Platelet-derived Growth Factor Isoforms Research Articles

Platelet-derived growth factor promotes endometrial epithelial cell proliferation

OBJECTIVE: Our purpose was to determine the effects of platelet-derived growth factor on the proliferation of endometrial epithelial cells. Platelet-derived growth factor and its receptors have been identified in the endometrium, and platelet-derived growth factor is a mitogen for endometrial stromal cells. Released from macrophages and platelets at sites of ectopic endometrial growth, platelet-derived growth factor could promote the progression of endometriosis and endometrial cancer. STUDY DESIGN: Endometrial epithelail cell lines were developed from proliferative-phase endometria from two patients without endometrial lesions. Cell lines were confirmed to be epithelial. Proliferation assays were conducted on both lines with recombinant human platelet-derived growth factor-AA, AB, and BB. Assays were also performed at different doses, times, and cell densities with platelet-derived growth factor-AB. RESULTS: All isoforms of platelet-derived growth factor were potent mitogens for both endometrial epithelial cell lines. The greatest proliferative responses were achieved at 10 ng/ml and at 24 hours. Responses decreased significantly in confluent cultures. CONCLUSIONS: These results suggest that endometrial epithelial cells have functional platelet-derived growth factor-α receptors that signal cell replication. The greater activity of platelet-derived growth factor in subconfluent cultures may indicate that receptor numbers or affinity are up-regulated when cell-cell contact is disrupted. These data support a role for platelet-derived growth factor in normal endometrial proliferation and in pathologic proliferation such as endometriosis and endometrial cancer.

Testicular development involves the spatiotemporal control of PDGFs and PDGF receptors gene expression and action.

Platelet-derived growth factors (PDGFs) are growth-regulatory molecules that stimulate chemotaxis, proliferation and metabolism primarily of cells of mesenchymal origin. In this study, we found high levels of PDGFs and PDGFs receptors (PDGFRs) mRNAs, and specific immunostaining for the corresponding proteins in the rat testis. PDGFs and PDGFRs expression was shown to be developmentally regulated and tissue specific. Expression of PDGFs and PDGFRs genes was observed in whole testis RNA 2 d before birth, increased through postnatal day 5 and fell to low levels in adult. The predominant cell population expressing transcripts of the PDGFs and PDGFRs genes during prenatal and early postnatal periods were Sertoli cells and peritubular myoid cells (PMC) or their precursors, respectively, while in adult animals PDGFs and PDGFRs were confined in Leydig cells. We also found that early postnatal Sertoli cells produce PDGF-like substances and that this production is inhibited dose dependently by follicle-stimulating hormone (FSH). The expression of PDGFRs by PMC and of PDGFs by Sertoli cells corresponds in temporal sequence to the developmental period of PMC proliferation and migration from the interstitium to the peritubulum. Moreover, we observed that all the PDGF isoforms and the medium conditioned by early postnatal Sertoli cells show a strong chemotactic activity for PMC which is inhibited by anti-PDGF antibodies. These data indicate that, through the spatiotemporal pattern of PDGF ligands and receptors expression, PDGF may play a role in testicular development and homeostasis.

Different effects of platelet derived growth factor isoforms on DNA synthesis and migration in human vascular smooth muscle cells.

Conference Article| November 01 1995 Different Effects Of Platelet Derived Growth Factor Isoforms On DNA Synthesis And Migration In Human Vascular Smooth Muscle Cells GERARD F. CLUNN; GERARD F. CLUNN 1Department of Clinical Pharmacology, St. Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, London, W2 1NY Search for other works by this author on: This Site PubMed Google Scholar JONATHON S. REFSON; JONATHON S. REFSON 1Department of Clinical Pharmacology, St. Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, London, W2 1NY Search for other works by this author on: This Site PubMed Google Scholar MICHAEL SCHACHTER; MICHAEL SCHACHTER 1Department of Clinical Pharmacology, St. Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, London, W2 1NY Search for other works by this author on: This Site PubMed Google Scholar ALUN D. HUGHES ALUN D. HUGHES 1Department of Clinical Pharmacology, St. Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, London, W2 1NY Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (4): 608S. https://doi.org/10.1042/bst023608s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation GERARD F. CLUNN, JONATHON S. REFSON, MICHAEL SCHACHTER, ALUN D. HUGHES; Different Effects Of Platelet Derived Growth Factor Isoforms On DNA Synthesis And Migration In Human Vascular Smooth Muscle Cells. Biochem Soc Trans 1 November 1995; 23 (4): 608S. doi: https://doi.org/10.1042/bst023608s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.

