PDGF-AA Binding Research Articles

Abstract C56: Characterization of hz515H7, a novel humanized anti-CXCR4 antibody: In vitro efficacy on CXCR4-associated signaling pathways and in vivo antitumor activity.

Abstract C56: Characterization of hz515H7, a novel humanized anti-CXCR4 antibody: In vitro efficacy on CXCR4-associated signaling pathways and in vivo antitumor activity.

TMEFF2 Is a PDGF-AA Binding Protein with Methylation-Associated Gene Silencing in Multiple Cancer Types Including Glioma

BackgroundTMEFF2 is a protein containing a single EGF-like domain and two follistatin-like modules. The biological function of TMEFF2 remains unclear with conflicting reports suggesting both a positive and a negative association between TMEFF2 expression and human cancers.Methodology/Principal FindingsHere we report that the extracellular domain of TMEFF2 interacts with PDGF-AA. This interaction requires the amino terminal region of the extracellular domain containing the follistatin modules and cannot be mediated by the EGF-like domain alone. Furthermore, the extracellular domain of TMEFF2 interferes with PDGF-AA–stimulated fibroblast proliferation in a dose–dependent manner. TMEFF2 expression is downregulated in human brain cancers and is negatively correlated with PDGF-AA expression. Suppressed expression of TMEFF2 is associated with its hypermethylation in several human tumor types, including glioblastoma and cancers of ovarian, rectal, colon and lung origins. Analysis of glioma subtypes indicates that TMEFF2 hypermethylation and decreased expression are associated with a subset of non-Proneural gliomas that do not display CpG island methylator phentoype.Conclusions/SignificanceThese data provide the first evidence that TMEFF2 can function to regulate PDGF signaling and that it is hypermethylated and downregulated in glioma and several other cancers, thereby suggesting an important role for this protein in the etiology of human cancers.

High-affinity Binding of Basic Fibroblast Growth Factor and Platelet-derived Growth Factor-AA to the Core Protein of the NG2 Proteoglycan

NG2 is a transmembrane chondroitin sulfate proteoglycan that is expressed by immature progenitor cells in several developmental lineages and by some types of malignant cells. In vitro studies have suggested that NG2 participates in growth factor activation of the platelet-derived growth factor-alpha receptor. In this study the ability of recombinant NG2 core protein to interact with several different growth factors (epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF)165 and transforming growth factor (TGF)-beta1) was investigated using two different assay systems: enzyme-linked immunosorbent assay-type solid-phase binding and an optical biosensor (BIAcore) system. High-affinity binding of bFGF and PDGF-AA to the core protein of NG2 could be demonstrated with both types of assays. Using both the BIAcore software analysis program and nonlinear regression analysis of the solid phase binding data, KD values in the low nanomolar range were obtained for binding of each of these growth factors to NG2. The results further indicate that NG2 contains at least two binding sites for each of these two growth factors. PDGF-BB, TGF-beta1, VEGF, and EGF exhibited little or no binding to NG2 in either type of assay. These data suggest that NG2 can have an important role in organizing and presenting some types of mitogenic growth factors at the cell surface.

Prostaglandin-E2 counteracts interleukin-1beta-stimulated upregulation of platelet-derived growth factor alpha-receptor on rat pulmonary myofibroblasts.

