Stable expression of human platelet-derived growth factor B chain by bovine aortic endothelial cells. Matrix association and selective proteolytic cleavage by thrombin.
The localization of platelet-derived growth factor (PDGF) B chain and the regulation of its release by thrombin were investigated in bovine aortic endothelial cells stably transfected with the cDNA for human PDGF B chain (c-sis). Northern blot analysis of c-sis transfected cells revealed increased expression of PDGF B mRNA and constitutive release of 5-10-fold greater amounts of PDGF than in control cells. Incubation with bovine alpha-thrombin further induced PDGF release into the conditioned medium. Metabolic labeling of bovine aortic endothelial cells overexpressing c-sis revealed an inefficient rate of constitutive PDGF release, with the majority of newly synthesized PDGF remaining extracellular matrix-associated. Thrombin treatment, however, led to a dramatic increase in the amount of PDGF released into the medium due to selective proteolytic cleavage of matrix-associated precursors. Incubation with a synthetic peptide representing residues 212-230 of precursor PDGF B chain, previously shown to induce the release of PDGF from cell- or matrix-associated heparan sulfate proteoglycans, led to the release of slightly larger species of PDGF which were susceptible to proteolytic cleavage by thrombin in vitro. In addition, PDGF precursors immunoprecipitated from cells were also cleaved by thrombin in vitro. We have demonstrated, using normal diploid endothelial cells overexpressing c-sis, that PDGF B chain is stably expressed as a matrix-associated protein which is either inefficiently released into the medium or cleaved by paracrine proteases such as thrombin. Modulation of PDGF release by selective cleavage of preformed, matrix-bound precursors may represent a significant mechanism for acute regulation of release of this growth factor independent of changes in the rate of synthesis.
Soyombo
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Department of Cell Biology, Cleveland Clinic Foundation, Ohio 44195.
DiCorleto
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PE
D000818:Animals; D001011:Aorta; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis; D013917:Thrombin / Q000378:metabolism*
D018076:DNA, Complementary; D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; D011498:Protein Precursors; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D013917:Thrombin