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.     

Different effects of homo- and heterodimers of platelet-derived growth factor A and B chains on human and mouse fibroblasts.

Binding sites for platelet-derived growth factor (PDGF) differ in their selectivity for the AA, AB and BB forms of PDGF. Human fibroblasts bind BB well and AA poorly, whereas Swiss 3T3 cells bind more similar quantities of each ligand. We found that AA PDGF was weakly mitogenic for human fibroblasts, but strongly mitogenic for 3T3 cells. Tyrosine phosphorylation of human fibroblast receptors was stimulated most by BB and least by AA, whereas the phosphorylation of 3T3 cell receptors was stimulated more uniformly by the three dimers. The receptor polypeptides that were phosphorylated were very similar. We suggest that phosphorylation of the receptor is proportional to the number of binding sites available for each ligand. Tyrosine phosphorylation of a number of other cell proteins was also proportional to receptor phosphorylation. In contrast, protein kinase C (PKC)-dependent serine and tyrosine phosphorylations were stimulated maximally by low level occupancy of PDGF binding sites, and phosphorylation of p36 required high occupancy. These data raise the possibility that differences in biological potency of AA, AB and BB forms of PDGF may be due simply to differences in the numbers of binding sites, rather than to different biochemical functions of their receptors.     

Differential regulation of the expression of proteinases/antiproteinases in fibroblasts. Effects of interleukin-1 and platelet-derived growth factor.

We have previously reported that polypeptide growth factors had an anti-inflammatory effect by decreasing the cytokine-enhanced expression of factor B (FB), an activator of the alternative complement pathway, in human fibroblasts. To further characterize the role of cytokines and growth factors in the inflammatory/repair continuum, we have studied the effects of interleukin-1 (IL-1) and platelet-derived growth factor (PDGF) on the expression of metalloproteinases/antiproteinases of the extracellular matrix in cultured human fibroblasts. Co-incubation of IL-1 and PDGF synergistically increased the expression of stromelysin and interstitial collagenase to 23-fold (for both proteins) over background, while PDGF decreased the IL-1-enhanced expression of FB by 82%. PDGF, but not IL-1, alone or in combination, increased the synthesis of tissue inhibitor of metalloproteinases. RNA blot analysis indicated that the changes in protein synthesis were regulated at a pretranslational level. Cycloheximide treatment indicated that the effects of PDGF on the metalloproteinases/antiproteinases were not protein-dependent, in contrast to results obtained for FB. The effect of the three dimeric forms of PDGF (AA, AB, and BB) on the synthesis of metalloproteinases and FB was also analyzed. The effects were qualitatively similar for each of the dimeric forms; however, the BB and AB isoforms had considerably greater effects than PDGF-AA. It has been reported that the PDGF receptors found in human fibroblasts have higher binding affinity for the BB and AB isoforms of the growth factor. The results presented in this paper are in accord with the possibility that differences in the biological activity of the three isoforms of PDGF are due to differences in the number or affinity of the binding sites of the target cells, rather than to different activation pathways of the receptor. Thus, PDGF increased cytokine effects on metalloproteinases, while decreasing cytokine effects or complement activator FB. The net effect of these changes may be to decrease inflammation and enhance remodeling early in repair and to enhance matrix stability later in the repair process.     

Comparison of PDGF-AA- and PDGF-BB-Induced Phosphoinositide Formation in Human and Mouse Fibroblasts

