RESUMO
Ultraviolet (UV) irradiation is a major environmental factor responsible for a high incidence of premature skin aging, referred to as photoaging, as well as skin cancer and melanoma. UVA irradiation represents 90% of the solar UV light reaching the earth's surface, and yet the mechanisms by which it exerts its biological effects are not clear. UVA penetrates into the skin tissue, reaching the basal layers of the active dividing cells and, therefore, the contribution of UVA to skin damage may be significant. The majority of UVA energy is absorbed by unidentified photosensitizers in the cells which are postulated to generate reactive oxygen species (ROS). It has been believed that both chronological aging and photoaging share the same molecular features and, as such, it is very common to utilize UV irradiation for induction of skin aging. To determine the involvement of protein kinase isoforms in chronological aging and photoaging, we utilized in vitro aging model systems of primary murine fibroblasts and primary fibroblasts isolated from PKC null mice. We show for the first time distinct involvement of PKC isoforms PKCdelta and PKCalpha in photoaging versus cellular senescence. While chronological aging is accompanied by overexpression and activation of PKCalpha, UV irradiation and ROS production are associated with photoaging accompanied by PKCdelta downregulation and nuclear translocation.
Assuntos
Proteína Quinase C-delta/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Catalase/metabolismo , Forma Celular , Células Cultivadas , Senescência Celular , Fibroblastos , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase C-alfa/deficiência , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/deficiência , Proteína Quinase C-delta/genética , Transporte Proteico , Superóxido Dismutase/metabolismoRESUMO
Activation of the STAT family of transcription factors is regulated by cytokines and growth factors. STAT tyrosine and serine phosphorylation are linked to the transcriptional activation and function of STAT. We have previously described a unique pathway inducing keratinocyte proliferation, which is mediated by insulin stimulation and depends on protein kinase C delta (PKCdelta). In this study, we assessed STAT3 activation downstream of this pathway and characterized the role of PKCdelta activation in STAT3 tyrosine and serine phosphorylation and keratinocyte proliferation. Following insulin stimulation, STAT3 interacted with PKCdelta but not with any other PKC isoform expressed in skin. Activated forms of PKCdelta and STAT3 were essential for insulin-induced PKCdelta-STAT3 activation in keratinocyte proliferation. Abrogation of PKCdelta activity inhibited insulin-induced STAT3 phosphorylation, PKCdelta-STAT3 association and nuclear translocation. In addition, overexpression of STAT3 tyrosine mutant eliminated insulin-induced PKCdelta activation and keratinocyte proliferation. Finally, overexpression of a STAT3 serine mutant abrogated insulin-induced STAT3 serine phosphorylation and STAT3-induced keratinocyte proliferation, whereas STAT3 tyrosine phosphorylation was induced and nuclear localization remained intact. This study indicates that PKCdelta activation is a primary regulator of STAT3 serine phosphorylation and that PKCdelta is essential in directing insulin-induced signaling in keratinocyte proliferation.
Assuntos
Hipoglicemiantes/farmacologia , Insulina/farmacologia , Queratinócitos/enzimologia , Proteína Quinase C-delta/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Hipoglicemiantes/metabolismo , Insulina/metabolismo , Queratinócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
In mammalian epidermis, alpha6beta4 integrin is expressed exclusively on the basal layer localized to the hemidesmosomes, where it interacts extracellularly with the laminin-5 ligand. During differentiation, loss of alpha6beta4 is associated with keratinocyte detachment from the basement membrane and upward migration. The protein kinase C (PKC) family of isoforms participates in regulation of integrin function and is linked to skin differentiation. Exposure of primary murine keratinocytes to PKC activators specifically downregulates alpha6beta4 expression. Utilizing recombinant adenoviruses, we selectively overexpressed skin PKC isoforms in primary keratinocytes. PKCdelta and PKCzeta induced downregulation of alpha6beta4 protein expression, leading to reduced keratinocyte attachment to laminin-5 and enhanced gradual detachment from the underlying matrix. In contrast, PKCalpha upregulated alpha6beta4 protein expression, leading to increased keratinocyte attachment to laminin-5 and to the underlying matrix. Altogether, these results suggest distinct roles for specific PKC isoforms in alpha6beta4 functional regulation during the early stages of skin differentiation.
