Antiinflammatory effects of polypeptide growth factors. Platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor inhibit the cytokine-induced expression of the alternative complement pathway activator factor B in human fibroblasts.

The synthesis of complement components in human fibroblasts is modulated by mediators of inflammation such as cytokines. In particular, interleukin-1 (IL-1) and tumor necrosis factor (TNF) induce time- and dose-dependent increases in the synthesis of complement proteins factor B (FB), C3, and factor H (FH). Polypeptide growth factors are also soluble mediators released during inflammation and able to modulate many fibroblast functions. We have studied the effects of polypeptide growth factors platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) on the synthesis of complement proteins in cultured human fibroblasts. PDGF, EGF, and FGF alone did not affect the level of synthesis of any of the complement proteins analyzed, but simultaneous incubation of PDGF, EGF, or FGF with IL-1 and TNF resulted in a dose-dependent inhibition of the cytokine-enhanced expression of FB. Inhibition of FB synthesis was observed between 4 and 8 h of exposure to PDGF and persisted for 4 h after the removal of the growth factor. Analysis of steady-state levels of specific FB mRNA suggested that PDGF-induced inhibition of FB synthesis is mediated at a pretranslational level and that it requires new protein synthesis. The effect of the growth factors was limited to FB, with marginal or no inhibition on the cytokine-enhanced synthesis of C3 and FH, excluding the possibility that the inhibitory effects of PDGF, EGF, and FGF on FB synthesis were due to a negative modulation of the growth factors on cytokine cell membrane receptors. Specific inhibition of cytokine-induced increases in FB synthesis by the growth factors may represent down regulation of the acute inflammatory process, further permitting progression to processes of tissue repair and remodeling. Study of the interactions between cytokines and growth factors in the regulation of synthesis of complement proteins may also provide a system for investigating mechanisms of signal transduction of both polypeptide growth factors and cytokines.     

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.     

Platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. Two different PDGF chains termed A and B encoded by different genes have been identified leading to three different PDGF isoforms, the AA and BB homodimers and the AB heterodimer. All three forms have been observed in vivo and possess biological activity in vitro with the AA homodimer being the poorest cellular mitogen. The availability of highly purified recombinant PDGF isoforms was the initial basis for comparative studies in order to specify the different spectra of activity of the various PDGF species. This review is particularly focused on the AB heterodimer as from the standpoint of heterologous gene expression, this species is the one with the highest demands concerning expression and purification protocols. This explains the fact that, in comparison to PDGF-BB, only very limited data on the in vivo application of PDGF-AB are available so far.     

Platelet-derived growth factor exerts disparate effects on odontoblast differentiation depending on the dimers in rat dental pulp cells

Platelet-derived growth factor (PDGF) has recently been demonstrated to control the expression of alkaline phosphatase and proteoglycan synthesis of odontoblastic cells in dental pulp tissues. Although PDGF appears to be closely related to dentinogenesis, much about the mode of action of PDGF on odontoblast differentiation remains unclear. In this study, we examined the effects of three PDGF dimers (PDGF AA, AB, and BB) on odontoblastic differentiation of dental pulp cells in long-term mineralized cultures. Dental pulp cells isolated from rat lower incisors were continuously treated with each of PDGF AA, AB, and BB in separate cultures for 20 days. The three PDGF dimers suppressed alkaline phosphatase activity, osteocalcin and calcium content, and the formation of dentin-like nodules. The expression of mRNA for dentin sialoprotein (DSP) in the cells was inhibited by PDGF AA treatment, whereas PDGF AB and BB treatment stimulated the expression of DSP, even though the dentin-like nodule formation was inhibited. Although the effects of PDGF on odontoblastic differentiation varied among the dimers, the cells expressed both PDGF and receptors, whose quantities were similar. These results suggest that PDGF exerts diverse effects on odontoblastic differentiation depending on its dimeric form. These in vitro findings explain, at least in part, the in vivo action of PDGF in dentinogenesis during the repair process of damaged dental pulp.This work was supported in part by grants-in-aid from the Ministry of Science, Education, and Culture of Japan     

Platelet-derived growth factor AA homodimer is the predominant isoform in human platelets and acute human wound fluid.