The platelet-derived growth factor (PDGF) alpha-receptor (PDGF-Ralpha) is upregulated during lung fibrogenesis, and induction of PDGF-Ralpha on cultured lung myofibroblasts by interleukin (IL)-1beta results in an increased mitogenic response to PDGF. Because IL-1beta stimulates prostaglandin (PG) E2 production, we investigated whether IL-1beta could upregulate PDGF-Ralpha via a PGE2-dependent mechanism. IL-1beta increased the production of PGE2 by rat lung myofibroblasts and the cyclooxygenase (COX) inhibitor indomethacin blocked IL-1beta-induced PGE2 production. However, indomethacin did not inhibit IL-1beta-stimulated upregulation of [125I]PDGF-AA binding sites, indicating that PDGF-Ralpha induction does not require PGE2 synthesis. Instead, PGE2 downregulated PDGF-Ralpha protein and messenger RNA expression, and counteracted the IL-1beta-stimulated increase in [125I]PDGF-AA binding. Pretreatment of cells with indomethacin or the COX-2 specific inhibitor NS-398 attenuated the suppressive effect of exogenous PGE2 on PDGF-Ralpha, indicating that endogenous PGE2 released by IL-1beta treatment also contributed to downregulation of PDGF-Ralpha. PDGF-Rbeta expression was not altered by IL-1beta or PGE2. Pretreatment of myofibroblasts with IL-lbeta increased PDGF-stimulated mitogenesis, and this effect was blocked by coincubation with PGE2. In contrast, PGE2 enhanced epidermal growth factor- or basic fibroblast growth factor-2-stimulated cell proliferation approximately 50%. Because IL-1beta upregulates both PGE2 production and PDGF-Ralpha expression, these data suggest that PGE2 functions in a negative feedback loop to limit expression of PDGF-Ralpha and suppress PDGF-stimulated myofibroblast proliferation.

RP-1551s, a Family of Azaphilones Produced by Penicillium sp., Inhibit the Binding of PDGF to the Extracellular Domain of Its Receptor.

Nine azaphilones designated RP-1551-1, -2, -3, -4, -5, -6, -7, -M1, and -M2 were isolated from the culture broth of Penicillium sp. SPC-21609 as inhibitors of PDGF binding to its receptor. RP-1551s inhibit the binding of PDGF AA to the extracellular domain of PDGF alpha-receptor with IC50 values ranging from 0.1 to 2 microM without affecting PDGF BB binding to the extracellular domain of PDGF beta-receptor. PDGF binding was not restored after the PDGF alpha-receptor extracellular domain was washed in an attempt to remove the RP-1551-1 bound to the receptor. This result suggests that RP-1551-1 may irreversibly interact with the PDGF alpha-receptor. Since many azaphilone compounds possess high reactivity with an amino group, RP-1551-1 may prevent PDGF AA binding by reacting with amino groups on the alpha-receptor extracellular domain.

Role of Nuclear Factor-κB and Mitogen-Activated Protein Kinase Signaling Pathways in IL-1β-Mediated Induction of α-PDGF Receptor Expression in Rat Pulmonary Myofibroblasts

AbstractInduction of the α-platelet-derived growth factor receptor (PDGF-Rα) by IL-1β in lung myofibroblasts enhances mitogenic and chemotactic responses to PDGF, and this could be a mechanism of myofibroblast hyperplasia during lung fibrogenesis. Since the regulation of many genes by IL-1β involves activation of NF-κB and mitogen-activated protein (MAP) kinases, we examined these signaling pathways in the control of PDGF-Rα expression by IL-1β in cultured rat lung myofibroblasts. Treatment of cells with pyrrolidine dithiocarbamate (PDTC), an antioxidant that inhibits NF-κB activation, completely blocked PDGF-Rα up-regulation by IL-1β as assayed by [125I]PDGF-AA binding and PDGF-Rα mRNA expression, suggesting a role for NF-κB. However, while IL-1β and TNF-α both induced nuclear binding of the Rel proteins p50 and p65 to an NF-κB consensus oligonucleotide in gel shift assays and caused transient degradation of inhibitor of NF-κB-α (IκB-α) in the cytoplasm of myofibroblasts, only IL-1β up-regulated PDGF-Rα. These results suggest that NF-κB activation alone is not sufficient for up-regulation of PDGF-Rα. An investigation of MAP kinase signaling pathways revealed that IL-1β or PDTC activated extracellular signal-regulated kinase-2 (ERK-2) and c-jun NH2 terminal kinase-1 (JNK-1) phosphorylation of PHAS-1 and c-Jun substrates, respectively. Pretreatment of cells with the MAP kinase kinase-1 (MEK1) inhibitor PD 98059 blocked IL-1β-induced activation of ERK-2 by more than 90% but enhanced IL-1β-stimulated induction of PDGF-Rα expression fourfold. Taken together, these data suggest that IL-1β activates both positive and negative signaling pathways that control the expression of PDGF-Rα. IL-1β appears to mediate its negative effects on PDGF-Rα expression via MAP kinase activation, while the factor(s) that mediate induction of PDGF-Rα remain to be elucidated.

