Cholesterol sulfate stimulates involucrin transcription in keratinocytes by increasing Fra-1, Fra-2, and Jun D

Lipids that are synthesized de novo in the epidermis, including fatty acids , oxysterols, 1,25-dihydroxvitamin Ds, and farnesol, can regulate the diffe rentiation of normal human keratinocytes (NHK), Cholesterol sulfate (CS), a n epidermal lipid that is produced in the upper nucleated la;vers of the ep idermis coincident with terminal differentiation, has been shown to play a role in the regulation of the late stages of keratinocyte differentiation, including formation of the cornified envelope. In the present study, we det ermined i) whether CS regulates involucrin (INV), an early keratinocyte dif ferentiation marker, and ii) the mechanism by which CS regulates differenti ation. mRNA and protein levels of INV, a precursor protein of the cornified envelope, increased 2- to 3-fold in NHK incubated in the presence of CS, I n contrast, cholesterol had no effect on INV protein or mRNA levels. Transc riptional regulation was assessed in NHK transfected with INV promoter-luci ferase constructs. CS increased luciferase reporter activity approximately 2- to 3-fold in NHK transfected with a 3.7-kb INV promoter construct. Delet ional analysis revealed a CS-responsive region of the INV promoter located between bp -2452 and -1880, A 5-base pair (bp) mutation of the AP-1 site (b p -2117 to -2111) within this responsive region abolished CS responsiveness , suggesting a role for the AP-1 complex in the regulation of INV transcrip tion by CS, Electrophoretic mobility shift analysis demonstrated increased binding of nuclear extracts isolated from CS-treated NHK to AP-1 DNA as com pared with vehicle-treated controls. Incubation of the nuclear extract with the appropriate antibodies showed that the AP-1 DNA-binding complex contai ned Fra-1, Fra-2, and Jun D, Western blots demonstrated that CS treatment i ncreased the levels of Fra-1, Fra-2, and Jun D, and Northern analyses revea led that CS increased mRNA levels for these same AP-1 factors. These data i ndicate that CS, an endogenous lipid synthesized by keratinocytes, regulate s the early stages of keratinocyte differentiation, and may do so through i ts ability to modulate levels of AP-1 proteins.