Differential Chemotactic Responses Mediated by Platelet-Derived Growth-Factor α- and β-Receptors

We have performed a modified Boyden chamber chemotaxis assay using NRK-49F cells under serum-free condition, in order to characterize the chemotactic activity of recombinant platelet derived growth factor (PDGF)-AA, -AB, and -BB. All three PDGF isoforms showed similar chemotactic activity on NRK cells expressing both PDGF α- and β-receptors. A checkerboard analysis revealed that PDGF-AA was a true chemoattractant for NRK cells, indicating that α-receptors could transduce signals for the chemotactic response. We then examined the inhibitory effects of PDGF isoforms in the upper chamber on NRK cell migration to PDGF in the lower chamber. When higher concentrations of PDGF-AB or -BB were present in the upper chamber, cell migration to any of the PDGF isoforms was completely blocked, whereas PDGF-AA in the upper chamber could not induce complete inhibition of the chemotactic migration to PDGF-AB or -BB. Even when 100 ng/ml of PDGF-AA was present in the upper chamber, NRK cell migration to 5 ng/ml of PDGF-AB in the lower chamber was not completely blocked. These findings suggest that the chemotactic response induced by ligand binding to PDGF β-receptor may be different from that induced by ligand binding to PDGF α-receptors.

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.

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.

Control of meningioma cell growth by platelet-derived growth factor (PDGF)

We have examined the possible involvement of PDGF and PDGF receptors in the growth control of five meningiomas, analyzing the biopsy specimens and the primary cultures derived from the same tumors. Light and electron microscopy demonstrated that MAbs against PDGF β-receptors immunodecorate meningioma cells in vivo and in vitro, while those against α-receptors gave negative results. The effects of PDGF isoforms AA, AB, BB and of PDGF neutralizing antibodies on meningioma cultures were examined using [ 3H]thymidine incorporation analysis. Only with PDGF-AB and -BB a mitogenic effect was observed, while PDGF-neutralizing antibodies produced a reduction of [ 3H]thymidine incorporation. The production of PDGF-like growth factors by meningioma cells was tested analyzing the effects of meningioma culture-conditioned media on the growth of Swiss 3T3 cells. In all cases meningioma conditioned media stimulated the in vitro growth of 3T3 fibroblasts and this stimulatory effect was strongly reduced by PDGF-neutralizing antibodies. Furthermore, Northern blot analysis demonstrated expression of c-sis/PDGF-B and PDGF β-receptors mRNA in all meningioma biopsies and in all the derived cultures. Our results provide strong evidence that PDGF-B chain and PDGF β-receptors are involved in growth control mechanisms of human meningiomas through autocrine and/or paracrine mechanisms.

Growth factor effects on the expression of collagenase and TIMP-1 in periodontal ligament cells.

The fibroblast is a prominent cellular component of the periodontal ligament. It is believed to play an important role in collagen metabolism in health and disease. The turnover of collagen in the periodontal ligament is believed to be controlled by the balance between collagen synthesis and degradation. The family of matrix metalloproteinases and their inhibitors is one of the mechanisms which regulates this balance. The factors that regulate the synthesis of collagenase and its inhibitor, TIMP-1, by the periodontal ligament cell are poorly understood. The present study was undertaken to assess the effect of interleukin-1 beta (IL-1 beta), platelet-derived growth factor (PDGF), and transforming growth factor-beta 1 (TGF-beta) on the expression of collagenase (MMP-1) and TIMP-1 mRNA in periodontal derived fibroblasts using reverse transcription polymerase chain reaction (RT-PCR). Early passage periodontal ligament derived fibroblasts were treated with IL-1 beta (10 and 100 pg/ml), two isoforms of PDGF, -AA and -BB (4 and 20 ng/ml) and TGF-beta (1 and 10 ng/ml). Treatment with growth factors from 2 to 24 hours revealed that the largest effects on MMP-1 mRNA occurred after 24 hours. IL-1 beta induced a 5 to 9 fold increase in MMP-1 mRNA. The two isoforms of PDGF had less of an effect (3 to 5 fold) on MMP-1 mRNA whereas TGF-beta induced a 25 to 50% decrease in the expression of this message. None of the growth factors had an effect on TIMP-1 mRNA expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific effects of platelet derived growth factor (PDGF) on fetal rat and human dopaminergic neurons in vitro.