PDGF-alpha receptor subunit expression down-regulated by IL-1beta in human periodontal ligament cells.

The responses of cells to the distinct PDGF isoforms have been correlated directly to the relative numbers of specific PDGF receptor subunits on the cell surface. The modulation of PDGF-alpha receptor subunits, the major subunit expressed in human periodontal ligament (PDL) cells, by cytokines present in the periodontal wound site, such as interleukin-1 (IL-1), may be an important factor influencing regenerative outcomes. The purpose of the present study was to examine the effects of IL-1 beta on PDGF-alpha receptor subunit expression in human PDL cells. Primary cultures of human PDL cells were treated with IL-1 beta over a range of concentrations. We assessed PDGF-alpha receptor subunits by examining the mitogenic responses of cells to PDGF-AA, specific binding of 125I-labeled PDGF-AA, immunofluorescent analysis of PDGF-alpha receptor subunits, and PDGF-alpha receptor subunit mRNA levels using Northern blot analysis. The results demonstrate a significant concentration-dependent decrease in 3H-thymidine incorporation in response to PDGF-AA following IL-1 beta treatment (p < 0.001). This decreased response correlated directly with IL-1-induced decreases in 125I-labeled PDGF-AA binding (p < 0.01), the numbers of immunolabeled PDGF-alpha receptor subunits, and in PDGF-alpha receptor subunit mRNA levels. However, when combined with TGF-beta, IL-1 beta did not show additional down-regulation in proliferative response to PDGF-AA or PDGF-alpha receptor subunits beyond that achieved with these factors individually. These experiments identify IL-1 beta, along with TGF-beta, as significant inhibitors of PDGF stimulation in human PDL cells, acting through the down-regulation of PDGF-alpha receptor subunit expression.

Role of Immunoglobulin-like Domains 2–4 of the Platelet-derived Growth Factor α-Receptor in Ligand-Receptor Complex Assembly

Platelet-derived growth factor (PDGF) is a dimeric protein that exerts its effects through tyrosine kinase alpha- and beta-receptors. The extracellular part of each receptor is composed of five Ig-like domains. Recombinant forms of alpha-receptor domains 1-4 (alphaRD1-4), 1-3 (alphaRD1-3), and 1 and 2 (alphaRD1-2) were prepared after expression in Chinese hamster ovary cells and were used to study the assembly of soluble ligand-receptor complexes. When incubated with micromolar concentrations of PDGF, both alphaRD1-3 and alphaRD1-4 formed complexes of 1:2 molar composition, i.e. one dimeric PDGF molecule bound two soluble receptors. alphaRD1-3, in contrast to alphaRD1-4, formed detectable 1:1 complexes under conditions of ligand excess. alphaRD1-4 displayed an increased ability to form 1:2 complexes as compared with alphaRD1-3 under conditions of limiting concentrations of ligand. We thus conclude that Ig-like domain 4-mediated receptor-receptor interactions contribute to 1:2 PDGF.alphaRD1-4 complex formation. Since alphaRD1-4 and alphaRD1-3 were equipotent in blocking binding of subnanomolar concentrations of PDGF to cell-surface receptors, we also conclude that this effect is predominantly achieved through formation of Ig-like domain 4-independent 1:1 ligand-receptor complexes. Finally, since alphaRD1-2 bound PDGF-BB with high affinity, whereas PDGF-AA was bound only with low affinity, we conclude that Ig-like domain 3 of the PDGF alpha-receptor contains epitopes of particular importance for PDGF-AA binding and that most of the PDGF-BB-binding epitopes reside in Ig-like domains 1 and 2.

