Ultraviolet irradiation induces cyclooxygenase-2 expression in keratinocytes.

We examined the effect of ultraviolet (UV) irradiation on the expression of cyclooxygenases in cultured HaCaT keratinocytes and in human skin in vivo. UVB irradiation (10 and 50 mJ/cm2) and hydrogen peroxide (200 micromol/L) increased cyclooxygenase-2 mRNA expression in HaCaT keratinocytes. No clear expression of cyclooxygenase-1 mRNA was detected in either control or stimulated HaCaT cells. Genistein, a tyrosine kinase inhibitor, suppressed both the basal and stimulated expression of cyclooxygenase-2 in HaCaT cells. UVB-induced cyclooxygenase-2 mRNA expression was partly inhibited by the antioxidant N-acetylcysteine and by H-7, a non-specific inhibitor of protein kinase C. Solar-simulated irradiation (40 mJ/cm2) was found to induce in vivo both cyclooxygenase-2 mRNA and protein expression in human skin, whereas the expression of cyclooxygenase-1 mRNA remained at the basal level. Our results show that cyclooxygenase-2 expression is induced by UV irradiation and suggest that tyrosine kinases and reactive oxygen intermediates are involved in this induction of cyclooxygenase-2.

UV irradiation induces downregulation of bcl-2 expression in vitro and in vivo.

Recently, the proto-oncogenes bcl-2 and bax have emerged as important regulators of the apoptotic form of cell death. We examined UV irradiation-elicited apoptosis and regulation of bcl-2 and bax expression both in vivo in human skin and in vitro in HeLa cells. Using flow cytometric analysis, HeLa cells were found to undergo apoptosis at the 12-h time-point after exposure to UVB irradiation (100 mJ/cm2). The expression of bcl-2 mRNA was found to decrease after a single dose of UVB radiation (doses 10-200 mJ/cm2). In contrast, the expression of bax mRNA was not significantly changed. When human skin was irradiated with a single dose of solar-simulated radiation (40 mJ/cm2), Bcl-2-positive cells were significantly reduced in the epidermis at the 3- and 6-h time-points. Our results suggest that UV irradiation downregulates bcl-2 expression both in vitro at the mRNA level and in vivo at the protein level, and that downregulation of bcl-2 constitutes a mechanism of potential importance in UV-induced apoptosis in human epidermis.

Differential regulation of the AP-1 family members by UV irradiation in vitro and in vivo.

We have examined the effect of UVB and solar-simulated irradiation on the expression of the AP-1 family of transcription factors and the cytokine IL-6 both in cell cultures and in human skin in vivo. UVB irradiation potently induced c-jun, junB and c-fos mRNA levels in vitro in HaCaT cells. IL-6 mRNA was induced in response to UVB irradiation 2-3 h later than c-jun, junB and c-fos mRNAs. In human skin in vivo, solar-simulated irradiation induced transiently junB expression. Genistein, a tyrosine kinase inhibitor, augmented the induction of c-jun and junB by UVB irradiation in HaCaT cells. The results of this study provide evidence that in addition to c-jun and c-fos, junB is also an essential component of the human UV-response. This study also suggests that UVB irradiation regulates the AP-1 family by several mechanisms and that the signalling mechanisms of UVB irradiation are considerably different from the ones used by UVC irradiation.

Regulation of copper/zinc and manganese superoxide dismutase by UVB irradiation, oxidative stress and cytokines.

We have examined the effects of UVB irradiation, oxidative stress and cytokines on the antioxidant enzymes copper/zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in HeLa cells. A single dose of UVB irradiation regulated dose-dependently the expression of the 4 kb transcript of MnSOD although it did not have any significant effect on MnSOD enzymatic activity. In contrast, UVB irradiation reduced both the enzymatic activity and the expression of the 0.7 and 0.9 kb mRNA transcripts of CuZnSOD. The cytokines TNF-alpha (1 ng ml-1 and 10 ng ml-1) and IL-6 (100 U ml-1) induced MnSOD activity, and TNF-alpha also upregulated MnSOD mRNA expression. Interestingly, genistein, a soy isoflavone and a tyrosine kinase inhibitor, was able to inhibit the induction of Mn-SOD activity and mRNA expression by TNF-alpha. Enzymatic CuZnSOD activity was depressed by a high dose of H2O2 while IL-6 or TNF-alpha had no effect on CuZnSOD activity. Our results demonstrate that, in addition to enzyme activity level, UVB irradiation can regulate the superoxide dismutases at the mRNA level. We also suggest that UVB irradiation, oxidative stress and cytokines regulate differentially CuZnSOD and MnSOD, and that the activities and expression of these antioxidant enzymes are controlled by distinct mechanisms.

Effects of prolonged EGF treatment on phospholipid turnover and DAG formation in murine keratinocytes.

In murine keratinocytes, the cellular diacylglycerol (DAG) content was considerably elevated following a 48-h exposure to epidermal growth factor (EGF), while formation of inositol phosphates (InsP) was not stimulated. A similar loss of InsP production upon stimulation of keratinocytes with 1.4 mM Ca2+ was seen after pretreatment with R59022, a DAG kinase inhibitor. These data suggest that accumulated endogenous DAG has an inhibitory feedback effect on PLC activity. To elucidate the possible phospholipid source of elevated DAG in keratinocytes, cells were first labeled with [3H]-choline and then exposed to EGF for 24 h or TPA, a protein kinase C activator, for 8 h. As expected, TPA increased [3H]-choline release into the culture medium, whereas EGF decreased the release, suggesting that EGF treatment does not result in sustained stimulation of phosphatidylcholine turnover. The release of [14C]-dihomo-gamma-linolenic acid (DHGLA), predominately bound to the 2-positions of phospholipids, was also stimulated by 8 h of TPA treatment but not by 24 h of EGF treatment. The distribution of DHGLA in various phospholipid subclasses was not influenced by EGF. These results indicate that prolonged EGF treatment does not markedly activate phospholipid A2 (PLA2) or lysophospholipase, and that the DAG accumulation after prolonged EGF exposure is apparently not associated with stimulated breakdown of any specific lipid pool. It is concluded that changes in keratinocyte lipid turnover induced by prolonged EGF treatment differ from those associated with short-term EGF exposure.