j. Cosmet. Scio, 54, 229-238 (May/June 2003) Inhibition of matrix metalloproteinase-1 and -2 expression using nitric oxide synthase inhibitors in UV-irradiated human dermal fibroblasts TAEBOO CHOE, BUMCHUN LEE, INCHUL PARK, and SEOKIL HONG, Division of Chemical and Biological Engineering, Konkuk University, Hwayang-dong l, Kwangjin-gu, Seoul 143-701 (T.C.), Hanbul Cosmetics, Samsung-myun, Umsung-kun, Chungbuk (B.L.), and Laboratory of Cell Biology, Korea Cancer Center Hospital, Seoul (I. P., S.H.), Korea. Accepted for publication March 4, 2002. Synopsis The production of matrix metalloproteinases (MMP) by UV-irradiated skin fibroblasts and the degradation of the extracellular matrix by these enzymes is known as one of the main causes of photoaging. Recently, the Fisher group showed that MMP expression is mainly regulated by members of the mitogen-activated protein kinase family such as extracellular signal-regulated kinase, c-Jun amino-terminal kinase, and p38, each of which forms a signaling pathway. In this work, we initially examined the effect of nitric oxide (NO) and nitric oxide synthase (NOS) inhibitors on the production of MMP-1 and MMP-2 by human dermal fibroblasts (HDF). NO is a multifunctional messenger molecule generated from L-arginine and can activate guanylate cyclase to increase cGMP. We found that treatment of HDF with an NO donor, sodium nitroprusside (50 microM), enhanced the expression ofMMP-1 and -2 by 153% and 243 %, respectively, and treatment by 8-Br-cGMP enhanced MMP-1 and -2 expression by 137% and 254%, respectively. When UV-irradiated HDF was treated with NOS inhibitors such as aminoguanidine (AG) and baicalein (BAC), there resulted a decrease in MMP production. When 20 microM of BAC was added in the culture media of UV-irradiated HDF, only 40% of MMP-1 and 42% of MMP-2 was produced, compared to the case without BAC. Taken together, we concluded that the production of MMP-1 and -2 by UV-irradiated HDF is regulated through the signaling pathway involving NO and that it can be downregulated using NOS inhibitors. INTRODUCTION Matrix metalloproteinases (MMPs) are a family of enzymes that are responsible for the degradation of extracellular matrix (ECM) components such as collagen, laminin, and proteoglycans. Among the MMPs, MMP-1 is an interstitial collagenase that degrades fibrillar collagens and proteoglycans, and MMP-2 is a gelatinase that degrades denatured collagens and elastin. The main reason for the connective tissue changes in the UV- irradiated skin has been clarified as the production of MMPs, including MMP-1 and MMP-2 (1-5), and the degradation of ECM components by these enzymes. The expres- 229

230 JOURNAL OF COSMETIC SCIENCE sion of MMPs in UV-irradiated fibroblasts is known to be initiated by singlet oxygen (6), alpha-melanocyte stimulating hormone produced by keratinocytes (7), or by the activation of cell surface growth factor and cytokine receptor, which mimics the actions of receptor ligands (8,9). Recently, Fisher et al. (10) showed that MMP expression is mainly regulated by members of the mitogen-activated protein (MAP) kinase family such as extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, each of which forms a signaling pathway. In some types of cells, the expression of MMP is mediated by nitric oxide (NO) (11,12). NO is a multifunctional messenger molecule generated from L-arginine by enzymes such as inducible nitric oxide synthase (iNOS), and it can activate guanylate cyclase, stimulating the production of intracellular cGMP and activation of cGMP-dependent protein kinase. The production and diffusion of NO in triggering the melanogenesis of melanocytes by the UV-irradiated keratino- cytes is well documented (13), but relatively little is known about the effect of NO on the production of MMP by the epidermal fibroblasts, which are also under the influence of cytokines released from keratinocytes. In this work, we examined the effect of NO and iNOS inhibitors on the production of MMP-1 and MMP-2 by UV-irradiated human derreal fibroblasts (HDF). We found that treatment of HDF with an NO donor, sodium nitroprusside (SNP), enhanced the expression of MMP-1 and -2 and that treatment with iNOS inhibitors such as aminoguanidine (AG) and baicalein (BAC) resulted in a decrease in MMP production. Taken together, we concluded that the production of MMP-1 and -2 by UV-irradiated HDF is regulated through the signaling pathway involving NO, and that it can be downregulated using iNOS inhibitors such as AG, BAC, and the extract of Scutellaria root containing large amounts of iNOS inhibitors. MATERIALS AND METHODS REAGENTS Sodium nitroprusside (SNP) (14,15), aminoguanidine (AG), nitro-L-arginine methyl ester (NAME), nitro-L-arginine (NAL), baicalein (BAC), and 8-Br-cGMP, anti-mouse IgG for the secondary antibody, were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-MMP-1 antibody (Ab-5), anti-MMP-2 antibody (Ab-3), and anti-mouse IgG conjugated with alkaline phosphatase were obtained from Cal-Biochem. Scutellaria root extract was obtained from Bioland. CULTURE OF HUMAN DERMAL FIBROBLASTS HDFs from newborn foreskin were acquired from Korea Cancer Center Hospital. HDFs were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS and kept in a humidified 5% CO 2 atmosphere were 37øC. HDFs from passages 6 to 10 were used in the experiments. UV IRRADIATION AND NO DETECTION HDFs (1.5 x 105/well) were seeded into 350 plates and cultured overnight. Prior to UV irradiation, the cells were washed twice with phosphate-buffered saline (PBS). The cells were irradiated from a distance of 15 cm by a UV source (UVA simulator, Jhonsam,