Molecular structure and transcriptional regulation of the gene for the platelet-derived growth factor alpha receptor in cultured vascular smooth muscle cells.

Abstract

PDGF has been shown to contribute to hypertrophy in vascular smooth muscle cells (VSMC). PDGF-AA differentially promotes protein synthesis in VSMC from spontaneously hypertensive rats (SHR) but not in those from Wistar-Kyoto rats (WKY). This observation has led us to postulate a role for PDGF a receptor (PDGFR-a) in the hypertensive hypertrophy of blood vessels. Western and Northern blot analyses demonstrated a high and specific expression of the PDGFR-a protein and mRNA in SHR cells but not in WKY cells. To clarify the mechanism of the differential expression of the PDGFR-cv gene, we isolated the promoter region of the gene. Studies on the promoter functions indicated that this promoter is active in SHR cells but not in WKY cells. The regulatory domain responsible for this difference was narrowed to the sequence between -246 and -139, which enhanced the promoter activity of SHR fivefold over the basal activity. DNase I footprinting and gel-shift assay indicated that this sequence specifically interact with nuclear proteins from VSMC through the binding site for CCAAT/ enhancer-binding proteins, and members of the C/enhancer-binding protein family play a significant role in the strain-specific transcription of the PDGFR-a gene. (J. Clin. Invest. 1995.96:558-567.)

Fibroblast Growth Factor-2 Represses Platelet-derived Growth Factor Receptor-α (PDGFR-α) Transcription via ERK1/2-dependent Sp1 Phosphorylation and an Atypical cis-Acting Element in the Proximal PDGFR-α Promoter

“…1A). To confirm these observations at the level of transcription, we co-transfected SMCs with CMV-Sp1 and pLuc-a2, a Firefly luciferase-based reporter construct driven by 1.3 kb of PDGFR-␣ promoter (23). The cells were also transfected with the Renilla luciferase-based construct to correct for transfection efficiency.…”

“…Plasmid Constructs-The plasmid pLuc-a2 (23), containing the promoter region of platelet-derived growth factor receptor-␣, was kindly donated by Dr Yutaka Kitami from Ehime University School of Medicine, Ehime, Japan. pLuc-a2.Sp1m3 and CMV-Sp1.mThr 453 /mThr 739 were produced using the QuikChange® XL site-directed mutagenesis kit (Stratagene) in accordance with the manufacturer's instructions.…”

Transcriptional Regulation of the Platelet-derived Growth Factor α Receptor Gene via CCAAT/Enhancer-binding Protein-δ in Vascular Smooth Muscle Cells

“…Each DNA fragment was labeled with [␣-32 P]dCTP using the random primer method. Total cellular RNA extraction from VSMC and Northern blot analysis were carried out by the methods described previously (19,20).…”

“…For competition experiments or supershift assay, a 100-fold molar excess of unlabeled probe or 1-2 l of antibodies against each subtype of the C/EBP family was added to nuclear extracts, respectively, and was incubated for 30 min at room temperature before addition of the labeled C/EBP probe. Then all reaction mixtures were analyzed by 5% polyacrylamide gel electrophoresis under nondenaturing conditions, and the gel was dried and processed as described previously (19).…”

“…Western Blotting-Western blotting was performed by the method described previously (17,19). Briefly, nuclear extracts (2.5 g) prepared from VSMC were directly subjected to Western blotting for C/EBP␣, -␤, and -␦.…”