BIPHASIC EFFECT OF EXOGENOUS NITRIC-OXIDE ON PROLIFERATION AND DIFFERENTIATION IN SKIN-DERIVED KERATINOCYTES BUT NOT FIBROBLASTS

Nitric oxide (NO) is known to exert cytotoxic and cytostatic effects i n various cells and tissues. Although NO formation in human skin has b een convincingly demonstrated, little is known about the NO-mediated e ffects in skin physiology and pathology. Here, we investigate the infl uence of NO on proliferation, differentiation, and apoptosis of primar y cultures of normal human keratinocytes and fibroblasts. Four differe nt NO donors at concentrations ranging from 0.01 to 5 mM were added ev ery 12 h or 24 h to primary cultures of human keratinocytes and fibrob lasts, and cells cultured for up to 3 d in the presence of these compo unds. Cultures were examined for necrosis or apoptosis using trypan bl ue exclusion and in situ nick-translation. Cultures were also screened for the expression of the proliferation marker Ki67 and for an increa se in cell numbers using neutral red staining. In addition, keratinocy tes were stained for cytokeratin 6 expression to assess differentiatio n. We find that both keratinocytes and fibroblasts are highly resistan t 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 g reater than or equal to 0.25 mM of the NO donor. In keratinocytes a bi phasic effect is found with increased proliferation at low concentrati ons ranging from 0.01 to 0.25 mM and cytostasis at concentrations of g reater than or equal to 0.5 mM. Conversely, expression of cytokeratin 6 is decreased at the lower NO donor concentrations and increased at h igher concentrations as an indication of induction of differentiation at higher NO concentrations. Collectively, our results demonstrate tha t 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.