Immunoglobulin-like Domain 4-mediated Receptor-Receptor Interactions Contribute to Platelet-derived Growth Factor-induced Receptor Dimerization

Platelet-derived growth factor (PDGF) is a dimeric growth factor that activates its tyrosine kinase receptor by inducing receptor dimerization. In this study, we investigated if receptor-receptor interactions, in addition to ligand-receptor interactions, contribute to the ligand-induced dimerization of the PDGF receptors. Analysis of two deletion mutants of the PDGF alpha-receptor indicated a role for Ig-like domain 4 in ligand-receptor or receptor-receptor interactions. When the fourth Ig-like domain of the PDGF alpha-receptor instead was replaced with the corresponding sequence of the stem cell factor receptor, the binding of PDGF-AA and -BB was not affected, nor was the ability to form homodimeric receptor complexes. This indicates that Ig-like domain 4 does not participate in ligand-receptor interactions. However, the chimeras did not form heterodimers with wild-type PDGF alpha- or beta-receptors. Together, these findings suggest that Ig-like domain 4 mediates specific receptor-receptor interactions. This notion was also supported by the finding that a soluble form of Ig-like domain 4 of the PDGF alpha-receptor acted as a PDGF alpha-receptor antagonist. We conclude that specific receptor-receptor interactions contribute to PDGF receptor dimerization in vivo and that complementary epitopes in Ig-like domain 4 mediate these interactions. Our experiments also identify Ig-like domain 4 as a target for PDGF antagonists.

Alveolar macrophages stimulated with titanium dioxide, chrysotile asbestos, and residual oil fly ash upregulate the PDGF receptor-alpha on lung fibroblasts through an IL-1beta-dependent mechanism.

Enhanced proliferation of fibroblasts is a primary characteristic of lung fibrosis. Macrophage-secreted platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for lung fibroblasts. The magnitude of the fibroblast PDGF response is dependent on the number of PDGF receptor alpha (PDGF-R alpha) relative to PDGF-R beta at the cell surface. We recently reported that upregulation of the PDGF-R alpha subtype by interleukin (IL)-1beta results in enhanced lung fibroblast proliferation in response to PDGF-AA, PDGF-AB, and PDGF-BB whereas transforming growth factor (TGF)-beta1 has the opposite effect. Both IL-1beta and TGF-beta1 are produced by particle-activated macrophages in vivo and in vitro. We studied the net effect of macrophage conditioned medium (MOCM), which contains both IL-1beta and TGF-beta1, on the expression of the lung fibroblast PDGF receptor system. MOCM obtained from unstimulated, titanium dioxide (TiO2)-, chrysotile asbestos-, or residual oil fly ash (ROFA)-exposed macrophages in vitro increased [125I]PDGF-AA binding 3-, 6-, 6-, and 20-fold, respectively. These increases correlated with increased PDGF-R alpha mRNA and protein expression as shown by northern and western assays. PDGF-AB and -BB-stimulated [3H]thymidine incorporation by fibroblasts was enhanced 5-, 5-, 10-, and 20-fold by pretreatment with MOCM from unstimulated, TiO2-, asbestos-, and ROFA-exposed macrophages, respectively. [125I]PDGF-AA binding experiments using the IL-1 receptor antagonist blocked the upregulatory effect of all MOCM samples. Latent TGF-beta1 present in MOCM was activated by acid treatment, inhibiting upregulation by approximately 60%, a result similar to experiments with IL-1beta and TGF-beta1 mixtures. Treatment with a TGF-beta neutralizing antibody restored full upregulatory activity to acidified MOCM. Thus activated macrophages increase lung fibroblast PDGF-R alpha primarily due to the secretion of IL-1beta. Intratracheal instillation of ROFA particles in rats induced a 2-fold increase in total lung PDGF-R alpha mRNA in vivo. These findings support the idea that macrophage-derived IL-1beta plays a key role in the initiation of a fibrotic response by increasing fibroblast PDGF-R alpha expression, thereby dramatically potentiating the mitogenic response to PDGF.

Basic fibroblast growth factor induces expression of the PDGF receptor-alpha on human bronchial smooth muscle cells.

