Destruction of collagen is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion, matrix metalloproteinase-1 (MMP-1), is induced by exposure to sunlight. To study the molecular trigger for this induction, human skin was ultraviolet-B (UVB)-irradiated and treated with liposome-encapsulated DNA repair enzymes. The photolyase-mediated DNA repair of epidermal UV damage was associated with a reduction of MMP-1 mRNA and protein expression in both the epidermal and dermal compartments of the skin. The role of the epidermal cells in MMP-1 induction in the fibroblasts was examined when human epidermal keratinocytes were irradiated with UVB and their media were transferred to unirradiated human dermal fibroblasts. Transfer of media from irradiated keratinocytes to unirradiated fibroblasts enhanced MMP-1 mRNA and protein. Thus, UV damage to keratinocytes of the epidermis may participate in the destruction of collagen in the dermis by release of soluble mediators that signal fibroblasts to release MMP-1. The MMP-1 induction was reduced when the keratinocytes were treated with DNA repair enzymes T4 endonuclease V or UV endonuclease prior to transfer of the media to fibroblasts. This implies that UVB, which deposits most of its energy on the chromatin of the epidermal keratinocytes and to a lesser extent in the upper dermis, has a significant role in photoaging. DNA damage in the keratinocytes initiates one of the signals for MMP-1 release, and enhancing DNA repair can reduce MMP-1 expression in human skin cells and tissue.
Ergothioneine (EGT) is a sulfur‐containing amino acid, and is presumed to function as a natural antioxidant. The purpose of this study was to identify the nature of the antioxidant activity and investigate the effects of EGT on UV‐induced cellular response. In chemical studies, EGT scavenged the superoxide anion radical (•O2–) and singlet oxygen (1O2). In cultured fibroblasts, EGT suppressed TNF‐α upregulation by UVB irradiation. In addition, in fibroblasts exposed to UV‐A, EGT suppressed the expression of matrix metalloproteinase 1 (MMP‐1) protein by nearly 50% and reduced MMP‐1 mRNA expression. From these results, we conclude that EGT scavenges reactive oxygen species generated by both type I and type II photosensitization and suppresses both TNF‐α expression and MMP‐1 at their transcriptional level. EGT may reduce skin anti‐aging effects after UV irradiation by the scavenging of •O2– and 1O2, and reducing signals for protease and inflammatory activity.
alpha-Tocopherol is a lipophilic vitamin that exhibits an antioxidative activity. The purpose of this study was to clarify the roles of alpha-tocopherol in the regulation of intracellular glutathione (GSH) levels in HaCaT keratinocytes. When HaCaT keratinocytes were cultivated with alpha-tocopherol for 24 h, the intracellular GSH was increased at every concentration of alpha-tocopherol tested. Furthermore, the HaCaT keratinocytes cultured with alpha-tocopherol at 50 microM for 24 h exhibited resistance against H2O2. However, a short exposure of HaCaT keratinocytes to alpha-tocopherol for 1 h did not influence either the GSH level or the resistance to H2O2. These findings suggest that GSH, which is inductively synthesized by alpha-tocopherol, effectively reduces exogenous oxidative stress. To evaluate the effect of alpha-tocopherol on the GSH level, BSO, which is a typical inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), was used. When BSO was added to HaCaT keratinocytes, no action of alpha-tocopherol on the GSH level was observed. On the other hand, alpha-tocopherol resulted in the up-regulation of gamma-GCS-HS (heavy subunit) mRNA. In addition, water soluble alpha-tocopherol derivatives (alpha-tocopherol phosphate and trolox) caused no changes in GSH level. From these results, it was concluded that alpha-tocopherol increases the intracellular GSH level of HaCaT keratinocytes through the up-regulation of gamma-GCS-HS mRNA.
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