In certain cells, such as human fibroblasts (AG 1523), there is a clear difference in the cell motility response induced by the different isoforms of platelet-derived growth factor (PDGF). PDGF-BB induces extensive actin reorganization and is a potent chemotactic agent, whereas PDGF-AA has a limited effect on actin reorganization and is not chemotactic. In the present study, we wanted to compare these effects on cell motility with the effects of the PDGF isoforms on phosphoinositide (PtdIns) turnover. We find that stimulation of serum-starved AG 1523 cells with PDGF-AA or PDGF-BB caused an initial increase of the phosphatidylinositol phosphate and bisphosphate (PtdInsP and PtdInsP₂) pools, suggesting that activation of the phosphoinositide kinases is an initial response to PDGF stimulation. Despite a lower number of PDGF α-receptors than β-receptors on these cells, the initial formation of PtdInsP and PtdInsP₂ appears to be stimulated to a similar degree by the two PDGF isoforms. In contrast, PtdInsP₂ hydrolysis, indirectly measured as formation of phosphatidic acid, was correlated to the number of receptors. During prolonged exposure to PDGF-BB the stimulated PtdIns turnover remained at a high level, whereas the effect of PDGF-AA appeared more transient. A marked increase in the synthesis of a component migrating as phosphatidylinositol trisphosphate (PtdInsP_a) was also detected after stimulation with PDGF-BB for 5 min. With PDGF-AA minor amounts were found, indicating that activation of the PtdIns 3′-kinase occurs also via the PDGF α-receptor. Stimulation with PDGF-BB, but not -AA, also induced a 50% decrease in lyso-PtdIns. In murine fibroblasts (Swiss 3T3), where the two PDGF isoforms have a similar effect on cell motility, the two PDGF isoforms also similarly induced PtdIns turnover, PtdInsP₃ formation, and a decrease in lyso-PtdIns. Thus, there seems to be a correlation between PDGF-induced PtdIns turnover and PDGF-induced actin reorganization. This is compatible with previous evidence suggesting the microfilament formation is directly linked to an increased turnover of polyphosphoinositides in stimulated cells.     

Biochemical and functional discrimination of platelet-derived growth factor alpha and beta receptors in BALB/c-3T3 cells.

Three biologically active isoforms of platelet-derived growth factor (PDGF) exist: PDGF-AB, the predominant form in human platelets; PDGF-BB, the product of the c-sis protooncogene; and PDGF-AA. PDGF-BB and PDGF-AB interact with two distinct PDGF receptors (termed alpha and beta) of similar size, whereas PDGF-AA binds alpha receptors only. To dissect alpha and beta receptor-mediated signals, we compared the biological activities of PDGF-AA and PDGF-BB in density-arrested BALB/c-3T3 cells, which possess a 4:1 ratio of beta to alpha receptors, and assessed the contribution of alpha receptors to PDGF-BB- and PDGF-AB-induced responses. In addition, we describe a convenient method for resolving alpha and beta receptors on one-dimensional protein gels. This protocol involves treatment of cells with neuraminidase, a desialylating agent, and subsequent in vitro autophosphorylation of solubilized cells, and was used to monitor the presence or absence of alpha and beta receptors under various experimental conditions. Our data show that although higher concentrations were required, PDGF-AA stimulated DNA synthesis to the same extent as did PDGF-BB. Both isoforms induced inositol phosphate formation, epidermal growth factor transmodulation, and PDGF receptor autophosphorylation; PDGF-AA, however, was less effective than was PDGF-BB even at doses causing maximal mitogenesis. Pretreatment of cells with PDGF-AA for 30-60 min at 37 degrees C effectively down-regulated alpha receptors as verified by the absence of desialylated alpha receptor phosphorylation. Depletion of alpha receptors did not affect the capacity of PDGF-BB or PDGF-AB to activate the beta receptor tyrosine kinase, as assessed by tyrosine phosphorylation of an endogenous substrate, or stimulate the formation of inositol phosphates. We suggest that alpha and beta receptors independently mediate similar biological responses in BALB/c-3T3 cells, and that alpha receptors are not required for responses induced by PDGF-BB or PDGF-AB.     

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.     

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.     