Platelet-derived growth factor (PDGF) is a dimeric protein composed of A- and B-chains (AA, AB, and BB). PDGF purified from human platelets has been shown to be composed primarily of the AB heterodimer. Immunoblots of total platelet extracts, cell-bound PDGF from the platelet extracts, and acute human wound fluid with PDGF A- and B-chain-specific antisera all demonstrate that the PDGF A-chain is the predominant peptide. Chemotactic and immunochemical assays of chromatographic fractions during PDGF isolation support these observations and demonstrate that PDGF AA can be separated from PDGF AB and BB by ion exchange chromatography. These studies indicate that the AA isoform constitutes the major PDGF dimer contained in human platelets and is the major form present at sites of injury during the acute phase of the wound repair response.     

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.     

PDGF BB stimulates proliferation and differentiation in cultured chondrocytes from rat rib growth plate.

The role of two isoforms (PDGF AA and PDGF BB) of platelet derived growth factor either alone or in combination with insulin-like growth factor I, on the regulation of proliferation and differentiation of rat rib growth plate chondrocytes was analyzed. PDGF BB increased DNA-synthesis in a dose dependent manner with a half maximal effect at 1 ng/ml. When PDGF BB was combined with IGF-I, an additive effect on DNA-synthesis was observed. PDGF AA and BB alone or combined with IGF-I had no appreciable effects on proteoglycan synthesis. Both homodimers caused an increase in AP-activity, indicating stimulation of cell differentiation. Cultured chondrocytes bound 125I-PDGF AA and 125I-PDGF BB and after stimulation with PDGF expressed c-fos protein. Thus, both homodimers play an important role in chondrocyte differentiation and together with IGF-I interact in the regulation of longitudinal bone growth.     

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.     

Effects of homo- and heterodimeric isoforms of PDGF on signalling events in rat renal mesangial cells.

Platelet-derived growth factor (PDGF) exists in three dimeric isoforms, AA, BB and AB. Mesangial cells exclusively bound the BB homodimer and responded only to the BB isoform in terms of DNA synthesis and phosphoinositide hydrolysis. PDGF-BB stimulated a dose-dependent formation of inositol trisphosphate (InsP3). Neither pertussis toxin nor short-term (10 min) treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited the PDGF-BB-evoked production of InsP3. In contrast, the response to PDGF-BB was attenuated in cells in which protein kinase C has been down-regulated by long-term (24 h) treatment with TPA. In parallel to the generation of InsP3, there was a biphasic increase in 1,2-diacylglycerol (DAG). The second peak of DAG generation was associated with a concomitant 2-fold increase in choline formation. In addition, PDGF-BB stimulated the accumulation of phosphatidylpropanol, produced by phospholipase D phosphatidyl transferase activity, when 1-propanol was added to mesangial cells. Stimulation of mesangial cells with PDGF-BB caused a dose-dependent formation of prostaglandin E2. Furthermore, mesangial cells secreted PDGF-AA into the culture supernatant.     

PDGF在大鼠断层供皮区创面愈合过程中表达变化的研究

实验研究已经证明Ｐｌａｔｅｌｅｔ－ｄｅｒｉｖｅｄｇｒｏｗｔｈｆａｃｔｏｒ（ＰＤＧＦ）能够促进各种类型的伤口愈合,然而在伤口愈合过程中内源性ＰＤＧＦ表达变化的研究却少有报道,为探讨ＰＤＧＦ对伤口愈合的影响,我们应用原位杂交、斑点杂交技术观察了内源性ＰＤＧＦ在大鼠创面愈合过程中的表达变化,结果发现：在创面愈合过程中,肉芽组织中的成纤维细胞,毛细血管内皮细胞及创缘真皮内的毛囊上皮细胞均能表达ＰＤＧＦ－ＢＢ基因,在伤后６天,组织修复的高峰期,ＰＤＧＦ－ＢＢ基因表达达到最强,伤后１２天,伤口完全上皮化,ＰＤＧＦ的基因表达也恢复正常,说明ＰＤＧＦ的基因表达和伤口愈合时间有密切的关系。提示ＰＤＧＦ在创面愈合过程中可能起着重要的调控作用。     

Mitogenic effect of transforming growth factor beta 1 on human fibroblasts involves the induction of platelet-derived growth factor alpha receptors

Platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), potent modulators of mesenchymal cell growth and differentiation, are often colocalizable in vivo. Previous in vitro studies in fibroblastic cell lines have shown variable, even antagonistic effects of TGF-beta on the mitogenic action of PDGF. This study demonstrates that in diploid human dermal fibroblasts, TGF-beta 1 is weakly mitogenic in the absence of serum or purified growth factors, and that TGF-beta 1 potentiates DNA synthesis in PDGF-stimulated fibroblasts with delayed kinetics when compared to stimulation with PDGF alone. TGF-beta 1 enhances mitogenic potency of all three PDGF isoforms and increases receptor binding of both 125I PDGF-AA and 125I PDGF-BB, consistent with the increased expression of the alpha type PDGF receptor. The induction of PDGF alpha receptor subunits by TGF-beta may play a role in enhancing the proliferative potential of human fibroblasts in certain physiologic and pathologic conditions.     

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.     

Immunochemical localization of the epitope for a monoclonal antibody that neutralizes human platelet-derived growth factor mitogenic activity.

A monoclonal antibody (mAb), sis 1, generated against human c-sis-encoded platelet-derived growth factor (PDGF) BB, was shown by enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis to recognize human PDGF BB and human platelet PDGF AB but not the human PDGF AA. This monoclonal antibody potently inhibited PDGF receptor-binding and mitogenic activities of both human PDGF BB and PDGF AB but had no effect on PDGF AA. Finally, we demonstrated that an immunoaffinity-purified anti-c-sis peptide antibody (anti-V4) which also blocked binding of PDGF BB to its cognate receptor and competed with mAb sis 1 for binding to PDGF BB. All of these results suggest that mAb sis 1 recognizes an epitope of the c-sis gene product, PDGF BB, that spatially overlaps the V4 surface domain of PDGF BB, immunochemically localizing a region of PDGF BB critical for PDGF receptor binding and activation.     

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.     

Effect of the different dimeric forms of the platelet-derived growth factor on cellular responses in mouse Swiss 3T3 fibroblasts

PDGF consists of two polypeptide chains, A and B, and all three possible dimers have been isolated from different sources. Human PDGF, essentially AB, porcine PDGF (BB) and recombinant PDGF-AA were tested on Swiss 3T3 fibroblasts for their ability to stimulate mitogenesis, phosphoinositide turnover and tyrosine phosphorylation of the PDGF receptor. When used in saturating amounts, the three isoforms were equally active in inducing mitogenesis. However, PDGF-AA was less active than AB and BB to induce the phosphorylation of the receptor and the turnover of phosphoinositides (30% and 50%, respectively). These findings suggest that, in Swiss 3T3 fibroblasts, PDGF receptors of the alpha-type are present in a slightly lower amount than beta-type. In addition, the two types of receptor appear to have similar physiological functions.     

Basic fibroblast growth factor modulates the mitogenic potency of the platelet-derived growth factor (PDGF) isoforms by specific upregulation of the PDGF alpha receptor in vascular smooth muscle cells.

Platelet-derived growth factor AA (PDGF AA), in contrast to PDGF AB and BB, is a poor mitogen for smooth muscle cells (SMC). However, together with basic fibroblast growth factor (bFGF) it acts synergistically on DNA synthesis of these cells. Northern blot analysis revealed that bFGF selectively increases the PDGF-receptor alpha subtype (PDGF-R alpha) mRNA level without a significant effect on the PDGF-R beta mRNA level. The amount of PDGF-R alpha protein is also selectively increased after stimulating SMC with bFGF as shown by immunoprecipitation of lysates from SMC with anti-PDGF-R alpha antibodies. The number of binding sites for 125I-PDGF AA is more than doubled after bFGF-treatment, whereas the specific binding for PDGF AB and BB increased only by approximately 30 and 20%, respectively. The increase in the number of PDGF-R alpha renders the SMC responsive for PDGF AA as demonstrated by the induction of the proto-oncogene c-fos as well as by an increased cell proliferation. The enhanced PDGF binding after bFGF treatment may in fact explain the observed synergistic behavior. These data are discussed with regard to a possible role of growth factor-induced transmodulation of receptor expression during atherogenesis.     

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.