Differential binding, biological and biochemical actions of recombinant PDGF AA, AB, and BB molecules on connective tissue cells.

We have compared the biological and biochemical properties of recombinant PDGF AA, AB, and BB using three types of fibroblastic cells: NIH/3T3, human skin fibroblast, and fetal bovine aortic smooth muscle. PDGF binding, receptor autophosphorylation, phosphatidyl inositol hydrolysis, as well as chemotactic and mitogenic responses of the cells were analyzed. PDGF-AB and PDGF-BB showed similar receptor binding, receptor autophosphorylation, and potent biological activity for all three of the cell types tested. In contrast, PDGF-AA was biologically active only for the NIH/3T3 cells in which binding sites for PDGF-AA were abundant, but was inactive for bovine aortic smooth muscle cells and human skin fibroblasts in which binding sites for PDGF-AA were absent. PDGF-AA could not induce any biochemical changes in the human skin fibroblasts or smooth muscle cells. Western blot studies with anti-Type alpha and beta PDGF receptor antibodies indicate that the NIH/3T3 cells contained both PDGF alpha and beta receptors, whereas the human skin fibroblasts and bovine smooth muscle cells contained only detectable levels of beta receptors. These results indicate that cells possessing high levels of PDGF beta receptors only are capable of responding equally well to either PDGF AB or BB.     

Platelet-derived growth factor isoforms AA, AB, and BB differentially activate poly r(I):r(C)-induced genes in human fibroblast FS4 cells.

Polyribocytidylic-polyriboinosinic acid [poly r(I):r(C)]-inducible genes were isolated by a differential screening procedure from a human fibroblast cell (FS-4) cDNA bank. Among yet unidentified genes (gene 274), one codes for a protein with multiple finger motifs and has previously been detected in endothelial cells after tumor necrosis factor-alpha (TNF-alpha) treatment (A20; Opipari et al., 1990), the second one codes for a variant of the I kappa B family (Haskill et al., 1991), and a third one for the Ca2+ ATPase (isoform 1). Platelet-derived growth factor (PDGF) isoforms (AA, AB, and BB) stimulated the expression of these immediate-early genes. But the extent of the respective induction correlated neither with the number of the two receptors alpha or beta nor with the level of PDGF-stimulated receptor autophosphorylation on tyrosine. Although alpha-receptors were less abundant than beta-receptors (12,500 binding sites were estimated for PDGF-AA, KD 0.03 nM; 20,000 for PDGF-AB, KD 0.03 nM; 35,000 for PDGF-BB KD 0.16 nM) and tyrosine phosphorylation induced by PDGF-AA was significantly less than that evoked by PDGF-BB, some of the investigated genes were more strongly induced by PDGF-AA. We discuss how the differences in the biological potency of the PDGF isoforms may reside in different functions of the two receptors by activation of alternative signaling pathways.     

Differential induction of the c-fos promoter through distinct PDGF receptor-mediated signaling pathways.

The multiple isoforms of PDGF induce fibroblastic mitogenesis through two distinct PDGF receptors, alpha and beta. The molecular mechanisms by which these alpha and beta PDGF receptors regulate gene expression are poorly understood. We present data which indicates that differential induction of c-fos gene expression by PDGF isoforms occurs through distinct PDGF alpha and beta receptor-mediated signaling pathways. Comparison of PDGF-AA with PDGF-BB stimulation showed that PDGF-BB induced prolonged expression of the c-fos gene in BALB/c-3T3 cells, but that PDGF-AA induced more potent activation of the serum response element (SRE) in transient transfection assays. PDGF-AA, which binds alpha but not beta PDGF receptors, could only induce the SRE through a protein kinase C (PKC)-dependent pathway, whereas PDGF-BB, which binds both alpha and beta PDGF receptors, could also induce the SRE through a PKC-independent pathway. These results suggest that PDGF alpha receptors activate the PKC-dependent signaling pathway while PDGF beta receptors also activate a PKC-independent pathway. In addition, we found that PDGF-BB could induce another c-fos promoter element within the -90 to +10 region, suggesting that the more potent mitogenic effect and prolonged c-fos gene expression induced by PDGF-BB may result from cooperativity between more than one c-fos promoter elements.     