Ligand activation causes a phosphorylation-dependent change in platelet-derived growth factor receptor conformation

The effect of ligand binding on platelet-derived growth factor (PDGF) receptor conformation was examined using peptide antibodies directed against specific receptor domains. Antiserum 83, which was directed to the receptor's carboxyl terminus (residues 934-951), preferentially immunoprecipitated the ligand-activated form of the PDGF receptor from 35S-labeled BALB/c 3T3 cells. By contrast, two antisera directed against other receptor sequences precipitated unactivated and activated receptors equally well. Denatured receptors were recognized equally by all antisera, even 83. Thus, ligand activation caused a change in PDGF receptor conformation that enhanced accessibility of the antibody to the carboxyl terminus. The activated receptor conformation was induced by three different forms of PDGF (AA and BB homodimers and AB heterodimers) and was reversed by suramin, a polyanionic compound that dissociates PDGF from the receptor. The inhibitory effect of suramin on receptor conformation was abolished by the phosphatase inhibitor, sodium orthovanadate, suggesting that receptor phosphorylation mediated the conformational change. In a cell-free assay, the change in receptor conformation was induced by PDGF only in the presence of ATP and was inhibited by adenyl-5'-yl imidodiphosphate, a nonhydrolyzable analog of ATP. The functional significance of receptor conformation was examined in Chinese hamster ovary fibroblasts transfected with wild-type or mutated forms of the PDGF receptor. When receptor tyrosine kinase activity was abolished by a mutation of the ATP binding site the receptor no longer underwent PDGF-induced conformational change and did not mediate PDGF-induced mitogenesis even though 125I-PDGF binding was normal. These findings show that ligand binding elicits a phosphorylation-dependent change in PDGF receptor conformation that may be important for receptor function.     

Binding of different dimeric forms of PDGF to human fibroblasts: evidence for two separate receptor types

The binding of the three dimeric forms of platelet-derived growth factor (PDGF), PDGF-AA, PDGF-AB and PDGF-BB, to human fibroblasts was studied. Cross-competition experiments revealed the existence of two different PDGF receptor classes: the type A PDGF receptor bound all three dimeric forms of PDGF, whereas the type B PDGF receptor bound PDGF-BB with high affinity and PDGF-AB with lower affinity, but not PDGF-AA. The sizes of the two receptors were estimated with affinity labeling techniques; the A type receptor appeared as a major component of 125 kd and a minor of 160 kd, and the B type receptor as two components of 160 and 175 kd. A previously established PDGF receptor monoclonal antibody, PDGFR-B2, was shown to react with the B type receptor only. The different abilities of the three dimeric forms of PDGF to stimulate incorporation of [3H]TdR into human fibroblasts indicated that the major mitogenic effect of PDGF is mediated via the B type receptor.     

Interrelationships of platelet-derived growth factor isoform-induced changes in c-fos expression, intracellular free calcium, and mitogenesis

Both increases in c-fos proto-oncogene expression and intracellular free calcium ([Ca2+]i) have been implicated as necessary components of the signal transduction pathway by which platelet-derived growth factor (PDGF) stimulates DNA synthesis in cultured BALB/c3T3 fibroblasts. To determine the interrelationship between PDGF-induced increases in c-fos proto-oncogene expression and [Ca2+]i, purified, recombinant BB and AA homodimeric isoforms of PDGF were used to evaluate the dose-response relationships and mechanisms of growth factor-induced changes in these two parameters as well as DNA synthesis. Concentration-dependent increases in [Ca2+]i, c-fos expression, and [3H]thymidine incorporation were observed with both BB and AA PDGF isoforms. BB PDGF was consistently more potent and efficacious than the AA isoform in eliciting a given response. The [Ca2+]i dependency of PDGF-induced increases in c-fos expression and DNA synthesis was determined by pretreatment of cells with agents that inhibit increases in [Ca2+]i: BAPTA, Quin-2, and TMB-8. Under these conditions, PDGF-induced DNA synthesis was blocked, whereas c-fos expression was enhanced. Conversely, in cells made deficient in protein kinase C (PKC) activity by prolonged treatment with phorbol ester, BB and AA PDGF-induced c-fos expression was inhibited by 75-80%, while PDGF-induced increases in [Ca2+]i and DNA synthesis were unaffected or enhanced. Additionally, the PKC-independent component of PDGF-stimulated c-fos expression was found to be independent of increases in [Ca2+]i. These data suggest that 1) both BB and AA PDGF isoforms elicit alterations in [Ca2+]i and c-fos proto-oncogene expression through the same or similar mechanisms in BALB/c3T3 fibroblasts, 2) PDGF-stimulated increases in [Ca2+]i are not required for c-fos expression, and 3) distinct pathways regulate PDGF-induced c-fos expression and mitogenesis, with c-fos expression being substantially PKC-dependent yet [Ca2+]i-independent, while mitogenesis is [Ca2+]i-dependent yet PKC-independent.     

