INHIBITION OF MATRIX METALLOPROTEINASES 233 0.6 fl" 0.5 '[] MMP1 •5•. * 0 0.4 :•-• 0 ............ • ........... • .............. • ................ • .... •- ,._t._,_. •! ........ • 0 5 10 15 20 30 UV irradiation (J/c•) Figure 1. The effect of UV irradiation on the production of MMP-1 and MMP-2 by human dermal fibroblasts. HDFs (1.5 x 105/well) were seeded into 35• plates and cultured overnight. The cells were irradiated from a distance of 15 cm by a UV so•ce for a given time. *n = 3. p 0.05 vs no UV ex•sure. by UV irradiation of HDFs is a result of the activation of cell surface growth factors and cytokine receptors, which have in common the requirement for dimerization to initiate signal transduction. UV radiation rapidly activates EGF receptors, followed by the activation of Ras, ERK, JNK, and p38 (18,19). These stress-activated MAP kinases then increase the proteins of c-Jun, c-Fos, and ATF. The dimerization of these proteins activates AP-1 DNA binding and, finally, the induction of MMPs. Fisher et al. showed that retinoic acid can inhibit the induction of MMPs by UV irradiation in human skin by blocking the DNA binding of AP-1 and c-Jun protein induction (20). EFFECT OF NITRIC OXIDE DONOR ON THE PRODUCTION OF MMPS To determine the effect of NO on the production of MMP-1 and -2, HDFs were treated with SNP. SNP is a donor of NO and it can mimic the cellular effects of NO. Treatment with SNP increased MMP-2 production acutely, to about 243% of untreated cells with 50 microM of SNP. The amount of secreted MMP-1 in the HDF culture medium was also increased with SNP treatment, but was less significant than that of MMP-2, about 153% (Figure 2A). The result of zymography also shows that gelatinase activity was increased with SNP treatment. The combined effect of SNP and UV treatments was also tested, as shown in Figure 2B. The null hypothesis was that the effect of UV irradiation is not mediated by NO, and that subsequently the addition of SNP to UV-irradiated HDF should increase MMP production to the same degree as in cells not treated with UV irradiation and more than in cells treated only with UV irradiation. In the UV- treated cells, 50 microM of SNP did not increase MMP-1 production significantly, while that of MMP-2 increased by 196% (Figure 2B). This result indicates that the effect of UV irradiation and SNP treatment on MMP-! production by HDF is not cumulative,

234 JOURNAL OF COSMETIC SCIENCE 0.8 :• 0.7 0.6 o 0.5 ß ,.., 0.4. (..1 = 0.3 o (• 0.2 .4,.,• •3 0.1 0 S Concentration of SNP(microM) Figure 2. The effect of sodium nitroprusside on the production of MMP-1 and MMP-2 by human dermal fibroblasts. A: without UV irradiation. B: with UV irradiation. *n = 3. p 0.05 vs no treatment. which means that the UV irradiation already produced a near-maximum increase in the production of MMP-1 and the addition of 50 microM of SNP could not increase the MMP-1 production further. On the other hand, the production of MMP-2 seems to be influenced by both UV irradiation and SNP treatment at the same time. In some biological systems, the effects of NO are mediated by cGMP this signaling cascade may involve activation of guanylate cyclase, up-regulation of cGMP, and activation of cGMP- dependent protein kinase (21). To determine the degree of involvement of cGMP in the production of MMPs, the effects of 8-Br-cGMP were measured. 8-Br-cGMP is a stable cell-permeable analog of cGMP that can mimic the cellular effects of cGMP. Treatment with 50 microM of 8-Br-cGMP also increased MMP-1 and -2 production by 137% and 254%, respectively, similar to the effect of SNP (Figure 3). This result indicates that the increase in MMP-1 and -2 production by SNP-treated HDF is not the result of a toxic effect of SNP on the cells, but is mediated by NO or related metabolites, and that NO and cGMP possibly mediate the UV-induced increase of MMP production. EFFECT OF UV ON THE PRODUCTION OF iNOS AND NO The production of nitric oxide in UV-irradiated keratinocytes was reported by Romero-