The neurotrophic effects of the BB isoform of platelet-derived growth factor (PDGF) on rat and human fetal mesencephalic dopaminergic neurons have been characterized in vitro. A dose-response analysis demonstrated maximal responses at 30 ng/ml of PDGF-BB. This concentration resulted in a marked increase in the survival and neurite outgrowth from rat and human tyrosine hydroxylase-(TH) positive, presumed dopaminergic neurons after 7 days in vitro. The effects of PDGF-BB on survival of TH-positive neurons were comparable to those of brain-derived neurotrophic factor (BDNF), whereas neurite outgrowth was more pronounced after addition of BDNF. The combination of BDNF and PDGF-BB yielded no additive effects. Double immunohistochemical staining of rat cultures demonstrated PDGF beta-receptors on about 90% of the TH-positive neurons. PDGF-BB treatment of rat mesencephalic cultures induced an upregulation of c-fos and TH mRNA with maximal levels after 0.5-2 h as assessed by quantitative PCR analysis. An increased number of Fos protein-positive cells was detected immunohistochemically after 4 h of PDGF-BB treatment. The present results provide further evidence for specific and direct effects of PDGF-BB on gene expression, survival and neurite outgrowth of mesencephalic dopaminergic neurons of rat and human origin.

Isoforms of platelet-derived growth factor and its receptors in epiretinal membranes: Immunolocalization to retinal pigmented epithelial cells

Epiretinal membranes (ERMs) form on the inner surface of the retina in conjunction with various ocular disease processes, but the factors controlling their development are not understood. The predominant cell types involved are retinal pigmented epithelial (RPE) cells and retinal glia. Cultured RPE cells secrete platelet-derived growth factor (PDGF), which is chemotactic and mitogenic for both RPE cells and retinal glia and, therefore, could be involved in the development of ERMs. In the present study, we performed immunohistochemical staining for PDGF A chain (PDGF-A), PDGF B chain (PDGF-B), and both types of PDGF receptors (PDGFr alpha and PDGFr beta) on ERMs associated with various disease processes. PDGF-A is detected in most ERMs, regardless of the associated disease process, and it appears to be localized predominantly in RPE cells, recognized by the presence of pigment and the immunohistochemical demonstration of some or all of the following RPE-associated epitopes: class III beta-tubulin, keratin, the 65-kDa microsomal protein recognized by the RPE9 antibody, and cellular retinaldehyde-binding protein. PDGF-B is found only in minor subpopulations of cells in about half of the ERMs evaluated and, with only occasional exceptions, appears to be localized almost entirely in blood-borne cells found in and around vessels in vascularized ERMs. Both PDGFr alpha and PDGFr beta are demonstrated in most ERMs with neither isotype consistently predominating: they are found predominantly on RPE cells with many cells expressing both receptor types. ERMs with little or no RPE cell component contain little or no PDGF and PDGF receptor, whereas those in which the RPE cell represents the major cell type, have widespread PDGF and PDGF receptor positivity. These findings show that RPE cells in ERMs produce PDGF-A and PDGF alpha and PDGF beta receptors and suggest that autocrine and paracrine stimulation with PDGF may be involved in ERM pathogenesis.

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.

Production and Secretion of Platelet‐Derived Growth Factor AB by Cultured Human Keratinocytes: Regulatory Effects of Phorbol 12‐Myristate 13‐Acetate, Etretinate, 1,25‐Dihydroxyvitamin D3, and Several Cytokines

Platelet-derived growth factor (PDGF) is a potent mitogen for several mesenchymal cells and plays an important role in wound repair. Three PDGF isoforms, PDGF-AA, PDGF-BB, and PDGF-AB, have been found to be generated in various tissues. PDGF-AB production by normal human keratinocytes (NHKs), by human squamous cell carcinoma cell line (HSC-1) cells, and by human dermal fibroblasts (HDFs) was studied in the presence of agents which influence cell growth. Both NHKs and HSC-1 cells spontaneously produced and secreted PDGF-AB. NHKs grown in keratinocyte growth medium produced more PDGF-AB than did those grown in keratinocyte basic medium. Phorbol 12-myristate 13-acetate inhibited PDGF-AB production in NHKs but promoted its production in HSC-1 cells. 1,25-dihydroxyvitamin D3 up-regulated PDGF-AB production, whereas etretinate did not. High levels of calcium in the culture medium induced little change in cellular PDGF-AB levels. Prostaglandin E1 slightly inhibited PDGF-AB production, transforming growth factor beta 1 promoted PDGF-AB production and interferon-gamma, interleukin-1 alpha, and tumor necrosis factor-alpha failed to exert any influence at all. Cultured HDFs did not produce any detectable PDGF-AB. These results suggest that keratinocytes are a major source of cutaneous PDGF and that this factor may therefore play an important role in wound repair and in certain proliferative skin diseases.

