PDGF-stimulated fibroblast proliferation is enhanced synergistically by receptor-recognized alpha 2-macroglobulin.
alpha-Macroglobulins derived from plasma or secreted by macrophages are platelet-derived growth factor (PDGF) binding proteins that compete with cell-surface receptors on fibroblasts for PDGF binding. alpha 2-Macroglobulin (alpha 2M) derived from bovine plasma was tested for its ability to modulate the PDGF-induced proliferation of primary passage rat lung fibroblasts (RLFs) and a human skin fibroblast cell line (CRL 1508). Fibroblasts were grown in 10% fetal bovine serum (FBS) for 24 hr, then washed with serum-free medium before adding serum-free defined medium (SFDM) containing insulin and transferrin. To this medium were added varying concentrations of human plasma-derived AB-PDGF and alpha 2 M, alone or in combination. Receptor-recognized alpha 2M was prepared by treatment with methylamine. Both native alpha 2M and the alpha 2M-methylamine (alpha 2M-MA) were tested for growth promoting activity in the absence or presence of PDGF. After 3 days, a concentration-dependent growth curve of fibroblast proliferation was demonstrated for PDGF alone, with near maximal stimulation reached at 15-20 ng/ml PDGF. alpha 2M and alpha 2M-MA alone had no effect on cell proliferation. However, alpha 2M-MA concentrations above 32 micrograms/ml synergistically enhanced PDGF-stimulated proliferation greater than 100% in the presence of 15 ng/ml PDGF. Native alpha 2M enhanced PDGF-stimulated growth 80-100% above PDGF controls only at low concentrations (32-64 micrograms/ml alpha 2M). High concentrations of native alpha 2M (128-256 micrograms/ml) either had no effect on growth or were inhibitory to PDGF-stimulated growth, depending on the cell type tested. Rat lung fibroblasts were shown to secrete a factor(s) that inhibited the trypsin-binding capacity of native alpha 2M. We further demonstrated that early passage RLFs possess specific cell-surface receptors for [125I]-PDGF and [125I]-alpha 2M-MA, and preincubation of RLFs with alpha 2M-MA increased the specific binding of [125I]-PDGF to the cell surface of these fibroblasts. Considered together, these data support the view that receptor-recognized alpha 2M synergistically enhances the proliferative capacity of PDGF. We postulate that receptor-recognized alpha Ms enhance PDGF-stimulated growth by increasing the local concentration of PDGF at the cell surface, where the PDGF could be released in close proximity to its own receptors.
Bonner
J C
JC
Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Badgett
A
A
Osornio-Vargas
A R
AR
Hoffman
M
M
Brody
A R
AR
D000818:Animals; D001667:Binding, Competitive; D002455:Cell Division; D005347:Fibroblasts / Q000166:cytology*; D006801:Humans; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008168:Lung / Q000166:cytology; D008744:Methylamines; D010982:Platelet-Derived Growth Factor / Q000378:metabolism / Q000502:physiology*; D011869:Radioligand Assay; D051381:Rats; D011956:Receptors, Cell Surface / Q000502:physiology; D011971:Receptors, Immunologic / Q000502:physiology; D017479:Receptors, Platelet-Derived Growth Factor; D012867:Skin / Q000166:cytology; D000511:alpha-Macroglobulins / Q000378:metabolism / Q000502:physiology*
D007457:Iodine Radioisotopes; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008744:Methylamines; D010982:Platelet-Derived Growth Factor; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins; C027451:methylamine; D017479:Receptors, Platelet-Derived Growth Factor