Chimeric alpha- and beta-platelet-derived growth factor (PDGF) receptors define three immunoglobulin-like domains of the alpha-PDGF receptor that determine PDGF-AA binding specificity

Binding of platelet-derived growth factor (PDGF) to its cell surface receptors stimulates a variety of biochemical and biological responses. Two receptor gene products (designated alpha and beta) have been identified, and the different binding affinities of various PDGF isoforms for these receptors are prime determinants of the spectrum of responses observed. The beta receptor binds PDGF-BB, but not PDGF-AA, while the alpha receptor binds PDGF-AA and PDGF-BB. We utilized these different ligand binding specificities to investigate the PDGF-AA binding site in the human alpha-PDGF receptor by constructing chimeric molecules between the human alpha- and beta-PDGF receptors. Our results demonstrate that amino acids 1-340 of the alpha-PDGF receptor comprise the region that confers PDGF-AA binding specificity. This region corresponds to immunoglobulin-like sub-domains 1, 2, and 3 of its external domain.     

The A-type receptor for platelet-derived growth factor mediates protein tyrosine phosphorylation, receptor transmodulation and a mitogenic response.

A clonal human glioma cell line, U-343 MGa 31L, which expresses the A-type but not the B-type receptor for platelet-derived growth factor (PDGF), was used in a functional study of the A-type receptor. PDGF-AA induced, in a dose- and time-dependent manner, phosphorylation on tyrosine residues of the receptor in metabolically labelled cells. The optimal dose was around 30 ng/ml; at 100 ng/ml, phosphorylation was maximal at 15 min and had almost returned to the control level after 60 min. The phosphorylation on tyrosine residues of the PDGF A-type receptor was stimulated by PDGF-AA, PDGF-AB and PDGF-BB; these isoforms also stimulated [3H]thymidine incorporation into U-343 MGa 31L cells. In addition, activation of the A-type PDGF receptor induced transmodulation of the epidermal growth factor receptor.     

Isoform-specific induction of actin reorganization by platelet-derived growth factor suggests that the functionally active receptor is a dimer.

Human platelet-derived growth factor (PDGF) occurs as three isoforms which are made up of disulfide-bonded A and B chains. The isoforms bind with different affinities to two different but structurally related cell surface receptors. The A type receptor binds all three isoforms (PDGF-AA, PDGF-AB, PDGF-BB) with high affinity, whereas the B type receptor binds PDGF-BB with high affinity, PDGF-AB with lower affinity but does not appear to bind PDGF-AA. We have utilized the differential effects of the three isoforms on actin reorganization and membrane ruffling in human foreskin fibroblasts to probe the idea that ligand-induced receptor dimerization is associated with receptor activation. Actin reorganization was found to be induced only by PDGF-AB and PDGF-BB and is therefore likely to be mediated by the B type receptor. Simultaneous addition of PDGF-AA, or downregulation of the A type receptor blocked the effect of PDGF-AB but not that of PDGF-BB. This is compatible with a model by which PDGF-AB binds to and dimerizes one A and one B type receptor; PDGF-AB therefore requires A type receptors in order to be functionally active at physiological concentrations. In cells with down-regulated A type receptors, high concentrations of PDGF-AB inhibited the effect of PDGF-BB on actin reorganization. We believe that this is due to a monovalent binding of PDGF-AB to the B type receptors which prevents PDGF-BB from dimerizing the receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The PDGF family: four gene products form five dimeric isoforms

Platelet-derived growth factors (PDGFs) were discovered more than two decades ago. Today the PDGF family of growth factors consists of five different disulphide-linked dimers built up of four different polypeptide chains encoded by four different genes. These isoforms, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD, act via two receptor tyrosine kinases, PDGF receptors alpha and beta. The classic PDGFs, PDGF-A and PDGF-B, undergo intracellular activation during transport in the exocytic pathway for subsequent secretion, while the novel PDGFs, PDGF-C and PDGF-D, are secreted as latent factors that require activation by extracellular proteases. The classical PDGF polypeptide chains, PDGF-A and PDGF-B, are well studied and they regulate several physiological and pathophysiological processes, mainly using cells of mesenchymal or neuroectodermal origin as their targets. The discovery of two additional ligands for the two PDGF receptors suggests that PDGF-mediated cellular signaling is more complex than previously thought.     

