Oxysterols induce differentiation in human keratinocytes and increase Ap-1-dependent involucrin transcription

Ligands and activators of the nuclear hormone receptor superfamily are impo rtant in the regulation of epidermal development and differentiation. Previ ously, we showed that naturally occurring fatty acids, as well as synthetic ligands for the peroxisome proliferator-activated receptor, induce keratin ocyte differentiation in vitro. Here we asked whether oxysterols, another c lass of lipids formed de novo in the epidermis and that activate liver X-ac tivated receptor, regulate keratinocyte differentiation. mRNA and protein l evels of involucrin and transglutaminase 1, markers of differentiation, inc reased 2- to 3-fold in normal human keratinocytes incubated in the presence of 25- or 22R-hydroxycholesterol in low calcium. In high calcium, which al one induces differentiation, mRNA levels were further increased by oxystero ls. Rates of cornified envelope formation, an indicator of terminal differe ntiation, also increased 2-fold with oxysterol treatment. In contrast, the rate of DNA synthesis was inhibited approximately 50% by oxysterols. Transc riptional regulation was assessed in keratinocytes transfected with either transglutaminase 1 or involucrin promoter-luciferase constructs. 22R-hydrox ycholesterol increased transglutaminase 1 and involucrin promoter activity 2- to 3-fold. Either deletion of the -2452 bp to -1880 bp region of the inv olucrin promoter, or mutation of the AP-1 site within this region, abolishe d oxysterol responsiveness. Moreover, increased AP-1 DNA binding was observ ed in oxysterol-treated keratinocytes by gel shift analyses. Finally, we de monstrated 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-depend ent transcription of the involucrin gene, an effect that may be mediated by liver X-activated receptor.