Platelet-derived growth factor and its role in health and disease

Platelet-derived growth factor (PDGF) was first discovered in platelets because they are the principal source of mitogenic activity in whole blood serum for mesenchymal cells in culture. PDGF is ubiquitous in that it can be formed by a large number of normal cells as well as many varieties of transformed cells. However, its expression and biological activity appear to be controlled at a number of different levels. The molecule consists of two peptide chains (termed 'A' and 'B') and is found as one of at least three possible isoforms, (AB, AA or BB). Each of these isoforms binds to a high-affinity cell-surface receptor that is composed of two different subunits, each of which has specificity for one or the other of the peptide chains of PDGF. The two receptor subunits are present in differing amounts on different cell types, and therefore the capacity of the different isoforms of PDGF to induce mitogenesis depends on the specific PDGF isoform and the relative numbers of receptor subunits present on the responding cell. In addition to inducing cell replication, PDGF elicits a number of intracellular signals related to mitogenesis, is chemotactic, is a vasoconstrictor, activates leukocytes, and modulates extracellular matrix turnover. This growth factor is probably involved in a number of biologically important events including wound repair, embryogenesis and development, and inflammation, leading to fibrosis, atherosclerosis and neoplasia.     

PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor

The term 'platelet-derived growth factor' (PDGF) refers to a family of disulphide-bonded dimeric isoforms that are important for growth, survival and function in several types of connective tissue cell. So far, three different PDGF chains have been identified - the classical PDGF-A and PDGF-B and the recently identified PDGF-C. PDGF isoforms (PDGF-AA, AB, BB and CC) exert their cellular effects by differential binding to two receptor tyrosine kinases. The PDGF alpha-receptor (PDGFR-alpha) binds to all three PDGF chains, whereas the beta-receptor (PDGFR-beta) binds only to PDGF-B. Gene-targeting studies using mice have shown that the genes for PDGF-A and PDGF-B, as well as the two PDGFR genes, are essential for normal development. Furthermore, overexpression of PDGFs is linked to different pathological conditions, including malignancies, atherosclerosis and fibroproliferative diseases. Here we have identify and characterize a fourth member of the PDGF family, PDGF-D. PDGF-D has a two-domain structure similar to PDGF-C and is secreted as a disulphide-linked homodimer, PDGF-DD. Upon limited proteolysis, PDGF-DD is activated and becomes a specific agonistic ligand for PDGFR-beta. PDGF-DD is the first known PDGFR-beta-specific ligand, and its unique receptor specificity indicates that it may be important for development and pathophysiology in several organs.     

Platelet-derived growth factor (PDGF) alpha receptor activation modulates the calcium mobilizing activity of the PDGF beta receptor in Balb/c3T3 fibroblasts.