Bronchial smooth muscle cell (SMC) hyperplasia is a key feature in the pathology of asthma. Platelet-derived growth factor (PDGF) isoforms are SMC mitogens. We investigated the effect of basic fibroblast growth factor (bFGF), transforming growth factor-beta 1 (TGF-beta 1), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha) on the PDGF receptor system on human bronchial SMC from three different donors. bFGF induced gene expression of the PDGF alpha-receptor (PDGF-R alpha) approximately threefold without altering the PDGF beta-receptor (PDGF-R beta). IL-1 beta and TNF-alpha did not affect the PDGF receptor system. TGF-beta 1 downregulated PDGF-R alpha mRNA approximately 60% without changing PDGF-R beta mRNA levels. Receptor assays showed that bFGF increased the [125I]PDGF-AA binding site approximately twofold, whereas TGF-beta 1 reduced [125I]PDGF-AA binding approximately 60%. TGF-beta 1, but not latent TGF-beta 1, counteracted the bFGF-induced increase in [125I]PDGF-AA binding. PDGF-AA-stimulated tyrosine phosphorylation on the PDGF-R alpha was enhanced after treatment with bFGF, bFGF pretreatment enhanced the mitogenic response of SMC to PDGF-AA and PDGF-AB. These findings suggest that upregulation of the PDGF-R alpha by bFGF could contribute to SMC hyperplasia during chronic airway inflammation in asthma.

Maximal PDGF-induced lung fibroblast chemotaxis requires PDGF receptor-alpha.

Alteration of the platelet-derived growth factor (PDGF) receptor system could be important in enhancing the mitogenic and chemotactic potential of lung fibroblasts during pulmonary fibrogenesis. We previously reported that interleukin-1 beta (IL-1 beta) upregulates the PDGF receptor-alpha (PDGFR-alpha) gene, and in this study we sought to establish the importance of the PDGFR-alpha relative to the PDGFR-beta in mediating a chemotactic response to PDGF-AA, -AB, and -BB. Pretreatment of fibroblasts for 24 h with IL-1 beta increased chemotaxis to all three PDGF isoforms. IL-1 beta pretreatment markedly increased the maximal number of 125I-labeled PDGF-AA binding sites but did not change the number of 125I-PDGF-AB or PDGF-BB sites. However, IL-1 beta increased 125I-PDGFR-AB affinity twofold. Neomycin (5 mM) was used as a PDGFR-alpha antagonist and completely blocked 125I-PDGF-AA binding and PDGF-AA-induced chemotaxis. The binding affinity of 125I-PDGF-AB and 125I-PDGF-BB was increased two-to threefold by neomycin, and chemotaxis to PDGF-AB and PDGF-BB was enhanced. These results define a role for the PDGFR-alpha as a regulatory receptor subtype that is necessary for PDGF isoforms to exert maximal chemotaxis.

Pro-inflammatory cytokines downregulate platelet derived growth factor-α receptor gene expression in human osteoblastic cells

Platelet derived growth factor (PDGF) is thought to play a significant role in bone repair and regeneration. We previously demonstrated that PDGF-AA binding can be modulated by interleukin-1 (IL-1). We now report that TNF-alpha significantly reduces PDGF-AA binding by decreasing the number of PDGF-alpha receptor subunits on the surface of normal human osteoblastic cells. This inhibition is likely due to a decrease in synthesis of PDGF-alpha receptors since TNF-alpha causes a relatively rapid decrease in PDGF-alpha receptor mRNA levels as determined by Northern blot analysis. The physiologic importance of this inhibition is demonstrated by a TNF-alpha induced decrease in PDGF-AA stimulated tyrosine kinase activity. When saturating concentrations of TNF-alpha were used, the addition of IL-1 further inhibited PDGF-AA binding and further decreased surface expression of PDGF-alpha receptors. In contrast, other mediators such as IL-6, PTH, 1,25(OH)2 vit D3, hydrocortisone, PGE2, bFGF, and IGF-1 had no effect. These results suggest that binding to the PDGF-alpha receptor is decreased by the strong pro-inflammatory cytokines such as IL-1 beta and TNF-alpha rather than as a general response to mediators important in bone resorption or bone formation. TNF-alpha and IL-1 are often co-expressed during destructive inflammatory processes. Thus, TNF-alpha and IL-1 may work in concert to limit the response of osteoblastic cells to PDGF-AA during periods of osseous inflammation.