Dimerization of B-type platelet-derived growth factor receptors occurs after ligand binding and is closely associated with receptor kinase activation

Platelet-derived growth factor (PDGF) was found to induce dimerization of purified B-type PDGF receptors, as analyzed by sodium dodecyl sulfate gel electrophoresis after covalent cross-linking using disuccinimidyl suberate. PDGF-BB was 20-fold more effective than PDGF-AB; PDGF-AA was without effect. The dimerization was dose-dependent and was maximal at 0.5-2 micrograms/ml PDGF-BB; at higher concentrations dimerization was less abundant. This indicates that dimerization occurred when one PDGF-BB molecule bound two receptor molecules. The dimerization correlated to activation of the tyrosine kinase of the receptor, determined as autophosphorylation, but was not dependent on phosphorylation reactions because it occurred also in the absence of ATP. Furthermore, dimerization of the receptor correlated with the ability to phosphorylate phosphofructokinase, an exogenous substrate. The complex of ligand and receptor dimer was stable; it resisted electrophoresis under nondenaturing conditions, as well as gel chromatography. The present data indicate that intermolecular mechanisms are involved in signal transduction from the external ligand binding domain to the internal effector domains of the B-type PDGF receptor.     

Platelet-derived growth factor (PDGF) stimulates PDGF receptor subunit dimerization and intersubunit trans-phosphorylation.

High affinity binding of platelet-derived growth factor (PDGF) has been proposed to involve the interaction of the dimeric PDGF ligand with two receptor subunits, designated alpha and beta. We have cloned and expressed a human PDGF receptor cDNA which differs in sequence from the beta-subunit and which has the PDGF binding properties and monoclonal antibody recognition, predicted for the alpha-subunit. Scatchard analysis indicated that PDGF-AA and PDGF-AB bound to transfected alpha-subunits with affinities of Kd = 0.06 and 0.05 nM, respectively. PDGF-BB bound with a significantly lower affinity (Kd = 0.4 nM). Nevertheless, this affinity is still great enough to mediate substantial PDGF-BB binding at physiological concentrations and would be considered to be "high affinity." We have used wild-type and kinase-inactive human beta-subunits to show that PDGF binding promotes receptor subunit dimerization in intact cells. In addition, we found that PDGF stimulates tyrosine phosphorylation of the kinase-inactive beta-subunit when it is expressed with alpha-subunits. The kinase-inactive beta-subunits were phosphorylated at tyrosine 857 and 751, the major phosphorylation sites of the wild-type beta-subunit, indicating either that intra- and intermolecular phosphorylation occurs on the same sites, or that a significant fraction of receptor tyrosine phosphorylation is intermolecular.     

Actions of platelet-derived growth factor isoforms in mesangial cells

Platelet-derived growth factor (PDGF) occurs as homodimers or heterodimers of related polypeptide chains PDGF-BB, -AA, and -AB. There are two receptors that bind PDGF, termed alpha and beta. The beta receptor recognizes PDGF B chain and is dimerized in response to PDGF BB. The alpha receptor recognizes PDGF B as well as A chains and can be dimerized by the three dimeric forms of PDGF AA, AB, and BB. To characterize PDGF receptor signaling mechanisms and biologic activities in human mesangial cells (MC), we explored the effects of the three PDGF isoforms on DNA synthesis, phospholipase C activation, and PDGF protooncogene induction. PDGF-BB homodimer and AB heterodimer induced a marked increase in DNA synthesis, activation of phsopholipase C, and autoinduction of PDGF A and B chain mRNAs, whereas PDGF-AA homodimer was without effect. The lack of response to PDGF AA could be accounted for by down regulation of the PDGF-alpha receptor since preincubation of MC with suramin restored PDGF AA-induced DNA synthesis. Ligand binding studies demonstrate specific binding of labeled PDGF BB and AB and to a lower extent PDGF AA isoforms to mesangial cells. These results are consistent with predominant expression of PDGF beta receptor in MC, which is linked to phospholipase-C activation. The potent biologic effects of PDGF-AB heterodimer in cells that express very few alpha receptors and do not respond to PDGF AA are somewhat inconsistent with the currently accepted model of PDGF receptor interaction and suggest the presence of additional mechanisms for PDGF isoform binding and activation. © 1994 Wiley-Liss, Inc.     

