Accumulation of PDGF B and cell-binding forms of PDGF A in the extracellular matrix.
PDGF is a powerful mitogen initially identified within platelets, but also shown to be produced by a wide variety of cell types. PDGF is encoded on two separate genes. These give rise to three polypeptides, PDGF B and two forms of PDGF A (SA and LA), resulting from alternative splicing of the PDGF A gene primary transcript. We report that in CHO cells transfected with PDGF gene constructs and producing moderate levels of PDGF homodimers, much of the PDGF LA and B produced, but little if any SA, is found in the matrix laid down beneath the cells. Immunoreactive PDGF in cells, and in matrix below expressing cells, was visualized by laser confocal microscopy. Western blotting of protein in matrix extracts, cell extracts, and secreted into the growth medium was used to demonstrate that the range of PDGF A polypeptides seen in the matrix was overlapping with those reported previously to be cell associated in cell types such as NIH3T3 and COS 7. This suggests that attachment to matrix or cell surface may be alternative fates for these polypeptides, with fate dependent on the characteristics of the producing cells. Immunoreactive PDGF A and B could be partially released by incubation of matrix material with heparin but not with other glycosaminoglycans. Digestion of matrix with chondroitin ABC lyase but not heparitinase or collagenase displaced some PDGF from its attachment sites. The results indicate attachment of PDGF to matrix proteoglycans, at least partly through the glycosaminoglycan moieties, and perhaps to additional components. The significance of matrix deposition for PDGF action is discussed.
CSIRO Division of Biomolecular Engineering, North Ryde, Sydney, Australia.
J L
JL
Kelly
A
A
Sánchez
G S
GS
Brown
C N
CN
Chesterman
M J
MJ
Sleigh
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005109:Extracellular Matrix / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis; D014162:Transfection
D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; C075278:platelet-derived growth factor A