Structural Role of Extracellular Domain 1 of α-Platelet-derived Growth Factor (PDGF) Receptor for PDGF-AA and PDGF-BB Binding

The purpose of this study was to bacterially express, purify, and refold combinations of the extracellular immunoglobulin (Ig)-like domains (2-3, 1-3, and 1-5) of the human alpha-platelet-derived growth factor receptor (alpha PDGFR) to characterize molecular interactions with its ligand, platelet-derived growth factor (PDGF). The far UV circular dichroism spectroscopy of the alpha-PDGFR extracellular domains (ECDs) revealed a predominantly beta-sheet protein, with a structure consistent with folded Ig-like domains. The addition of PDGF-BB to these ECD types changed the conformation of all three types with a decrease in mean residue ellipticity in the following rank order: 1-5 = 1-3 > 2-3. In striking contrast, addition of PDGF-AA to these ECD types markedly changed the conformation of ECD 2-3, by an increased mean residue ellipticity but no changes were observed for ECDs 1-3 and 1-5. PDGF-AA bound to the immobilized ECD types 2-3, 1-3, and 1-5 at concentrations of 20, 11, and 7.5 nM, respectively. In contrast, PDGF-BB bound the ECD types 2-3, 1-3, and 1-5 at concentrations of 3, 3, and 2.2 nM, respectively. Scatchard analysis of binding studies using labeled ECDs indicated that PDGF-BB bound ECD 1-3 and ECD 2-3 with KD values of 74 and 72 nM, respectively. While, PDGF-AA bound ECD 1-3 and ECD 2-3 with KD values of 33 and 87 nM, respectively. Therefore, our results indicated that the loss of ECD 1 impaired the binding affinity of alpha PDGFR ECD 1-3 toward PDGF-AA without having a similar effect on PDGF-BB binding. Together all of our data suggest that ECD 1 is differentially required for proper orientation of PDGF-AA but not PDGF-BB binding determinant within ECDs 2 and 3.

Interleukin 1 beta (IL-1 beta) and the IL-1 beta-alpha 2-macroglobulin complex upregulate the platelet-derived growth factor alpha-receptor on rat pulmonary fibroblasts.

Fibroblasts are the primary proliferating cell type in pulmonary fibrosis. We previously showed that inorganic, fibrogenic particles alter the platelet-derived growth factor (PDGF) receptor system on rat lung fibroblasts (Bonner, J. C., et al. 1993, J. Clin. Invest 92:425-430). In lung fibroblasts, PDGF is the most potent proliferative cytokine, and the responses to PDGF isoforms depend on the relative amounts of two PDGF receptors (PDGF-R alpha and PDGF-R beta). Interleukin 1 beta (IL-1 beta) production by lung macrophages is increased following exposure to fibrogenic particles. We have examined the role of IL-1 beta in regulating the lung fibroblast PDGF receptor system. IL-1 beta induced a 10-fold increase in the number of binding sites for [125I]PDGF-AA, caused a 2-fold increase in affinity of [125I]PDGF-AB, but it had no effect on [125I]PDGF-BB binding. PDGF-R alpha gene expression was increased 5-fold after 4 h of IL-1 beta treatment. IL-1 beta increased the proliferative and chemotactic response to PDGF isoforms in the following order of potency: AA > AB > BB. IL-1 beta was tested for its ability to cause increased [125I]PDGF-AA binding when complexed to its binding protein, alpha 2-macroglobulin (alpha 2M). IL-1 beta bound covalently to fast methyl-amine-activated alpha 2M (alpha 2M-MA). IL-1 beta-alpha 2M-MA or alpha 2M-MA alone possessed minimal activity for inducing an increase in [125I]PDGF-AA binding. However, treatment of the IL-1 beta-alpha 2M complex with thioredoxin, which released bioactive IL-1 beta that was covalently bound to alpha 2M, maximally increased [125I]PDGF-AA binding to the same extent as free IL-1 beta. These results indicate that the fibroblast response to PDGF isoforms is modulated by a complex interaction involving IL-1 beta, alpha 2M, and thioredoxin, all of which are produced in vivo by activated macrophages.