Regulation of proliferation and differentiation of myoblasts derived from adult mouse skeletal muscle by specific isoforms of PDGF

The expression of receptors and the mitogenic response to PDGF by C2 myoblasts, derived from adult mouse skeletal muscle, was investigated. Employing 125I-PDGF binding assays, we showed that the cells exhibit high level binding of PDGF-BB (approximately 165 x 10(3) molecules/cell at saturation) and much lower binding of the PDGF-AA and PDGF-AB (6-12 x 10(3) molecules/cell at saturation). This indicates that the C2 myoblasts express high levels of PDGF receptor beta-subunits and low levels of alpha-subunits. PDGF-BB enhances the proliferation of C2 cells maintained in 2% FCS by about fivefold. PDGF-AB had a moderate effect on cell proliferation (less than twofold) and PDGF-AA had no effect. Inverse effects of PDGF isoforms on the frequency of differentiated myoblasts were observed; the frequency of myosin-positive cells was reduced in the presence of PDGF-BB while PDGF-AA and PDGF-AB had no effect. PDGF may thus act to increase the number of myoblasts that participate in muscle regeneration following muscle trauma by stimulating the proliferation and by inhibiting the differentiation of myogenic cells.     

Cellular response to platelet-derived growth factor (PDGF)-AB after down-regulation of PDGF alpha-receptors. Evidence that functional binding does not require alpha-receptors.

Platelet-derived growth factor (PDGF) and its receptor exist in multiple forms. PDGF exists in three dimeric combinations of A and B subunit chains, which are the products of separate genes. The PDGF receptor is similarly encoded by genes for two distinct receptor proteins, alpha and beta. A recent model proposed PDGF binding involves the association of the two receptor proteins into three possible dimeric forms. An essential prediction of that model is that PDGF alpha-receptors are required for cells to bind and respond to the heterodimeric AB isoform of PDGF. In contrast, we found both binding and functional response to PDGF-AB was retained in Balb/c-3T3 cells after PDGF alpha-receptors had been down-regulated by PDGF-AA pretreatment. The observation that PDGF-AB could still elicit these responses suggests that at 37 degrees C, PDGF-AB may bind directly to beta-receptors in either monomeric or dimeric forms and that initial receptor activation may occur independently of the formation of alpha beta-receptor heterodimers.     

Chick limb bud mesodermal cell chondrogenesis: inhibition by isoforms of platelet-derived growth factor and reversal by recombinant bone morphogenetic protein.

Platelet-derived growth factor (PDGF) influences the proliferation and differentiation of a variety of cells. In this study, we have investigated the effect of PDGF isoforms on chondrogenesis by stage 24 chick limb bud mesoderm cells in culture. Synthesis of sulfated proteoglycans, an index of chondrogenesis, was inhibited by all three PDGF isoforms (PDGF-AA, PDGF-AB, and PDGF-BB). Application of PDGF isoforms during the first 2 days of culture, before the cells were overtly differentiating, resulted in decreased synthesis of sulfated proteoglycans. This was similar to when PDGF isoforms were present throughout the culture period. However, application of PDGF isoform during only the last 2 days of culture, did not inhibit cartilage matrix production. When chondrogenic and nonchondrogenic cells were separated from the cultures and replated, PDGF-AB and PDGF-BB inhibited the incorporation of sulfate by the chondrogenic cells. Recombinant bone morphogenetic protein 2B reversed the inhibitory effects of PDGF on sulfated proteoglycan synthesis and DNA synthesis. PDGF receptor binding analysis indicated that beta-receptors were predominant receptors present on the chondrogenic and nonchondrogenic cells of the stage 24 mesoderm. PDGF isoforms increased thymidine incorporation by 48 h in both high and low density cultures. However, at later periods, cell proliferation was inhibited by PDGF-AA and PDGF-AB but not by PDGF-BB. PDGF acted as a bifunctional modulator of mesodermal cell proliferation and thus may regulate chondrogenesis during limb differentiation and morphogenesis.     

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.     

Structural and functional aspects of the receptors for platelet-derived growth factor

