RESUMEN
BACKGROUND: Dishevelled-3 (Dvl3) is a multivalent scaffold essential to cell signaling in development. Dsh/Dvls enable a myriad of protein-protein interactions in Wnt signaling. In the canonical Wnt/ß-catenin pathway specifically, Dvl3 polymerizes to form dynamic protein aggregates, so-called "signalsomes", which propagate signals from the Wnt receptor Frizzled to downstream elements. RESULTS: Very large Dvl3-based supermolecular complexes form in response to Wnt3a. These complexes are identified by steric-exclusion chromatography, affinity pull-downs, proteomics, and fluorescence correlation microscopy (fcs). In the current work, the roles of Dvl3 phosphorylation and of Axin in the assembly of Dvl3-based supermolecular complexes in response to Wnt3a are probed in totipotent mouse F9 teratocarcinoma cells. Point mutations of phosphorylation sites of Dvl3 which interfere with Lef/Tcf-sensitive transcriptional activation by Wnt3a are shown to interfere more proximally with the assembly of Dvl3-based supermolecular complexes. Axin, a Dvl-interacting protein, plays a central role in organizing the beta-catenin destruction complex. The assembly of Dvl3-based supermolecular complexes is blocked either by depletion of Axin or by mutation of Axin sites necessary for polymerization in response to Wnt3a. CONCLUSION: These data demonstrate that Wnt3a activation of the canonical pathway requires specific phosphorylation events as well as Axin to assemble very large, Dvl3-based supermolecular complexes; these complexes are a prerequisite to activation of Lef/Tcf-sensitive transcription.
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
Reversible protein acetylation modulates higher-order chromatin structure and transcription activity of the genome. The reversible acetylation is executed by the intrinsic acetylase and deacetylase activities of co-regulators associated with the regulatory regions. Compounds capable of inhibiting deacetylase activity are a powerful tool for dissecting the role of protein acetylation in gene function. The ability of the deacetylase inhibitors to preferentially affect the homeostasis of transformed cells has also prompted studies for their clinical application. We present evidence that deacetylase inhibition with trichostatin A (TSA) affects the normal epidermal tissue architecture and pattern of expression by a mechanism(s) that does not correlate directly with the hyperacetylated histone status. While promoting abnormal differentiation, TSA specifically represses transcription initiation of the differentiation marker profilaggrin. Multiple factors, among which we have identified decreased Sp1 binding, a local decrease in acetylation activity, and enhanced synthesis and recruitment of a repressor histone demethylase, alter the chromatin configuration over the promoter, ultimately blocking its activation by c-jun. As compromised profilaggrin production leads to epidermal and consequently allergic disorders, our findings emphasize the need for a detailed investigation of the role deacetylase inhibitors may play in the maintenance of epidermal homeostasis in order to optimize their clinical applicability.
Asunto(s)
Diferenciación Celular/fisiología , Epidermis/fisiología , Inhibidores de Histona Desacetilasas , Histonas/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Queratinocitos/fisiología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Células Epidérmicas , Proteínas Filagrina , Regulación de la Expresión Génica/efectos de los fármacos , Histona Desacetilasas/fisiología , Histona Demetilasas , Homeostasis/fisiología , Humanos , Ácidos Hidroxámicos/farmacología , Proteínas de Filamentos Intermediarios/genética , Queratinocitos/citología , Masculino , Oxidorreductasas N-Desmetilantes/fisiología , Proteínas Quinasas/fisiología , Proteínas Proto-Oncogénicas c-jun/fisiologíaRESUMEN
Transcriptional repression often depends on the action of recruited co-repressor complexes with intrinsic enzymatic activities. The composition of these complexes depends on the nicotine amide dinucleotide co-factors and is thus directly reflective of the metabolic state of the cells. This study provides evidence that an enzyme, hRoDH-E2, with cytoplasmic phosphorylated and reduced forms of NAD-dependent retinol dehydrogenase activity may function in the nucleus as a transcriptional repressor. By using the promoter of the epidermal late differentiation marker profilaggrin as a model, we show that both in vivo and in vitro the protein is recruited over the promoter. hRoDH-E2 represses profilaggrin promoter activity by altering the function of other activators, such as Sp1. The repressive function is associated with the ability of nuclear hRoDH-E2 to modulate the acetylation/deacetylation activity in the vicinity of transcription initiation site. These findings add hRoDH-E2 to the small group of metabolic enzymes, which, by being recruited over promoter regions, could directly link the cytoplasmic and nuclear functions within the cell.