Transforming growth factor beta 1 downregulates the platelet-derived growth factor alpha-receptor subtype on human lung fibroblasts in vitro.

Fibroblasts are the central target cell in pulmonary fibrotic diseases, and their proliferation is mediated largely by platelet-derived growth factor (PDGF) isoforms secreted by activated lung macrophages. Several other macrophage-derived cytokines that are increased during fibrogenesis, including interleukin-1 beta and transforming growth factor-beta 1 (TGF-beta 1), could potentially modulate the mitogenic and chemotactic activity of PDGF by altering the expression of cell-surface PDGF receptors on fibroblasts. The PDGF receptor system on fibroblasts from a variety of tissues shows heterogeneous responses to TGF-beta 1. Lung fibroblasts have not been investigated in this regard. TGF-beta 1 downregulated the gene expression of the 6.5 kb PDGF-alpha receptor (PDGF-R alpha) transcript in normal human lung fibroblasts in a concentration-dependent fashion that was maximal at 3 ng/ml TGF-beta 1; this corresponded with a decrease in cell-surface PDGF-R alpha as measured by radioligand binding assays using [125I]PDGF-AA. The TGF-beta 1-induced down-regulation of the PDGF-R alpha gene was rapid (maximal suppression by 2 h post-treatment) and preceded the decrease in cell-surface alpha-receptor (maximal reduction by 6 h post-treatment). TGF-beta 1 treatment did not alter the rate of PDGF-R alpha mRNA degradation following the inhibition of transcription using actinomycin D, indicating that TGF-beta 1 increases PDGF-R alpha transcription. Scatchard analysis of saturation binding data showed that TGF-beta 1 decreased the number of [125I]PDGF-AA binding sites 5-fold without affecting receptor affinity. [125I]PDGF-AB binding sites were downregulated approximately 25%, and the number of [125I]PDGF-BB binding sites was not changed by TGF-beta 1 treatment, indicating that the PDGF-beta receptor was not affected. TGF-beta 1 reduced the mitogenic and chemotactic response to PDGF-AA by > 90%, whereas these biologic response to PDGF-AB and PDGF-BB were inhibited 50% to 80%. The proliferative and chemotactic responses of fibroblasts during tissue remodeling or during lung fibrosis are likely controlled by a complex network involving PDGF isoforms and cytokines that modify the PDGF receptor system.

Receptor binding of PDGF-AA and PDGF-BB, and the modulation of PDGF receptors by TGF-beta, in human periodontal ligament cells.

The growth factors PDGF-AA and PDGF-BB have previously been shown to be potent mitogens for human periodontal ligament (hPDL) cells in vitro. Additionally, the mitogenic response to PDGF-AA has been shown to be specifically inhibited by TGF-beta. The purpose of the present investigation was to examine the binding of PDGF-AA and PDGF-BB, and the modulation of PDGF binding by TGF-beta, in hPDL cells. Scatchard analysis identified an average of 32,000 PDGF-AA high-affinity binding sites per cell with a dissociation constant (Kd) of 0.66 nM and an average of 36,000 PDGF-BB binding sites per cell with a dissociation constant (kd) of 0.44 nM. After treatment with TGF-beta, the receptor number for PDGF-AA was found to specifically decrease by approximately 50%, with no change in binding affinity. This reduced number of binding sites was shown to correlate with both a decrease in levels of receptor tyrosine phosphorylation and a decreased number of alpha receptor subunits. Northern blot analysis identified the TGF-beta-mediated decrease in PDGF alpha receptor subunit mRNA levels. PDGF-BB showed little change in the number of binding sites or in the binding affinity with TGF-beta treatment, and the data were consistent with an increase in the number of beta receptor subunits. These results demonstrate nearly equivalent numbers of receptors for both PDGF-AA and PDGF-BB in hPDL cells. Also, modulation of PDGF binding, by TGF-beta, was shown to result in a reduced number of alpha receptor subunits with an increase in the number of beta receptor subunits.