Platelet-derived growth factor (PDGF) is a 30 kDa dimer of disulfide-bonded A and B chains. Three isoforms of PDGF have been isolated (PDGF-AA, PDGF-AB and PDGF-BB). These bind with different affinities and specificities to two structurally related cell surface receptors, viz. the α-receptor and the β-receptor. The receptors are transmembrane proteins with an intracellular, ligand-stimulatable protein tyrosine kinase domain. Activation of the receptors is intimately associated with receptor dimerization, and available data suggest that PDGF is a divalent ligand such that one molecule of PDGF binds and dimerizes two receptor molecules. Stimulation of PDGF receptors leads to a cascade of cellular events, which have been shown to require an intact receptor tyrosine kinase activity. However, ligand-induced internalization and degradation of the β-receptor occur essentially independent of the receptor kinase activity. Receptor activation leads to the phosphorylation on tyrosine residues of three enzymes, probably by direct phosphorylation: phospholipase C-γ, phosphatidylinositol 3′ kinase and Raf-1. In certain cells, PDGF β-receptor expression is inducible such that cells in normal tissue in vivo do not express receptors; only in inflammatory lesions or when cells are explanted in vitro, are receptors being expressed. Transformation by the v-sis oncogene is mediated by an autocrine PDGF-like growth factor. Although both the α- and β-receptors are structurally related to the v-fms and v-kit oncogenes, it is not known if the PDGF receptors have a transforming potential. In conclusion, the finding of three isoforms of PDGF that interact with two structurally related receptors implies a finely tuned regulatory network, the role of which in cell growth and transformation remains to be clarified.     

Heparin amplifies platelet-derived growth factor (PDGF)- BB-induced PDGF alpha -receptor but not PDGF beta -receptor tyrosine phosphorylation in heparan sulfate-deficient cells. Effects on signal transduction and biological responses

Platelet-derived growth factor (PDGF) induces mitogenic and migratory responses in a wide variety of cells, by activating specific receptor tyrosine kinases denoted the PDGF alpha- and beta-receptors. Different PDGF isoforms bind in a distinct manner to glycosaminoglycans, particularly heparan sulfate. In the present study, we show potentiation by exogenous heparin of PDGF-BB-induced PDGF alpha-receptor tyrosine phosphorylation in heparan sulfate-deficient Chinese hamster ovary (CHO) 677 cells. This effect was not seen for PDGF-AA treatment, and heparin lacked a potentiating effect on PDGF-BB stimulation of the PDGF beta-receptor. Heparin did not affect the affinity of PDGF-BB binding for the PDGF receptors on CHO 677 cells. The PDGF-BB-stimulated PDGF alpha-receptor phosphorylation was enhanced in a dose-dependent fashion by heparin at low concentration. The effect was modulated by 2-O- and 6-O-desulfation of the polysaccharide. Maximal induction of PDGF alpha-receptor tyrosine phosphorylation (6-fold) in CHO 677 cells was achieved by treatment with a heparin decasaccharide, but shorter oligosaccharides consisting of four or more monosaccharide units were also able to augment PDGF alpha-receptor phosphorylation, albeit at higher concentrations. Heparin potentiated PDGF-BB-induced activation of mitogen-activated protein kinase and protein kinase B (Akt) and allowed increased chemotaxis of the CHO 677 cells toward PDGF-BB. In conclusion, heparin modulates PDGF-BB-induced PDGF alpha-receptor phosphorylation and downstream signaling, with consequences for cellular responsiveness to the growth factor.     

Expression of biologically active human platelet-derived growth factor (PDGF) B-receptor in Xenopus laevis oocytes

Platelet-derived growth factor (PDGF) consists of three different isoforms, PDGF-AA, PDGF-AB and PDGF-BB, which bind to at least two types of receptors: the B-receptor, to which only PDGF-BB binds, and the A/B receptor, to which all three isoforms bind. Microinjection of synthetic mRNA in Xenopus laevis oocytes was used to obtain cell-surface expression of the human PDGF B-receptor. The production of receptor molecules of correct size (190 kd) was demonstrated by specific immunoprecipitation; the binding properties of the membrane- associated PDGF B-receptor were investigated with highly purified recombinant [125I] labeled human PDGF-BB and -AA. Unlike Swiss mouse 3T3 cells, which possess both B- and A/B-receptors and, therefore, bind both isoforms with high affinity, the mRNA-injected oocytes bound only the BB isoform. Mock-injected oocytes showed no specific binding.     

