mechanisms of action of nitric oxide (NO) in the skin, we attempted to identify genes that are regulated by NO in keratinocy tes. Using the human keratinocyte cell line HaCaT as a model system, we ide ntified a Cu/Zn superoxide dismutase (SOD) that was strongly induced by hig h concentrations (500 mu M) of NO-donating: agents (S-nitrosoglutathione, s odium nitroprusside and (2)-1-[2(2-aminoethyl)-N-(2-ammonioethyl)amino]d 1- ium-1,2-diolate (DETA-NO)), but not by serum or by single recombinant growt h factors and inflammatory cytokines or by treatment with superoxide anions . Furthermore, endogenously produced NO increased the expression of Cu/Zn S OD mRNA in keratinocytes. Moreover, treatment of HaCaT cells with NO was as sociated with a biphasic effect on cell proliferation, because low doses (1 00 mu M) of different NO donors (S-nitrosoglutathione and DETA-NO) mediated a proliferative signal to the cells, whereas high concentrations (500 mu M ) were cytostatic. To determine a possible correlation between the close re gulation of Cu/Zn SOD expression and proliferation by NO in keratinocytes, we established a cell line (psp1CZ1N) carrying a human Cu/Zn SOD cDNA under the control of a ponasterone-inducible promoter construct. Ponasterone-ind uced overexpression of Cu/Zn SOD caused a cytostatic effect in proliferatin g psp1CZ1N cells. We therefore suggest that the up-regulation of Cu/Zn SOD expression by NO establishes an inhibitory mechanism on keratinocyte prolif eration.