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

Abstract

Abstract-Prohferatlon of cultured neonatal vascular smooth muscle (VSM) cells 1~ enhanced by exposure to cychc mechanical strain, m part through autocrme action of secreted platelet-denved growth factor (PDGF) We examined transcrlptlon factors and DNA response elements that may participate m the mductlon of PDGF-A gene transcnptlon by mechanical strain. PDGF-A mRNA increased gradually over 4 to 24 hours exposure to cychc (1Hz) strain This was due, at least m part, to increased transcnptlon smce a full length (890bp) PDGF-A promoter reporter construct was induced 3 5-fold m transfected VSM cells exposed to strain for 24 hours A series of PDGF-A promoter truncation reporter constructs was used to ldentl@ potential regons of the promoter involved m regulation by strain Strain-responsive regions were found between -262bp and -92bp and between -92bp and -4lbp of the promoter Since these regions are GC-rich and contain response elements for Egr-1 and Sp-1, we exammed expression of these transcnptlon factors m response to strain mRNA for both factors increased over 0 5 to 4 hours of stram, while protein expression for both increased gradually over a 24 hours penod Gel shift assays with a probe specific for Egr-1 demonstrated at least 1 promment new shifted band after 4 to 12 hours exposure to strain An Sp-1 probe demonstrated constltutlve shifted bands that did not change m response to strain Thus, GC-rich regons m the proximal 92bp of the PDGF-A promoter contam mechanical strain-responsive elements that bmd Egr-1 and possibly Sp-1 (Hypertension. 1998;31[part 2]:170-175.)Key Words: vascular smooth muscle w mechanical strain n transcrlptlon factors n reporter genes n growth factors T he artenal wall 1s exposed to significant mechanical strain during the cardiac cycle ' Mechanical forces may partlclpate m the normal development of blood vessels' and m the pathological changes observed m hypertension '-' Recently, mechanical forces have been found to elicit a wide range of phenotyplc responses both m intact tissues and m cultured cells ' 6Our laboratory has previously shown that the apphcatlon of cychc (1 Hz) mechanical strains of 10% to 20% to neonatal VSM cells cultured on deformable &one elastomer membranes leads to increased prohferatlon, most likely via autocnne action of secreted PDGF' The magnitude of cychc strain necessary to induce PDGF m this m vitro system 1s comparable to the strain found m many vessels m situ ' Both PDGF-A and -B appear m the medium after 24 hours exposure to strain, PDGF-A' and -B8 RNAs are also increased by strain This response to mechanical stram observed m vitro may be relevant to the pathophyslology of hypertension, as several mvestlgators have demonstrated increased expression of PDGF m blood vessels of hypertensive animals 9 Negoro et al showed that antlhypertenslve drugs reduce expression of PDGF-A m vessel walls of hypertensive SHR rats 4The PDGF-A promoter has been cloned "I'~ and many of its regulatory elements have been identified 4 'z-l5 These studies suggest that a GC rich regio...

Rapid Induction and Translocation of Egr-1 in Response to Mechanical Strain in Vascular Smooth Muscle Cells

“…39 In additional preliminary studies, we could show that the SP-1 protein is induced by strain, possibly also implicating SP-1 sites in the strain response. 6 Recently, a functional interplay between Egr-1 and SP-1 in the PDGF-A-chain promoter has been elegantly demonstrated in bovine aortic endothelial cells in response to shear stress. 12 In these studies, the binding of Egr-1 to the PDGF-A-chain promoter was induced by shear stress, which displaced SP-1 from their overlapping recognition elements.…”

“…This observation supports the hypothesis that Egr-1 may contribute to the induction of PDGF-A transcription and ultimately secretion of PDGF in response to strain. 4,6 On the other hand, c-jun mRNA transcription and c-jun amino terminal kinase are activated on collagen, FN, and LN. This pattern of activation might reflect a matrix-independent, more general response to extracellular stress in VSM cells.…”

“…11,12 In cardiac myoctes, Egr-1, c-jun, and c-fos expression are increased by mechanical forces. [13][14][15] On the basis of VSM cells transfected with various PDGF-A-chain promoter truncations and subjected to strain, data from this laboratory identified a 92-bp region of the promoter that is responsive to strain 6 This region contains a binding site for the Egr-1 protein.…”

Matrix-Dependent Gene Expression of Egr-1 and PDGF A Regulate Angiotensin II–Induced Proliferation in Human Vascular Smooth Muscle Cells

“…27 Recent work from our laboratory has suggested a role for Egr-1 in the induction of PDGF-A by continuous cyclic mechanical strain in neonatal rat VSM cells. 28 We hypothesized that the Ang II-induced increase in PDGF A expression and release observed in the present study in human VSM cells might also be related to activation of Egr-1. We showed that steady-state mRNA of Egr-1 increased in response to Ang II in human VSM cells at 15 minutes; peak expression occurred at 30 minutes and levels returned to normal after 2 hours, at which time an increase in PDGF A expression was first observed.…”

Molecular Mechanism of Fibronectin Gene Activation by Cyclic Stretch in Vascular Smooth Muscle Cells

“…In a previous study, the GC-rich region of the platelet-derived growth factor-A gene promoter, which can bind Egr-1 and Sp1, is reported to be a stretch-responsive element in VSMCs (36). EMSA showed that a new Egr-1 binding activity was induced after exposure to cyclic stretch, while an Sp-1 probe disclosed constitutive shifted bands that did not change in response to stretch (36). Thus, the stretching seems to turn on the Egr-1 pathway to activate the platelet-derived growth factor-A gene promoter.…”