Isoform-specific induction of the low-density lipoprotein receptor gene by platelet-derived growth factor.

We investigated the effect of recombinant platelet-derived growth factor (PDGF) isomers on low-density lipoprotein (LDL) receptor gene expression and compared this with two indexes of cell growth response: expression of the immediate early gene c-myc and the rate of DNA synthesis. In human skin fibroblasts and NIH 3T3 cells, the PDGF-BB homodimer was more effective in inducing the LDL receptor gene and cell growth response compared with the PDGF-AA homodimer. The second messenger pathways utilized by PDGF receptors for enhancing LDL receptor gene response could, however, be dissociated from those utilized for enhancing c-myc gene response and were insensitive to inhibitors of tyrosine phosphorylation. Inhibition of tyrosine kinase activity inhibited c-myc gene response to PDGF-BB at 10(-8) M but had little effect on LDL receptor gene response. Such inhibition increased expression of the LDL receptor gene in the presence of the PDGF-AA isomer. Our results indicate that the response of the LDL receptor gene to PDGF isoforms reflects cellular growth response. However, different transduction pathways are utilized for PDGF activation of the c-myc and LDL receptor genes in mesenchymal cells.

Serum fractions and related agonists with calcium-mobilizing activity in the bovine growth plate chondrocyte.

Longitudinal growth of bone involves a complex sequence of cellular events in the cartilaginous epiphysis. Whole blood serum has been shown previously to be a potent stimulus to the cells of the growth plate, as demonstrated by its ability to activate the inositol phosphate-calcium second messenger system, resulting in a rise in intracellular Ca2+. By manipulating the preparation of serum to functionally separate it into its constituent parts, we have shown that the processes of platelet lysis and activation of the clotting cascade are responsible for the generation of factors that stimulate this signaling mechanism in isolated bovine growth plate chondrocytes. Through a subsequent trial of bioactive agents generated in these processes, we identified and partially characterized several novel agonists of growth plate chondrocytes:adenosine triphosphate and adenosine diphosphate, the purine energy substrates, and bradykinin, the bioactive peptide generated in a side reaction of the clotting cascade, each induces a rise in intracellular Ca2+ via release from intracellular ion stores. Additionally, the three distinct isoforms of platelet-derived growth factor (AA, AB, and BB), also released on platelet lysis, were compared with respect to their ability to stimulate the inositol phosphate-calcium second messenger system in growth plate chondrocytes.

Comparison of Effects of Platelet-Derived Growth Factor Isoforms on Signaling and DNA Synthesis of Human Cultured Saphenous Vein Cells

We examined the effect of platelet-derived growth factor (PDGF)-AA, PDGF-AB, and PDGF-BB isoforms on DNA synthesis, Ca2+ mobilization, and tyrosine phosphorylation in cultured human saphenous vein cells cultured by an explant technique; confluent cells derived from passage 3 were used for all studies. DNA synthesis was measured by [methyl3H]thymidine uptake, intracellular [Ca2+] ([Ca2+]i) was measured with the Ca(2+)-sensitive indicator fura-2, and tyrosine phosphorylation was measured by Western blotting techniques. All three isoforms of PDGF stimulated [methyl3H]thymidine uptake concentration dependently, with similar potency. PDGF-AB induced significantly greater [methyl3H]-thymidine uptake than PDGF-BB, and PDGF-AA was much less effective than either PDGF-AB or PDGF-BB. The effects of all three isoforms were inhibited by tyrphostin-23, a selective inhibitor of tyrosine kinases PDGF-AB and PDGF-BB increased [Ca2+]i, although the maximum response to PDGF-AB was significantly less than that to PDGF-BB. Both isoforms increased [Ca2+]i by stimulating influx and intracellular release of Ca2+. PDGF-AA had no measurable effect on [Ca2+]i. All three isoforms increased tyrosine phosphorylation of a 170-kDa protein detected by Western blotting. Quantitative densitometry indicated that PDGF-BB induced greater tyrosine phosphorylation than PDGF-AB and both PDGF-BB and PDGF-AB induced markedly more tyrosine phosphorylation than PDGF-AA. PDGF isoforms have differing efficacies in terms of DNA synthesis, Ca2+ mobilization, and tyrosine phosphorylation by human saphenous vein cells.

