title: Keratinocyte-specific retinoid regulation of human cellular retinoic acid binding protein-II (hCRABPII) gene promoter requires an evolutionarily conserved DR1 retinoic acid-responsive element.
abstract: Transcription of the hCRABPII gene is retinoid inducible in human skin keratinocytes (KC) but, surprisingly, not in cultured cells. The promoter for the gene harbors three putative nuclear receptor binding sites: DR5, upstream 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. In reporter gene assays, no significant retinoid response was observed in the promoter in cultured KC; however, overexpression of retinoid receptor heterodimers RARgamma x RXRalpha restored the response. Gel supershift assays showed that endogenous RARgamma x RXRalpha levels are much lower in cultured KC than in skin in vivo. Ligand-binding assays showed that cultured KC contain only one-third of the level of retinoic acid receptor (RAR) and one-eighth 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 RARgamma-RXRalpha and conferred RAR-selective retinoid responsiveness on a heterologous promoter. These data indicate that: (i) the previously reported lack of retinoid regulation of endogenous hCRABPII gene transcription in cultured KC is likely due to insufficient levels of RARgamma x RXRalpha, but not their cofactors; (ii) the conserved DR1d site is the major functional target in RARgamma x RXRalpha regulation of hCRABPII in KC; (iii) the DR1p site is nonfunctional due to its lack of affinity for RARgamma x RXRalpha, although its half-sites share high sequence homology with the consensus retinoid receptor-binding half-site.
authors.affiliation: Department of Dermatology, University of Michigan, Ann Arbor 48109, USA.
authors.forename: W
authors.initials: W
authors.lastname: Di
authors.forename: X Y
authors.initials: XY
authors.lastname: Li
authors.forename: S
authors.initials: S
authors.lastname: Datta
authors.forename: A
authors.initials: A
authors.lastname: Aström
authors.forename: G J
authors.initials: GJ
authors.lastname: Fisher
authors.forename: P
authors.initials: P
authors.lastname: Chambon
authors.forename: J J
authors.initials: JJ
authors.lastname: Voorhees
authors.forename: J H
authors.initials: JH
authors.lastname: Xiao
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D017124:Conserved Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D019281:Dimerization; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D047488:Retinoid X Receptors; D012176:Retinoids / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics; D014212:Tretinoin / Q000378:metabolism*
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D012176:Retinoids; D014157:Transcription Factors; C082928:retinoic acid binding protein II, cellular; D014212:Tretinoin