Importance of alternative exon usage for the binding of PDGF-AA to heparin-like glycosaminoglycans

Importance of alternative exon usage for the binding of PDGF-AA to heparin-like glycosaminoglycans

Structural coincidence of alpha PDGFR epitopes binding to platelet-derived growth factor-AA and a potent neutralizing monoclonal antibody.

We have generated two groups of deletion mutants of the alpha PDGFR and one group of chimeras between alpha PDGFR and beta PDGFR to further investigate the structural requirements of the alpha PDGFR for binding to platelet-derived growth factor (PDGF)-AA and to a monoclonal antibody against alpha PDGFR (designated as mAb-alpha R1). The mAb-alpha R1 has recently been reported to block high affinity binding of PDGF-AA to alpha PDGFR. The first group of mutants were carboxyl-terminal deletion mutants encoding the first two immunoglobulin (Ig)-like domains (alpha R1-216), the first four Ig-like domains (alpha R1-415), or all five Ig-like domains (alpha R1-530) of the alpha PDGFR. Since these mutants lacked transmembrane domains, their expression in NIH/3T3 cells resulted in secretion of the truncated alpha PDGFRs. Using conditioned medium from NIH/3T3 transfectants, we showed that mAb-alpha R1 was able to immunoprecipitate each of these secreted form of alpha PDGFRs, suggesting that the epitope recognized by mAb-alpha R1 is located within Ig-like domains 1 and 2 of the alpha PDGFR. Furthermore, PDGF-AA exhibited detectable binding to alpha R1-415 or alpha R1-530 but failed to interact with alpha R1-216, suggesting that the first two Ig-like domains of the alpha PDGFR are not sufficient for PDGF-AA binding. The second group of alpha PDGFR mutants were internal deletion mutants lacking Ig-like loop 1 (alpha R delta 49-100), Ig-like loop 2 (alpha R delta 150-189), Ig-like loop 3 (alpha R delta 235-290), or part of Ig-like loops 4 and 5 (alpha R delta 375-450). The internal deletion mutants were transfected into 32D cells which lack both alpha PDGFR and beta PDGFR. PDGF-AA bound with high affinity to 32D cells expressing alpha R delta 375-450 but not to 32D cells expressing the other three internal deletion mutants, suggesting that the region required for PDGF-AA binding should be within the first three Ig-like domains of the alpha PDGFR. In addition, mAb-alpha R1 bound to 32D cells expressing alpha R delta 235-290 but failed to bind 32D cells transfected with alpha R delta 49-100 or alpha R delta 150-189.(ABSTRACT TRUNCATED AT 400 WORDS)

Density-dependent inhibitory effect of transforming growth factor-beta 1 on human fibroblasts involves the down-regulation of platelet-derived growth factor alpha-receptors.

We have previously found that transforming growth factor-beta 1 (TGF-beta 1) inhibits the mitogenic activity of platelet-derived growth factor (PDGF) in cultures of human neonatal fibroblasts in a density-dependent fashion. In the present investigation we determined the effect of TGF-beta 1 on the PDGF alpha-receptor, which binds all PDGF isoforms, as well as on the beta-receptor, which binds only PDGF-BB with high affinity. We found that the inhibitory effect of TGF-beta 1 on PDGF-AA-induced mitogenesis was density-dependent; when dense cell cultures were preincubated with TGF-beta 1, there was an complete inhibition of 3H-thymidine incorporation, whereas the effect was less in sparse cultures. A similar density-dependent effect of TGF-beta 1 was seen in PDGF-BB treated cells, although less pronounced. The binding of 125I-labeled PDGF-AA and PDGF-BB to the alpha-receptor was significantly reduced after treatment with TGF-beta 1 in dense cultures, whereas the sparse cultures were less affected. A decrease of alpha-receptor mRNA was also seen. The levels of beta-receptor protein and mRNA were unaffected. We conclude that the growth inhibitory effect of TGF-beta 1 is cell density-dependent and involves down-regulation of PDGF alpha-receptors.