Chrysotile asbestos stimulates platelet-derived growth factor-AA production by rat lung fibroblasts in vitro: evidence for an autocrine loop.

We have investigated the mitogenic and chemotactic role of platelet-derived growth factor (PDGF) in pulmonary fibrogenesis induced by chrysotile asbestos. Since fibroblasts phagocytize asbestos in the lung interstitium, we have sought to learn whether the fibers alter the production of PDGF-like molecules by rat lung fibroblasts or induce mitogenesis of these fibroblasts in vitro. Conditioned medium as well as cell lysates from fibroblasts exposed to asbestos contained approximately 4-fold more PDGF than unexposed cells as detected by Western blot. Two distinct molecular weight forms of PDGF (36 and 18 kD) were detected by Western blotting. We postulate that these PDGF-like molecules are homologues of human PDGF-AA since we could not detect any PDGF in a sensitive enzyme immunoassay that recognized only PDGF-BB and PDGF-AB. Furthermore, PDGF-A chain mRNA was readily detected by Northern analysis, whereas PDGF-B chain mRNA was not detected by conventional Northern analysis. However, message amplification using a reverse transcriptase polymerase chain reaction allowed detection of the B-chain message. A significant dose-dependent mitogenic effect of asbestos was found by using both a cell proliferation assay and nuclear labeling with bromodeoxyuridine when fibroblasts were exposed under serum-free conditions. This mitogenesis induced directly by asbestos was blocked almost entirely with an anti-PDGF antibody that neutralized all three PDGF isoforms. Thus, these data support our hypothesis that an autocrine loop for PDGF-AA is operative in vitro following exposure to asbestos in lung fibroblasts, and we suggest that this signaling pathway could be significant in the pathogenesis of pulmonary fibrosis.

Recycling of histamine in mouse mastocytoma cell line P-815

Regulation of platelet-derived growth factor isoform-mediated expression of prostaglandin G/H synthase in mesangial cells. Incubation of rat renal mesangial cells with platelet-derived growth factor (PDGF) -AB or -BB led to a transient increase in prostaglandin G/H synthase-2 (PGHS-2) mRNA expression with a maximum after two hours. Expression of PGHS-1 mRNA remained unchanged during short term incubation, but was enhanced about twofold after 8 to 12 hours incubation with PDGF-AB or -BB. Enhanced PGHS activity was still observed after 24 hours. Nevertheless, PGE2 release from mesangial cells was not enhanced by PDGF, hinting to the availability of arachidonic acid as rate-limiting step. PDGF receptors are coupled to multiple signaling pathways, among them phospholipase Cγ. PDGF-BB rapidly phoshorylated PLCγ, while phosphorylation by PDGF-AB was barely detectable. The differential effect of PDGF-BB and PDGF-AB was also seen with respect to calcium signaling: PDGF-BB but not PDGF-AB induced release of Ca2+ from internal stores. Activation of PLC and the resulting transient release of Ca2+ were not considered to be essential for PGHS-2 mRNA induction as both PDGF isoforms were equally effective in mRNA induction. Both PDGF isoforms led to a Ca2+ influx resulting in a long lasting elevation of [Ca2+]i. Enhanced [Ca2+]i seemed to be related to PGHS-2 mRNA expression, because PDGF-induced PGHS-2 mRNA was significantly reduced under Ca2+ free conditions. Diacylglycerol, liberated by PLC, is an activator of protein kinase C (PKC). Down-regulation of PKC by overnight incubation with phorbol ester (0.1 μm) attenuated PGHS-2 mRNA induction by PDGF-AB and -BB. Involvement of PKC was substantiated by the PKC inhibitor H7, which interfered with PDGF-mediated PGHS-2 mRNA expression, while HA1004, a considerably specific inhibitor of protein kinases A and G, was without effect. Taken together, signaling pathways other than PLCγ seem to be involved in activation of PKC and elevation of [Ca2+]i, which were shown to be essential elements of PDGF-mediated induction of PGHS-2 mRNA expression in mesangial cells.

Regulation of Fibronectin by Platelet-Derived Growth Factors in Cultured Rat Thoracic Aortic Smooth Muscle Cells.

