Project description:There is increasing evidence that autophagy contributes to the epidermal differentiation; however, the role of autophagy in epidermal tight junction (TJ) barrier remains unclear. To evaluate the role of autophagy in the maintenance of skin TJ barrier, we knocked out autophagy in human primary keratinocytes by infecting cells with autophagy-related gene 3 (Atg3) C264S mutant adenovirus.
Project description:Autophagy is a mechanism that regulates cellular metabolism and clearance of damaged macromolecules and organelles. Impaired degradation of modified macromolecules contributes to cellular dysfunction and is observed in aged tissue and senescent cells. We have inactivated Atg7, an essential autophagy gene, in murine keratinocytes and have found in an earlier study that this resulted in increased baseline oxidative stress and reduced capacity to degrade crosslinked proteins after oxidative ultraviolet stress. To investigate whether autophagy deficiency would promote cellular aging, we studied, how Atg7 deficient (KO) and Atg7 bearing cells (WT) would respond to stress induced by Paraquat (PQ), an oxidant drug commonly used to induce cellular senescence.
Project description:Glut1 is highly expressed in basal cells of keratinocytes, but the regulation of Glut1 and expression of additional glucose transporters in the skin has not been explored, here we specifically ablate Glut1 in epidermal keratinocytes to elucidate the role of glucose transport in the keratinocytes. we performed microarray analysis in WT and Glut1 deficient primary keratinocytes to determine the pathways might contribute to the impaired proliferation in Glut1 deficient keratinocytes.
Project description:Background: The excessive accumulation of melanosomes within keratinocytes contributes to skin hyperpigmentation and disrupts normal epidermal proliferation. Glabridin, an isoflavone derived from Glycyrrhiza glabra L., is a potent skin-lightening agent with tyrosinase-inhibitory activity. This study aims to further elucidate the skin-lightening mechanism of glabridin by investigating its effects on melanosome degradation. Methods: We investigated the effect of glabridin on melanosome degradation by measuring melanin content and PMEL17 protein levels. Autophagy activity was evaluated by mRNA sequencing, Western blot, and immunofluorescence. In vivo depigmenting efficacy was examined using epidermal skin equivalents and zebrafish embryos. Additionally, ATP content and mitochondrial membrane potential were measured to determine whether glabridin ameliorates melanosome-induced mitochondrial dysfunction. Results: Glabridin promoted melanosome degradation in keratinocytes and exhibited depigmenting effects in both pigmented epidermal skin equivalents and zebrafish embryos. Mechanistically, glabridin activated autophagy, and pharmacological inhibition of autophagy blocked its depigmenting effects. Additionally, we found that melanosome accumulation impaired mitochondrial function in keratinocytes, an effect that was reversed by glabridin. Conclusions: These findings demonstrate that glabridin promotes melanosome degradation via autophagy activation, which is associated with improved mitochondrial function, providing mechanistic insight into its skin-lightening effects.
Project description:Hair shafts are formed by terminal differentiation of hair keratinocytes (trichocytes) in which keratins and keratin-associated proteins accumulate and undergo cross-linking. Here we tested the hypothesis that maturation of the hair shaft also involves the coordinated degradation of other proteins and, specifically, that this degradation is mediated by autophagy. To this end, we deleted the non-redundant autophagy regulator Atg7 in keratinocytes of the epidermis and skin appendages, including hair, and determined the proteome of hair shafts from fully autophagy-competent and epithelial autophagy-deficient mice. The abrogation of autophagy led to significantly increased abundance of proteins regulating house-keeping functions of the cell and a decrease of cytoskeletal proteins. Translation factors, tRNA-ligases, ribosomal proteins and the components of proteasomes were particularly elevated in the absence of autophagy, indicating a central role of autophagy in regulating multiple steps of protein turnover in hair keratinocytes. These results demonstrate that hair keratinocytes depend on autophagy for establishing the mature protein composition of hair.
Project description:To determine the role of autophagy in the maintenance of genome stability and nucleic acid metabolism, the chromatin-bound proteins in autophagy-deficient ATG7-/- HEK293 cells were compared with autophagy-proficient ATG7+/+ HEK293 cells by Data-independent acquisition mass spectrometry (DIA-MS).
Project description:During re-epithelialization, keratinocytes have to undergo specific gene expression changes that grant them increased motility. Diabetic keratinocytes have been observed to portray an impairment in this aspect. We used microarrays to identify the changes in the expression patterns of both normal and diabetic migrating keratinocytes during re-epithelialization.