involucrin and 25-hydroxycholesterol

Skin photoaging due to UV irradiation is a degenerative process that appears more and more as a growing concern. Lipids, including oxysterols, are involved in degenerative processes; as skin cells contain various lipids, the aim of our study was to evaluate first, changes in keratinocyte lipid levels induced by UV exposure and second, cellular effects of oxysterols in cell morphology and several hallmarks of keratinocyte differentiation. Our mass spectrometry results demonstrated that UV irradiation induces changes in lipid profile of cultured keratinocytes; in particular, ceramides and oxysterols, specifically 25-hydroxycholesterol (25-OH), were increased. Using holography and confocal microscopy analyses, we highlighted cell thickening and cytoskeletal disruption after incubation of keratinocytes with 25-OH. These alterations were associated with keratinocyte differentiation patterns: autophagy stimulation and intracellular calcium increase as measured by cytofluorometry, and increased involucrin level detected by immunocytochemistry. To conclude, oxysterol deregulation could be considered as a common marker of degenerative disorders. During photoaging, 25-OH seems to play a key role inducing morphological changes and keratinocyte differentiation.

Topics: Autophagy; Cell Differentiation; Cell Line; Cell Survival; Ceramides; Culture Media; Cytoskeleton; Flow Cytometry; Humans; Hydroxycholesterols; Keratinocytes; Light; Lipids; Microscopy, Confocal; Necrosis; Oxysterols; Protein Precursors; Skin; Skin Aging; Ultraviolet Rays

Oxysterols, via activation of liver X receptor (LXR), regulate keratinocyte differentiation by stimulating transglutaminase cross-linking of several constituent proteins leading to the formation of the cornified envelope. We previously reported that oxysterols increase the expression of one of these cross-linked proteins, involucrin, and that this effect can be abolished by mutations of the distal activator protein (AP)-1 response element in the involucrin promoter. Furthermore, oxysterols increase AP-1 binding in an electrophoretic gel mobility shift assay and increase the expression of an AP-1 reporter. In this study, we describe the individual components of the AP-1 complex that are involved in the oxysterol-mediated AP-1 activation and stimulation of keratinocyte differentiation. We identified Fra-1 within the AP-1 DNA binding complex by supershift analysis of nuclear extracts from oxysterol-treated, cultured keratinocytes and confirmed that oxysterol treatment increased the levels of Fra-1 by western blot analysis. Additionally, on Western and Northern analysis, oxysterol treatment increased two other AP-1 proteins, Jun-D and c-Fos, whereas Fra-2, Jun-B, and c-Jun were not changed. Similar alterations in AP-1 proteins occurred when 25-OH-cholesterol or non-steroidal LXR agonists (GW3965, TO-901317) were used. These results indicate that oxysterols induce specific AP-1 proteins, thereby activating involucrin, one of the genes required for epidermal differentiation.

Topics: Cells, Cultured; DNA-Binding Proteins; Epidermal Cells; Gene Expression; Humans; Hydroxycholesterols; Keratinocytes; Liver X Receptors; Orphan Nuclear Receptors; Protein Precursors; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Transcription Factor AP-1

Ligands and activators of the nuclear hormone receptor superfamily are important in the regulation of epidermal development and differentiation. Previously, we showed that naturally occurring fatty acids, as well as synthetic ligands for the peroxisome proliferator-activated receptor, induce keratinocyte differentiation in vitro. Here we asked whether oxysterols, another class of lipids formed de novo in the epidermis and that activate liver X-activated receptor, regulate keratinocyte differentiation. mRNA and protein levels of involucrin and transglutaminase 1, markers of differentiation, increased 2- to 3-fold in normal human keratinocytes incubated in the presence of 25- or 22R-hydroxycholesterol in low calcium. In high calcium, which alone induces differentiation, mRNA levels were further increased by oxysterols. Rates of cornified envelope formation, an indicator of terminal differentiation, also increased 2-fold with oxysterol treatment. In contrast, the rate of DNA synthesis was inhibited approximately 50% by oxysterols. Transcriptional regulation was assessed in keratinocytes transfected with either transglutaminase 1 or involucrin promoter-luciferase constructs. 22R-hydroxycholesterol increased transglutaminase 1 and involucrin promoter activity 2- to 3-fold. Either deletion of the -2452 bp to -1880 bp region of the involucrin promoter, or mutation of the AP-1 site within this region, abolished oxysterol responsiveness. Moreover, increased AP-1 DNA binding was observed in oxysterol-treated keratinocytes by gel shift analyses. Finally, we demonstrated the presence of liver X-activated receptor alpha and beta mRNAs, and showed that oxysterols stimulate a liver X-activated receptor response element transfected into keratinocytes. These data suggest that oxysterols induce keratinocyte differentiation, in part through increased AP-1-dependent transcription of the involucrin gene, an effect that may be mediated by liver X-activated receptor.

Topics: Calcium; Cell Differentiation; DNA; DNA-Binding Proteins; Gene Expression Regulation; Humans; Hydroxycholesterols; Keratinocytes; Liver X Receptors; Mevalonic Acid; Orphan Nuclear Receptors; Promoter Regions, Genetic; Protein Precursors; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Response Elements; RNA, Messenger; Transcription Factor AP-1; Transcription, Genetic; Transglutaminases