Regulation of human profilaggrin promoter activity in cultured epithelial cells by retinoic acid and glucocorticoids

Vitamin A and other retinoids profoundly inhibit both morphological and bio chemical aspects of epidermal differentiation in vitro. Profilaggrin, like most other markers of keratinocyte differentiation, is negatively regulated by retinoic acid in vitro, both at the level of mRNA synthesis and by inhi biting the activity of endoproteases that convert profilaggrin to filaggrin . Profilaggrin is an abundant component of keratohyalin granules and forms the precursor of filaggrin, the keratin associated protein of the stratum c orneum. In this report, we identify a region of the human profilaggrin prom oter that is involved in the transcriptional regulation of expression by re tinoic acid (RA). A series of promoter deletions linked to the chlorampheni col acetyl transferase (CAT) reporter gene were prepared and analyzed by tr ansfection into Hela cells and keratinocytes. We also cotransfected vectors expressing retinoic acid receptor and cultured the transfected cells in th e presence and absence of ligand. The region responsive to retinoic acid wa s localized to a 53 bp sequence between - 1109 and - 1056 (relative to the mRNA start site at + 1) that contains a cluster of five retinoic acid respo nse elements with variable spacing and orientation. In vitro gel shift anal ysis demonstrated that nuclear retinoid receptors do not bind directly to t he identified sequence, suggesting that the mode of regulation by RA may be indirect or that binding requires another cofactor in addition to retinoid receptors. Whereas in keratin genes retinoic acid and glucocorticoid respo nsive sequences frequently coincide, the glucocorticoid response element in the profilaggrin promoter was located downstream of the RARE cluster betwe en - 965 and - 951. These studies demonstrate that RA and glucocorticoids r egulate profilaggrin expression at least in part by transcriptional mechani sms, via a region of the promoter that contains both retinoid and glucocort icoid responsive elements. (C) 2001 Elsevier Science Ireland Ltd. All right s reserved.