Keratinocyte-specific retinoid regulation of human cellular retinoic acid binding protein-II (hCRABPII) gene promoter requires an evolutionarily conserved DR1 retinoic acid responsive element

Transcription of the hCRABPII gene is retinoid inducible in human skin kera tinocytes (KC) but, surprisingly, not in cultured cells. The promoter for t he gene harbors three putative nuclear receptor binding sites: DR5, upstrea m of the transcription start site; DR1 (DR1d), distal to the site; and DR1 (DR1p), a proximal variant. DR1d, but not DR1p, is conserved between human and mouse. Although DR5 has been found to be a retinoid receptor target in COS-1 cells, the function of DR1 remains unknown, We examined the functions of these DR in retinoid regulation of the hCRABPII promoter in human KC. I n reporter gene assays, no significant retinoid response was observed in th e promoter in cultured KC; however, overexpression of retinoid receptor het erodimers RAR gamma.RXR alpha restored the response. Gel supershift assays showed that endogenous RAR gamma.RXR alpha levels are much lower in culture d KC than in skin in vivo. Ligand-binding assays showed that cultured KC co ntain only one-third of the level of retinoic acid receptor (RAR) and one-e ighth of the level of retinoid X receptor found in KC in skin. Deletion of the DR1d or DR5 sites reduced retinoid-induced promoter activity by 63% and 27%, respectively. Isolated DR1d and DR5 sites, but not DR1p, efficiently bound RAR gamma.RXR alpha and conferred RAR-selective retinoid responsivene ss on a heterologous promoter, These data indicate that: (i) the previously reported lack of retinoid regulation of endogenous hCRABPII gene transcrip tion in cultured KC is likely due to insufficient levels of RAR gamma.RXR a lpha, but not their cofactors; (ii) the conserved DR1d site is the major fu nctional target in RAR gamma.RXR alpha regulation of hCRABPII in KC; (iii) the DR1p site is nonfunctional due to its lack of affinity for RAR gamma.RX R alpha, although its half-sites share high sequence homology with the cons ensus retinoid receptor-binding half-site.