Biphasic Effect of Exogenous Nitric Oxide on Proliferation and Differentiation in Skin Derived Keratinocytes but Not Fibroblasts

Abstract

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.

Tumor necrosis factor receptor 1-mediated signaling is required for skin cancer development induced by NF-κB inhibition

“…A critical role of neutrophils in driving the hyperproliferation in the flaky skin mouse mutant has been demonstrated, and it is possible that neutrophils will prove to play a similar role in K5-I B␣ mice (22). It is known that neutrophils produce reactive oxygen species, lipid mediators, and proteolytic enzymes that potentially can affect keratinocyte proliferation (23)(24)(25). Of interest is also the infiltration of B220(ϩ) Pax5(Ϫ) cells with dendritic morphology, which we identify as plasmacytoid dendritic cells.…”

p68 DEAD Box RNA Helicase Expression in Keratinocytes

“…We could convincingly demonstrate the potency of exogenous NO to induce p68 expression in keratinocytes. Nevertheless, although lower amounts of NO (250 M) were also capable of increasing cellular p68 levels, a potent induction of p68 was only achieved with NO concentrations that have been shown to be cytostatic for cultured keratinocytes (26,36). By contrast, typical wound-related keratinocyte mitogens appeared to potently trigger an increase in p68 in the cells in vitro.…”

“…p68 RNA Helicase and Keratinocytes 44879 low concentrations of NO (26,36). However, it is not particularly known how NO exerts its unique actions on keratinocytes.…”

Modelling Blood Flow Regulation by Nitric Oxide in Psoriatic Plaques

“…This raises immediate and currently unanswered questions about the possible role for NO in onset of psoriasis; our modelling does not address this at all, considering only NO dynamics within a pre-existing plaque. The potential for NO having a causal effect in plaque formation arises from the many aspects of skin biology that are regulated by NO, including epidermal cell division and differentiation (Krischel et al, 1998), endothelial cell growth and migration (Ziche et al, 1994), and extracellular matrix turnover (Okamoto et al, 1997;Maeda et al, 1998). In a separate study (Savill et al, 2002), we have used mathematical modelling to investigate this last effect, suggesting that the NO-dependence of matrix degradation may help to explain the elongated rete pegs observed in psoriatic plaques.…”