Growth-promoting effect of platelet-derived growth factor on rat cardiac myocytes

Platelet-derived growth factor (PDGF) is a dimeric molecule consisting of disulfide-bonded A- and B-polypeptide chains. Homodimeric (PDGF-AA, PDGF-BB) as well as heterodimeric (PDGF-AB) isoforms exert their effects on target cells by binding with different specificities to two structurally related protein tyrosine kinase receptors, denoted alpha- and beta-receptors. PDGF stimulates growth in various cell types, but little is known about its effect on mammalian cardiomyocytes. Therefore, growth-promoting effect of PDGF on rat cardiomyocytes was investigated. Primary culture of neonatal rat ventricular myocytes was prepared and cellular growth was estimated by [3H]-leucine incorporation assay. Tyrosine-phosphorylated PDGF-beta receptor of cardiomyocytes was determined by immunoblotting analysis after immunoprecipitation. PDGF-beta receptor, extracellular signal-regulated kinase (ERK) 1/2 and phosphorylated ERK1/2 of cardiomyocytes were measured by immunoblotting analysis. [3H]-leucine incorporation into the cultured myocytes was increased in a time- and dose-dependent manner after PDGF-BB stimulation. Phosphorylation of PDGF-beta receptor and ERK1/2 in cardiomyocytes was increased after short-term stimulation of PDGF-BB. Protein expression of PDGF-beta receptor and ERK1/2 was increased after long-term stimulation of PDGF-BB. [(3)H]-leucine incorporation into the cultured myocytes induced by PDGF-BB was partly blocked by mitogen-activated ERK-activating kinase (MEK) inhibitor PD98059, phospholipase C (PLC) inhibitor U73122, and protein kinase C (PKC) inhibitor staurosporin aglycone, respectively. Therefore, PDGF beta receptor, ERK1/2, PLC and PKC are involved in the signal transduction of PDGF-induced growth response of rat cardiac myocytes.     

Calcium and growth responses of hyperresponsive airway smooth muscle to different isoforms of platelet-derived growth factor (PDGF)

Airway smooth muscle (ASM) mass is likely to be an important determinant of airway responsiveness. Highly inbred Fisher rats model innate hyperresponsiveness, and also have more ASM in vivo than control Lewis rats. Platelet derived growth factor (PDGF) is an important endogenous growth factor for ASM, and partially purified PDGF-AB causes enhanced growth of Fisher rat ASM cells, compared to Lewis cells. The aim of the present study was to determine the mitogenic effects of all three recombinant PDGF isoforms on ASM cells, and investigate the mechanisms of enhanced Fisher ASM growth responses. The potential mechanisms assessed include PDGF receptor expression and activation (tyrosine phosphorylation), and intracellular calcium (Ca2+) responses to PDGF isoforms. Fisher ASM cells had a greater mitogenic response to PDGF-AB and -AA, and a greater Ca2+ response to -BB than Lewis ASM cells. A Ca2+ response was not necessary for a mitogenic response, and the effects of PDGF isoforms on Ca2+ were not associated with their effects on growth. Therefore, we suggest that enhanced Fisher mitogenic response to PDGF-AA and -AB is not mediated by differences in Ca2+ signalling. Western analysis of the PDGF receptor indicated a similar expression of beta-PDGF receptor in ASM cells from the two rat strains, but a greater expression of alpha-PDGF receptor in Fisher cells; however, phosphorylation of the PDGF receptor following growth stimulation did not differ between strains. This suggests a role for post-receptor signals, in addition to enhanced receptor expression, in the enhanced growth response of Fisher ASM cells to PDGF-AA and -AB.     

Differential regulation of expression of two platelet-derived growth factor receptor subunits by transforming growth factor-beta

The binding of three radiolabeled isoforms of platelet-derived growth factor (PDGF), 125I-PDGF-AA, 125I-PDGF-AB, and 125I-PDGF-BB, is differentially affected by exposure of quiescent 3T3 cells to transforming growth factor-beta (TGF-beta). By 24 h after exposure to TGF-beta, binding of 125I-PDGF-AA and 125I-PDGF-AB is almost completely lost, whereas binding of 125I-PDGF-BB is reduced by only 40%. The loss of PDGF-binding sites caused by TGF-beta is time- and concentration-dependent and reflects a change in the pattern of expression of receptor subunits; the number of alpha-subunits decreases, and the number of beta-subunits increases. The loss of binding sites for PDGF-AA is accompanied by a decreased mitogenic response to PDGF-AA but not to PDGF-AB or PDGF-BB. These results suggest that TGF-beta may differentially regulate the expression of PDGF-binding sites and the mitogenic responsiveness toward the three PDGF isoforms. TGF-beta did not stimulate synthesis of PDGF A-chain mRNA or PDGF-AA protein, and PDGF-AA receptors could not be restored by the presence of suramin, suggesting that the loss of binding sites may result from direct effects on receptor expression rather than autocrine down-regulation by PDGF-AA.