Induction of fibronectin (FN) gene expression by platelet-derived growth factor (PDGF) isoforms in rat thoracic aortic smooth muscle cells (SMC) was examined. PDGF-BB enhances FN levels in SMC cultures in a time- and concentration-response fashion. PDGF-AA and PDGF-AB show no effect on FN levels. The effects of insulin and insulin-like growth factor-I (IGF-I) on PDGF-BB-induced FN levels were examined. No additivity of FN levels is observed between PDGF-BB and insulin and/or IGF-I. Experiments also show that PDGF-BB enhances FN mRNA levels, implying that acquisition of additional FN mRNA units accounts for the increase in FN levels. Induction of FN and FN mRNA levels by PDGF-BB could be one of the initial events in vascular SMC proliferation and extracellular matrix expansion, leading to atherosclerosis and hypertension. Copyright 1995 S. Karger AG, Basel

Platelet-derived growth factor (PDGF) B and A homodimers transform murine fibroblasts depending on the genetic background of the cell.

The endogenously overexpressed c-sis (platelet-derived growth factor (PDGF) B-chain) proto-oncogene and its viral counterpart v-sis effectively transform NIH 3T3 cells by an autocrine mechanism, whereas the PDGF A-chain gene does not. However, PDGF B continuously cultured with confluent NIH 3T3 cells fails to induce phenotypic transformation as would be predicted by the autocrine model. We now have established conditions in which subconfluent, logarithmically growing NIH 3T3 cells are efficiently transformed by exogenous PDGF B but not PDGF A. Clonally selected cells from transformed foci remain transformed when continuously cultured with PDGF B but revert to the non-transformed phenotype without PDGF B or if PDGF A is substituted for PDGF B, suggesting that PDGF B complements a genetically stable NIH 3T3 cell phenotype to induce transformation and initiates a signal different from that of PDGF A required to induce transformation. Neither PDGF A nor PDGF B transform normal rat kidney (NRK) cells under identical conditions of culture. However, we now demonstrate that exogenous PDGF A and PDGF B independently transform NRK cells that endogenously overexpress the anti-apoptotic bcl-2 gene. These results suggest that both PDGF A and PDGF B effectively transform murine fibroblasts with a compatible genotype, that transformation by the PDGF isoforms requires complementation with a compatible genotype in a multistep transformational process, and that the autocrine mechanism is required but not sufficient for transformation of murine fibroblasts by PDGF.

Differences in signal transduction between platelet-derived growth factor (PDGF) alpha and beta receptors in vascular smooth muscle cells. PDGF-BB is a potent mitogen, but PDGF-AA promotes only protein synthesis without activation of DNA synthesis.

Cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats express both alpha and beta isoforms of the platelet-derived growth factor (PDGF) receptors at high levels (100,000 and 240,000 sites/cell, respectively). In this cell type, PDGF-BB elicited a mitogenic response; however, PDGF-AA increased only protein synthesis without activating DNA synthesis. Protein kinase C (PKC) was activated by PDGF-AA as well as PDGF-BB with concomitant translocation from cytosol to membrane fractions. However, the hypertrophic effect of PDGF-AA was not affected by depletion of cellular PKC, whereas the mitogenic action of PDGF-BB was partially attenuated by the depletion. Following incubation with PDGF-AA or -BB, phospholipase C-gamma 1 (PLC-gamma 1) and phosphatidylinositol 3-kinase were tyrosine phosphorylated; however, the phosphorylation of Ras-GTPase-activating protein was induced only by PDGF-BB. Both PDGF isoforms resulted in a prompt and transient increase in the level of 1,2-diacylglycerol (DAG), presumably through the action of PLC-gamma 1. After returning to basal levels, the rate of DAG synthesis steadily increased for at least 15 min due to activation of phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC). Incubation with PDGF-BB-activated phospholipase D (PLD) in a PKC-dependent manner resulting in the formation of phosphatidic acid (PA). PA was also formed by the sequential reactions of PC-PLC and DAG kinase in the PDGF-BB-stimulated VSMC, and these sequential reactions were not affected by PKC depletion. In contrast, PDGF-AA stimulation did not result in increased PA synthesis as neither PLD nor DAG kinase activities were affected. PA may be a significant second messenger in the activation of DNA synthesis by PDGF-BB. These results indicate that signaling mechanisms of the PDGF-alpha and -beta receptors in VSMC are distinctly different in signal transduction in VSMC and that the alpha receptor promotes cellular hypertrophy (but not hyperplasia), whereas a mitogenic response is mediated only through the beta receptor.