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.
Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
J C
JC
Bonner
A
A
Badgett
A R
AR
Osornio-Vargas
M
M
Hoffman
A R
AR
Brody
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