Keratinocyte-Specific Retinoid Regulation of Human Cellular Retinoic Acid Binding Protein-II (hCRABPII) Gene Promoter Requires an Evolutionarily Conserved DR1 Retinoic Acid-Responsive Element  Wen Di, Xiao-Yan Li, Anders Åström, Pierre Chambon, John J. Voorhees, Jia-Hao Xiao  Journal of Investigative Dermatology  Volume 111, Issue 6, Pages 1109-1115 (December 1998) DOI: 10.1046/j.1523-1747.1998.00455.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Locations of putative RARE in the hCRABPII gene promoter region and related CAT reporter gene constructs. Structure of reporter gene constructs containing the hCRABPII promoter regions or isolated DR. Names of the constructs are shown to the right. DNA fragments containing different hCRABPII promoter regions were linked to the bacterial CAT gene in the constructs. Numbers below each construct indicate positions of DR and the 5′-end of promoter fragments in the constructs. Dashes represent deletion of a region containing DR1d and DR1p. In thetk-CAT constructs, isolated DR were linked to the minimal promoter (–105) from the HSVtk gene. Journal of Investigative Dermatology 1998 111, 1109-1115DOI: (10.1046/j.1523-1747.1998.00455.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Increasing levels of RARγ and RXRα in cultured KC restores induction of human and mouse CRABP gene promoters by RAR-selective retinoids. Cultured KC were transfected with reporter gene constructs hCRABPII/-6.3 kb-CAT or mCRABPII/-2.3 kb-CAT with or without expression vectors for RARγ and RXRα. Eighteen hours after transfection, cells were treated with vehicle (VEH, 0.1% ethanol) or RAR-selective retinoids (0.1 μM)tRA or CD367 for 48 h. They-axis shows relative CAT activity, which is expressed as fold induction over the basal CAT activity in the absence of ligands and expression vectors. Data represent means ± SEM (n = 2–9). Journal of Investigative Dermatology 1998 111, 1109-1115DOI: (10.1046/j.1523-1747.1998.00455.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Levels of endogenous retinoid receptors are lower in KC in culture than in skin. (a) Gel mobility supershift analysis of retinoid receptors in KC nuclear extracts from skin (lanes 1–4) andin vitro tissue culture (lanes 5–8). DR5 from hCRABPII was used as a probe. Types of receptor-specific antibodies used in the post-binding reactions are shown above the gels. Positions of RARγ·RXRα-bound and free probes are indicated to the left.Lanes 5′–8′ correspond to a longer exposure of the same gel containinglanes 5–8. (b) Direct ligand-binding analysis of total RAR and RXR in nuclear extracts from cultured KC (culture) or KCin vivo (skin). Data for receptors in skin were taken fromFisheret al. (1994). They-axis shows levels of receptors expressed as receptors per cell. Data represent means ± SEM (n = 6). Journal of Investigative Dermatology 1998 111, 1109-1115DOI: (10.1046/j.1523-1747.1998.00455.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Deletion of a DR1-containing region severely impairs regulation of the hCRABPII gene promoter by RARγ·RXRα heterodimers in cultured KC. Cultured KC were transfected with constructs –6.3 kb-CAT, –5.8 kb-CAT, –5.8 kb(Δ)-CAT, or –5.6 kb-CAT (Figure 1). RARγ + RXRα, cells cotransfected with expression vectors for RARγ and RXRα. Cells were treated with vehicle (VEH) or 0.1 μMtRA before being harvested for measuring CAT activity. They-axis represents relative CAT activity expressed as percentage of maximal activity obtained with construct –5.8 kb-CAT. Data represent means ± SEM (n = 2–5). Journal of Investigative Dermatology 1998 111, 1109-1115DOI: (10.1046/j.1523-1747.1998.00455.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Deletion of the distal DR1-containing region abolishes regulation of the hCRABPII gene promoter by RARγ·RXRα heterodimers in cultured KC. Cells were transfected with constructs –1.0 kb-CAT or –0.5 kb-CAT and treated with vehicle (VEH) or 0.1 μMtRA. RARγ + RXRα, cells cotransfected with expression vectors for RARγ and RXRα. They-axis shows CAT activity expressed as average fold induction over average basal activity obtained with corresponding constructs in the absence of ligands and receptor expression vectors. Data represent means ± SEM (n = 3). Journal of Investigative Dermatology 1998 111, 1109-1115DOI: (10.1046/j.1523-1747.1998.00455.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 DR1d and DR5, but not DR1p, confer retinoid responsiveness to a heterologous minimal promoter from the HSVtk gene in cultured KC. Cells were transfected withtk-CAT constructs containing isolated DR1p, DR1d, or DR5 from the hCRABPII promoter (Figure 1), or DR1 from the mCRABPII promoter (Durandet al. 1992). RARγ + RXRα, cells cotransfected with expression vectors for RARγ and RXRα. After transfection, cells were treated with vehicle (VEH) or 0.1 μM CD367. They-axis shows CAT activity expressed as average fold induction over average basal activity obtained with corresponding constructs in the absence of ligands and receptor expression vectors. Data represent means ± SEM (n = 3–7). Journal of Investigative Dermatology 1998 111, 1109-1115DOI: (10.1046/j.1523-1747.1998.00455.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 DR1d and DR5, but not DR1p, strongly bind RARγ·RXRα overexpressed in cultured KC or endogenous RARγ·RXRα in skin KC. (a) Autoradiograph of gel mobility shifts showing that DR5, DR1d, and DR1p directly bind RARγ·RXRα overexpressed in cultured KC with distinct affinities. Types of radiolabeled probes are indicated above the gel. Positions of RARγ·RXRα-bound or free probes are indicated to the left. (b) Competition of DR1d, but not DR1p, with DR5 for binding endogenous RARγ·RXRα in KC nuclear extracts from skin.Upper panel, autoradiograph of a representative gel shift of three competition experiments. Radiolabeled DR5 from hCRABPII was incubated with 6 μg of KC nuclear extracts from skin. Types and amounts of unlabeled competitors are indicated above the gel. The position of RARγ·RXRα-bound DR5 probes is indicated to the right.Lower panel, quantitation of results from the competition experiments. They-axis represents average values of the remaining amounts of RARγ·RXRα-bound probes, which are expressed as percentage of total binding activity in the absence of competitors. Data represent means ± SEM (n = 3). (c) Autoradiograph of a gel mobility supershift showing direct binding of endogenous RARγ·RXRα in KC nuclear extracts prepared from skin to DR1d. Radiolabeled DR1d probes were incubated with 6 μg of the extracts. Types of mouse monoclonal antibodies are indicated immediately above the gel. Positions of RARγ·RXRα-bound or RARγ·RXRα-free DR1d are indicated to the left. Journal of Investigative Dermatology 1998 111, 1109-1115DOI: (10.1046/j.1523-1747.1998.00455.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions