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(Hypertension. 1998;31:170.)
© 1998 American Heart Association, Inc.

Workshop on Vascular Biology & Hypertension: From Molecules to Humans

Strain-Responsive Regions in the Platelet-Derived Growth Factor-A Gene Promoter

Emily Wilson; Franklin Vives; Tucker Collins; Harlan E. Ives

From the Cardiovascular Research Institute, Division of Nephrology, University of California, San Francisco (EW, FV, HEI), and the Department of Pathology, Brigham and Women’s Hospital, Boston, (TC)

Correspondence to Harlan E. Ives, MD, Box 0532 HSE 672, University of California, San Francisco, CA 94143. E-mail: ives{at}itsa.ucsf.edu

Proliferation of cultured neonatal vascular smooth muscle (VSM) cells is enhanced by exposure to cyclic mechanical strain, in part through autocrine action of secreted platelet-derived growth factor (PDGF). We examined transcription factors and DNA response elements that may participate in the induction of PDGF-A gene transcription by mechanical strain. PDGF-A mRNA increased gradually over 4 to 24 hours exposure to cyclic (1Hz) strain. This was due, at least in part, to increased transcription since a full length (890bp) PDGF-A promoter reporter construct was induced 3.5-fold in transfected VSM cells exposed to strain for 24 hours. A series of PDGF-A promoter truncation reporter constructs was used to identify potential regions of the promoter involved in regulation by strain. Strain-responsive regions were found between -262bp and -92bp and between -92bp and -41bp of the promoter. Since these regions are GC-rich and contain response elements for Egr-1 and Sp-1, we examined expression of these transcription factors in response to strain. mRNA for both factors increased over 0.5 to 4 hours of strain, while protein expression for both increased gradually over a 24 hours period. Gel shift assays with a probe specific for Egr-1 demonstrated at least 1 prominent new shifted band after 4 to 12 hours exposure to strain. An Sp-1 probe demonstrated constitutive shifted bands that did not change in response to strain. Thus, GC-rich regions in the proximal 92bp of the PDGF-A promoter contain mechanical strain-responsive elements that bind Egr-1 and possibly Sp-1.

Key Words: vascular smooth muscle • mechanical strain • transcription factors • reporter genes • growth factors

Abbreviations: VSM = vascular smooth muscle • PDGF = platelet-derived growth factor • Hz = Hertz • SHR = spontaneously hypertensive rat • GAPDH = glyceraldehyde 3-phosphate dehydrogenase • dCTP = deoxycytosine trisphosphate • ECL = enhanced chemiluminescence • RSV = Rous sarcoma virus • EDTA = ethylenediaminetetraacetic acid • DTT = dithiothreitol • dIdC = poly deoxyinosine-deoxycytosine • CAT = chloramphenicol acetyltransferase • SRE = serum response element • BSA = bovine serum albumin

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