Retinoic acid inducibility of the human PDGF-a gene is mediated by 5'-distal DNA motifs that overlap with basal enhancer and vitamin D response elements

Abstract

Retinoic acid (RA) upregulates expression of PDGF ligands and receptors in neonatal rat lung fibroblasts, a process likely to promote maturation of the lung alveolus and possibly microstructures of other organs. A mutational analysis of the gene encoding the PDGF-A ligand has identified a complex retinoic acid response element (RARE) located far upstream of the transcription start site, in a 5'-distal enhanceosome region previously shown to harbor basal and vitamin D-inducible enhancer activity. Maximal RA responsiveness (fourfold) was conferred by nucleotide sequence located between -7064 and -6787, with a variety of deletion and point mutations revealing the importance of at least three nuclear receptor half-sites within the enhancer region (-6851 to -6824), as well as nucleotides located further upstream. Recombinant human retinoic acid receptor/retinoid-X receptor heterodimers bound with high affinity and sequence specificity to multiple regions within the RARE, as demonstrated by electrophoretic mobility shift and DNase I footprinting assays. The addition of RARE activity to previously described functions of the 5'-distal enhanceosome underscores the importance of this region as a key integration point for regulatory control of PDGF-A expression.

Figure 1

Retinoic acid induces expression of the endogenous PDGF-A gene. Neonatal rat lung fibroblasts were treated with all-trans retinoic acid for 24 h at the concentrations indicated. Values represent the fold-change in the ratio of PDGF-A to GAPDH mRNA concentrations following RA treatment, as determined by real-time PCR and described in Materials and Methods. The results shown are representative of duplicate experiments that demonstrate retinoic acid inducibility of PDGF-A mRNA expression.

Figure 2

Localization of a retinoic acid response element (RARE) between nucleotides −7064 and −6787 in the 5′-distal enhanceosome region of the PDGF-A gene. All results were obtained by transient transfection of CV-1 cells and represent relative luciferase activity obtained after correction for interwell variation in transfection efficiency with a cotransfected plasmid directing consitutive expression of Renilla luciferase. Results are expressed as the mean (±SEM) of at least six replicate transfected wells obtained over three independent transfection experiments. (A) CV-1 cells were transfected with luciferase reporter plasmids containing either a −7294 to +8 fragment of the PDGF-A gene, or a 5′-truncated fragment (−6787 to +8), as shown below the figure. Other treatments indicated are the presence or absence (+ or −) of expression vectors for RARβ and RARP, and RA administration (10−⁶ M) for 24 h. Fold-induction in luciferase activity is presented for treatment pairs in which a significant induction (p < 0.05, denoted with asterisk) from RA treatment was observed. (B) 5′-Endpoint deletional analysis of the −7064 to −6787 region. Represented at left is the series of 5′-endpoint deletions employed, within which four nuclear receptor-binding half-sites (A–D) and the ternary complex factor (TCF) binding motif within the ACE66 enhancer are represented with open and gray-filled boxes, respectively. An additional nuclear receptor-binding half-site (denoted α) located further upstream is also represented. Also shown is the construct pBL-TK-PRE, which contains an RARE from the RARP gene in fusion with the minimal HSV thymidine kinase (TK) promoter. (C) 3′-Endpoint deletional analysis of the −7064 to −6787 region, with deleted areas represented by dotted lines. (D) Mutational analysis of nuclear receptor-binding half-sites. Half-site motifs that contain inactivating mutations are identified with black boxes. Transfection efficiency was assessed by cotransfection with a β-galactosidase expression vector. Means not sharing an identical superscript are significantly different, as determined by ANOVA and the Fisher’s PLSD post hoc test for mean separation (p < 0.05).

Figure 3

Nucleotide sequence of the 5′-distal enhanceosome and neighboring regions from the PDGF-A gene. Shown is the sequence spanning the region between −7294 and −6787, which contains the basal enhancer element ACE66 (−6852 to −6787, outlined in gray box) at the 3′-terminus. Nucleotides are numbered relative to the transcription start site as determined previously (5,38). Relevant restriction endonuclease sites (XbaI, SstI, BspMI, and EcoRI) are underlined, while half-site motifs for nuclear receptor binding (a, A, B, C, and D), and binding sites for VDR (VDRE), AP1 and ternary complex factors (TCF) are boxed. Arrows denote locations of 5′-endpoints (left arrows) and 3′-endpoints (right arrows) employed in construction of deletion mutants for localization of the RARE.

Figure 4

Induction of 5′-distal enhanceosome activity by retinoic acid is not cooperative with vitamin D inducibility. Results represent fold-change in relative luciferase activity obtained with the −7294 to −6787 region in response to the treatments summarized below the graph. Treatments consisted of plasmid-directed overexpression of RARα and/or VDR, and administration of 1,25-(OH)₂D₃ (VitD, 10⁻⁷ M) and/or RA (10⁻⁶ M).

Figure 5

Retinoic acid receptor α/retinoid-X receptor α (RARα/RXRα) heterodimers bind with high affinity and nucleotide sequence specificity to the 5′-distal RARE: electrophoretic mobility shift assay (EMSA). (A) SDS-PAGE analysis of affinity-purified recombinant RARα and RXRα. (C) Binding of RARα/RXRα heterodimers to the −7064 to −6787 fragment of the PDGF-A gene: titration of increasing RARα protein (0−75 ng) into a fixed concentration of RXRα (12.5 ng). EMSA was conducted with a fixed amount (15 pmol, 20,000 cpm) of ³²P-labeled −7064 to −6787 probe (Pr). Binding reactions were conducted in the presence of bovine serum albumin (BSA) to minimize inert binding of recombinant proteins in very low concentrations to plastic surfaces. (C) RARα/RXRα heterodimers bind to the −7064 to −6787 fragment with much higher affinity than either protein alone. (D) Mutations in nuclear receptor-binding motifs A, B, and C result in diminished binding affinity for the RARα/RXRα heterodimer in vitro. Assays were conducted with 12.5 ng of both RARα and RXRα, ³²P-labeled −7064 to −6787 probe, and the indicated amounts (50–200-fold molar excess relative to probe) of unlabeled competitor DNAs (Compet.) corresponding to the −7064 to −6787 region. Competitors were constructed in wild-type (wt) form or containing substitution mutations within consensus nuclear receptor-binding motifs A–C as indicated.

Figure 6

RARα/RXRα binds to multiple sites within the −7064 to −6787 fragment: DNase I footprinting analysis. Analyses were conducted in the absence (−) or presence (0.4 or 1.0 μg) of RARα/RXRα. Double-stranded, ³²P-labeled −7064 to −6787 fragment was used as the binding substrate for analyses performed with labeled coding (left panel) or noncoding (right panel) strands. Identified with arrows to the left of each panel are mobilities of bands corresponding to nuclear receptor binding sites A–D and α, while regions of footprinting (FP, open boxes) and DNase I hypersensitivity (HS, shaded boxes) are indicated to the right of each panel.