In order to determine whether distinct platelet-derived growth factor (PDGF) receptors (alpha and beta) can modulate the activity of one another, PDGF isoform (AA, BB, and AB)-stimulated changes in Ca2+i were monitored by digitized video microscopy in single cells upon sequential addition of PDGF isoforms. In Balb/c 3T3 fibroblasts, all PDGF isoforms were capable of stimulating increases in Ca2+i of 200-600% above basal levels, although with different potencies: BB greater than or equal to AB greater than AA. All cells were BB-PDGF-responsive, but only 74% of cells examined responded to AA-PDGF. The Ca2+i response elicited by BB-PDGF was inhibited by 60-75% in cells stimulated 10 min earlier with the AA isoform. The half-life of this inhibition was 22 min. In cells in which the alpha receptor was down-regulated by prolonged incubation with AA-PDGF, BB-induced Ca2+i responses were not inhibited. Pretreatment of cells with phorbol ester did not inhibit BB-PDGF-induced increases in Ca2+i, yet down-regulation of PKC activity prevented the AA-PDGF inhibition of BB-PDGF-induced Ca2+i responses. An increase in Ca2+i induced by AlF(4-)-stimulated IP3 generation did not inhibit a subsequent BB-PDGF Ca2+i response; however, attenuation of AA-PDGF-induced extracellular Ca2+ influx with EGTA prevented the inhibition of BB-PDGF-induced Ca2+i increases. Readdition of Ca2+ to the medium after removal of EGTA restored the inhibition of the BB-PDGF Ca2+i response. The inhibition of the BB-PDGF Ca2+i response by AA-PDGF was not caused by inhibition of PDGF receptor tyrosine autophosphorylation, which was unchanged after pretreatment with AA-PDGF. These results demonstrate: (a) that only a subpopulation of cells possess a functional alpha receptor-mediated response as assessed by AA-PDGF-induced increases in Ca2+i, whereas all cells possess the beta receptor-mediated responses; and (b) AA-PDGF and its associated alpha receptor can modulate the activity of the beta receptor through a mechanism that is dependent upon Ca(2+)-influx which may be controlled in part by PKC activation.     

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.     

Platelet-derived growth factor AA homodimer stimulates protein synthesis rather than DNA synthesis in vascular smooth muscle cells from spontaneously hypertensive rats but not from normotensive rats.

Platelet-derived growth factor (PDGF) AB and BB isoforms were potent mitogens for cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). PDGF-AA promotes protein synthesis in a dose-dependent manner in SHR cells, whereas DNA synthesis was stimulated only slightly. However, this isoform did not activate either DNA or protein synthesis in WKY cells. PDGF-AA stimulated tyrosine phosphorylation of its receptor protein and phospholipase C-gamma 1 in SHR cell but not in WKY cells. These results indicate that vascular smooth muscle cell of SHR is uniquely responsive to PDGF-AA, presumably due to abnormality in receptor expression, in its hypertrophic response.     

Early changes in inositol lipids and their metabolites induced by platelet-derived growth factor in quiescent Swiss mouse 3T3 cells.

Inositol lipid turnover was studied in quiescent Swiss mouse 3T3 cells stimulated by platelet-derived growth factor (PDGF). Stimulation of the cells by PDGF for 10 min at 37 degrees C induced the following changes in lipids: in cells prelabelled with [32P]Pi, a 28% decrease in [32P]phosphatidylinositol 4,5-bisphosphate, a 41% decrease in [32P]phosphatidylinositol 4-phosphate and a 1.7-fold increase in the 32P-labelling of phosphatidic acid; in cells prelabelled with [3H8]arachidonic acid, a 17.9-fold increase in [3H]phosphatidic acid, a 20% decrease in [3H]phosphatidylinositol (PtdIns), an 8.6-fold increase in [3H]arachidonic acid released into the medium, a 57-fold increase in [3H]prostaglandin E2 in the medium, and a 5.3-fold increase in [3H]monoacylglycerol released into the medium (the last was identified as the 2-acyl derivative); in cells prelabelled with [2-3H]glycerol, a 1.7-fold increase in [3H]diacylglycerol, a 6.7-fold increase in [3H]phosphatidic acid, a 1.6-fold increase in [3H]lysophosphatidylcholine (lysoPtdCho), a 9% decrease in [3H]PtdIns, and a 1.6-fold increase in [3H]monoacylglycerol released into the medium. PDGF stimulated the formation of inositol tris-, bis- and mono-phosphates in the cells prelabelled with myo-[2-3H]inositol. These results indicate that, in Swiss 3T3 cells stimulated by PDGF, diacylglycerol produced by the hydrolysis of inositol lipids is partly degraded to 2-acylglycerol and partly converted into phosphatidic acid. The increase in lysoPtdCho indicates that a portion of arachidonic acid released from the stimulated cells is formed by the hydrolysis of PtdCho with a phospholipase A2. Different values of half-maximal doses of the partially purified PDGF used in this study were found for the various responses of quiescent Swiss 3T3 cells to PDGF. The values for half-maximal doses suggest that activation of a fraction of the cell-surface receptor for PDGF is sufficient for mitogenesis and for an increase in the cytoplasmic free Ca2+ concentration, and that the PGDF-stimulated lipid metabolism is probably proportional to the number of receptor sites activated by PDGF.     

