Nitric oxide (NO) is known to exert cytotoxic and cytostatic effects in various cells and tissues. Although NO formation in human skin has been convincingly demonstrated, little is known about the NO-mediated effects in skin physiology and pathology. Here, we investigate the influence of NO on proliferation, differentiation, and apoptosis of primary cultures of normal human keratinocytes and fibroblasts. Four different NO donors at concentrations ranging from 0.01 to 5 mM were added every 12 h or 24 h to primary cultures of human keratinocytes and fibroblasts, and cells cultured for up to 3 d in the presence of these compounds. Cultures were examined for necrosis or apoptosis using trypan blue exclusion and in situ nick-translation. Cultures were also screened for the expression of the proliferation marker Ki67 and for an increase in cell numbers using neutral red staining. In addition, keratinocytes were stained for cytokeratin 6 expression to assess differentiation. We find that both keratinocytes and fibroblasts are highly resistant towards necrosis- or apoptosis-inducing effects of NO. In both cell types NO modulates cell growth, albeit in a cell-type specific pattern: cytostasis becomes significant in fibroblasts at concentrations of > or = 0.25 mM of the NO donor. In keratinocytes a biphasic effect is found with increased proliferation at low concentrations ranging from 0.01 to 0.25 mM and cytostasis at concentrations of > or = 0.5 mM. Conversely, expression of cytokeratin 6 is decreased at the lower NO donor concentrations and increased at higher concentrations as an indication of induction of differentiation at higher NO concentrations. Collectively, our results demonstrate that NO modulates proliferation and differentiation in human skin cells, a finding that will help to explain the pathophysiology of human skin diseases. Moreover, these findings suggest that NO generation in human skin diseases is not directly associated with local cell destruction, in contrast to findings in several other human diseases.

A variety of toxic and modulating events induced by UVA exposure are described to cause cell death via apoptosis. Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). We have now searched for the role of iNOS expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the onset of apoptosis. This protection was completely abolished in the presence of the iNOS inhibitor L-N 5 -(1-iminoethyl)-ornithine (0.25 mM). Additionally, preincubation of cells with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate at concentrations from 10 to 1000 M as an exogenous NO-generating source before UVA irradiation led to a dose-dependent inhibition of both DNA strand breaks and apoptosis. In search of the molecular mechanism responsible for the protective effect, we find that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. In conclusion, we present evidence for a protective role of iNOS-derived NO in skin biology, because NO either endogenously produced or exogenously applied fully protects against UVA-induced cell damage and death. We also show that the NO-mediated expression modulation of proteins of the Bcl-2 family, an event upstream of caspase activation, appears to be the molecular mechanism underlying this protection.

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