RESUMEN
The cellular defense system against harmful levels of reactive oxygen species consists of antioxidant enzymatic activities and small nonenzymatic molecules. L-ergothioneine has long been recognized as a potent and stable low-molecular-weight antioxidant that humans consume with diet and that accumulates in cells normally subjected to high levels of oxidative stress. As L-ergothioneine is plasma membrane-impermeative, its protective function is restricted to cells that express the L-ergothioneine-specific receptor/transporter OCTN1. Here we report for the first time that both as resident skin cells and in culture, epidermal keratinocytes synthesize OCTN1, which enables them to internalize and accumulate L-ergothioneine. This accumulation confers upon the cells an increased antioxidant potential. Consequently, it reduces the levels of reactive oxygen species and DNA, protein, and lipid damage in keratinocytes subjected to solar-simulating UV oxidative stress. Our results suggest that L-ergothioneine not only prevents oxidative damage but also may enable DNA repair in the UV-irradiated cells. The diminished oxidative damage to cellular constituents limits the apoptotic response and results in increased cell viability. The cells' ability to take up, accumulate, and utilize the potent antioxidant L-ergothioneine positions this naturally occurring amino acid and its receptor/transporter as an integral part of the antioxidative defense system of the skin.
Asunto(s)
Antioxidantes/farmacología , Ergotioneína/farmacología , Fibroblastos/fisiología , Queratinocitos/fisiología , Proteínas de Transporte de Catión Orgánico/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Células Cultivadas , Citoprotección/efectos de los fármacos , Citoprotección/fisiología , Citoprotección/efectos de la radiación , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Prepucio/patología , Humanos , Recién Nacido , Queratinocitos/efectos de los fármacos , Queratinocitos/patología , Masculino , Proteínas de Transporte de Catión Orgánico/genética , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Transporte de Proteínas , Traumatismos por Radiación , SimportadoresRESUMEN
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.
Asunto(s)
Daño del ADN/fisiología , Metaloproteinasa 1 de la Matriz/metabolismo , Piel/efectos de la radiación , Rayos Ultravioleta , Adolescente , Adulto , Anciano , Línea Celular , Células Cultivadas , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/metabolismo , Desoxirribonucleasa (Dímero de Pirimidina)/química , Desoxirribonucleasa (Dímero de Pirimidina)/metabolismo , Dermis/metabolismo , Dermis/efectos de la radiación , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Epidermis/metabolismo , Epidermis/efectos de la radiación , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Expresión Génica/efectos de la radiación , Humanos , Immunoblotting , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Liposomas/química , Metaloproteinasa 1 de la Matriz/genética , Persona de Mediana Edad , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Dímeros de Pirimidina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citología , Piel/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Adulto JovenRESUMEN
BACKGROUND: L-ergothioneine (EGT) is a stable antioxidant found in food plants as well as in animal tissue undergoing relatively high levels of oxidative stress. Idebenone is a stable analog of the antioxidant coenzyme Q(10). All are potent antioxidants found in skincare products, but their relative potencies are not well described. AIMS: To establish the physiological relevance of EGT by examining transcription of the EGT transporter gene OCTN-1 and production of the receptor protein in skin fibroblasts. In addition, to compare the inhibition of lipid peroxide formation by coenzyme Q(10) and EGT. Furthermore, to compare the peroxide-scavenging abilities of EGT and idebenone in both simple solution and in cell cultures exposed to ultraviolet A (UVA). METHODS: OCTN-1 expression and production in cultured fibroblasts were measured through real-time reverse transcription-PCR and Western blotting, respectively. Alloxan-induced lipid peroxidation in liposomes was used to evaluate the inhibition of lipid peroxide formation. The abilities of EGT and idebenone to directly scavenge hydroxyl radicals produced by H(2)O(2 )were determined. Finally, we irradiated fibroblasts with UVA340 radiation and compared antioxidant capabilities to scavenge free radicals. RESULTS: We found that OCTN-1 is expressed and readily detectable in cultured human fibroblasts. EGT was more efficient in inhibiting lipid peroxide formation than coenzyme Q(10) or idebenone. Samples treated with EGT had significantly less peroxide than those treated with idebenone 120 min after adding the antioxidants to H(2)O(2). EGT acted significantly quicker and more efficiently in capturing reactive oxygen species (ROS) after UVA340 irradiation. CONCLUSIONS: EGT is a natural skin antioxidant, as evidenced by the presence of the EGT transporter in fibroblasts. EGT is a more powerful antioxidant than either coenzyme Q(10) or idebenone due to its relatively greater efficiency in directly scavenging free radicals and in protecting cells from UV-induced ROS.