Mitogen response and cell cycle kinetics of Swiss 3T3 cells in defined medium: differences from human fibroblasts and effects of cell density

The mitogen requirement and proliferative response of Swiss 3T3 cells in serum-free, chemically defined culture medium were compared with those of early-passage human diploid fibroblasts. The effects of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, transferrin, and dexamethasone on cell-cycle parameters were measured using 5'-bromo-deoxyuridine-Hoechst flow cytometry. Swiss 3T3 cells differ from human fibroblasts in several ways: (1) Swiss 3T3 cells showed a much higher dependence on PDGF than human fibroblasts; the growth of the latter, but not of the former, could be stimulated by the combination of EGF, insulin, and dexamethasone to the full extent of that when PDGF was present; (2) in the absence of PDGF, insulin was an absolute requirement for Swiss 3T3 cells to initiate DNA synthesis, while a substantial proportion of human fibroblasts could enter DNA synthesis without exogenous insulin or IGF-I; and (3) in the absence of PDGF, increasing insulin concentration increased the cycling fraction of Swiss 3T3 cells without an appreciable effect on the rate of cell exit from G0/G1, while under similar culture conditions, insulin showed its major effect on regulation of the G1 exit rate of human fibroblasts, without much effect on the cycling fraction. In addition, the proliferative response of high-density versus low-density, arrested Swiss 3T3 cells showed that the interaction of mitogens varied with cell density. At high cell density, the PDGF requirement was consistent with the "competence/progression" cell-cycle model. This growth response was not seen, however, when cells were plated at low density.     

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.     

Expression of PDGF and their receptors in human retinal pigment epithelial cells and fibroblasts: regulation by TGF-beta

Platelet derived growth factors (PDGF) are known to be associated with vitreoretinal disorders such as proliferative vitreoretinopathy (PVR). We have studied the expression of PDGF and their receptors in human retinal pigment epithelial cells (HRPE) and choroid fibroblasts (HCHF), and the regulation of PDGF and its receptors by various cytokines and growth factors. RT-PCR analyses showed enhanced expression of PDGF-A and PDGF-B mRNA in HRPE treated with TGF-beta, but not with other cytokines. A minimal increase was observed in PDGF-A mRNA in TGF-beta treated HCHF cells. PDGF-R alpha mRNA, which was expressed prominently in HCHF and at very low levels in HRPE, was not affected by any of the agents. PDGF-R beta was not detectable in either HRPE or HCHF. HRPE secreted PDGF-AA and AB constitutively, and this secretion was significantly enhanced by TGF-beta. In contrast, HCHF cultures did not secrete detectable levels of any of the three isoforms of PDGF (AA, AB, BB). All three human recombinant PDGF isoforms enhanced HCHF cell proliferation significantly, while only a minimal increase was observed in HRPE. PDGF isoforms also induced HCHF cell elongation and promoted migration of HCHF in an in vitro wound assay. The results presented in this study demonstrate that TGF-beta activated RPE cells produce PDGF that may act on fibroblasts and other mesenchyme derived cells which express PDGF receptors. These studies indicate that the promotion of the proliferation and migration of mesenchymal cells by RPE cell derived PDGF may facilitate the formation of fibrovascular tissues associated with PVR.