We analyzed the transcriptional program elicited by stimulation of normal human fibroblasts with plateletderived growth factor (PDGF) using cDNA microarrays. 103 significantly regulated transcripts that had not been previously linked to PDGF signaling were identified. Among them, a cluster of genes involved in fatty acid and cholesterol biosynthesis, including stearoyl-CoA desaturase (SCD), fatty acid synthase, and hydroxymethylglutaryl-CoA synthase (HMGCS), was up-regulated by PDGF after 24 h of treatment, and their expression correlated with increased membrane lipid production. These genes are known to be controlled by sterol regulatory element-binding proteins (SREBP). PDGF increased the amount of mature SREBP-1 and regulated the promoters of SCD and HMGCS in an SREBP-dependent manner. In line with these results, blocking SREBP processing by addition of 25-hydroxycholesterol blunted the effects of PDGF on lipogenic enzymes. SREBP activation was dependent on the phosphatidylinositol 3-kinase (PI3K) pathway, as judged from the effects of the inhibitor LY294002 and mutation of the PDGF receptor tyrosines that bind the PI3K adaptor subunit p85. Fibroblast growth factors (FGF-2 and FGF-4) and other growth factors mimicked the effects of PDGF on NIH3T3 and human fibroblasts. In conclusion, our results suggest that growth factors induce membrane lipid synthesis via the activation SREBP and PI3K.
Ligand induced activation of the beta‐receptor for platelet‐derived growth factor (PDGF) leads to activation of Src family tyrosine kinases. We have explored the possibility that the receptor itself is a substrate for Src. We show that Tyr934 in the kinase domain of the PDGF receptor is phosphorylated by Src. Cell lines expressing a beta‐receptor mutant, in which Tyr934 was replaced with a phenyalanine residue, showed reduced mitogenic signaling in response to PDGF‐BB. In contrast, the mutant receptor mediated increased signals for chemotaxis and actin reorganization. Whereas the motility responses of cells expressing wild‐type beta‐receptors were attenuated by inhibition of phosphatidylinositol 3′‐kinase, those of cells expressing the mutant receptor were only slightly influenced. In contrast, PDGF‐BB‐induced chemotaxis of the cells with the mutant receptor was attenuated by inhibition of protein kinase C, whereas the chemotaxis of cells expressing the wild‐type beta‐receptor was less affected. Moreover, the PDGF‐BB‐stimulated tyrosine phosphorylation of phospholipase C‐gamma was increased in the mutant receptor cells compared with wild‐type receptor cells. In conclusion, the characteristics of the Y934F mutant suggest that the phosphorylation of Tyr934 by Src negatively modulates a signal transduction pathway leading to motility responses which involves phospholipase C‐gamma, and shifts the response to increased mitogenicity.
Activation of the b-receptor for platelet-derived growth factor (PDGF) by its ligand leads to autophosphorylation on a number of tyrosine residues. Here we show that Tyr763 in the kinase insert region is a novel autophosphorylation site, which after phosphorylation binds the protein tyrosine phosphatase SHP-2. SHP-2 has also previously been shown to bind to phosphorylated Tyr1009 in the PDGF b-receptor. Porcine aortic endothelial (PAE) cells transfected with a PDGF breceptor in which Tyr763 and Tyr1009 were mutated to phenylalanine residues failed to associate with SHP-2 after ligand stimulation. Morover, PDGF-BB-induced Ras GTP-loading and Erk2 activation were severely compromised in the receptor mutant. Whereas the mitogenic response to PDGF-BB remained at the same level as in cells expressing wild-type PDGF b-receptor, chemotaxis induced by PDGF-BB was signi®cantly decreased in the case of the Y763F/Y1009F mutant cells, suggesting an important role for SHP-2 in chemotactic signaling.
Objective. Systemic sclerosis (SSc) is a severe connective tissue disease of unknown etiology, characterized by fibrosis of the skin and multiple internal organs. Recent findings suggested that the disease is driven by stimulatory autoantibodies to platelet-derived growth factor receptor (PDGFR), which stimulate the production of reactive oxygen species (ROS) and collagen by fibroblasts. These results opened novel avenues of research into the diagnosis and treatment of SSc. The present study was undertaken to confirm the presence of anti-PDGFR antibodies in patients with SSc.Methods. Immunoglobulins from 37 patients with SSc were purified by protein A/G chromatography. PDGFR activation was tested using 4 different sensitive bioassays, i.e., cell proliferation, ROS production, signal transduction, and receptor phosphorylation; the latter was also tested in a separate population of 7 patients with SSc from a different research center.Results. Purified IgG samples from patients with SSc were positive when tested for antinuclear autoantibodies, but did not specifically activate PDGFR␣ or PDGFR in any of the tests. Cell stimulation with PDGF itself consistently produced a strong signal.Conclusion. The present results raise questions regarding the existence of agonistic autoantibodies to PDGFR in SSc.
Tissue factor (TF) is the cellular receptor for factor FVIIa (FVIIa), and the complex is the principal initiator of blood coagulation. The effects of FVIIa binding to TF on cell migration and signal transduction of human fibroblasts, which express high amounts of TF, were studied. Fibroblasts incubated with FVIIa migrated toward a concentration gradient of PDGF-BB at approximately 100 times lower concentration than do fibroblasts not ligated with FVIIa. Anti-TF antibodies inhibited the increase in chemotaxis induced by FVIIa/TF. Moreover, a pronounced suppression of chemotaxis induced by PDGF-BB was observed with active site-inhibited FVIIa (FFR-FVIIa). The possibility that hyperchemotaxis was induced by a putative generation of FXa and thrombin activity was excluded. FVIIa/TF did not induce increased levels of PDGF β-receptors on the cell surface. Thus, the hyperchemotaxis was not a result of this mechanism. FVIIa induced the production of inositol-1,4,5-trisphosphate to the same extent as PDGF-BB; the effects of FVIIa and PDGF-BB were additive. FFR-FVIIa did not induce any release of inositol-1,4,5,-trisphosphate. Thus, binding of catalytically active FVIIa to TF can, independent of coagulation, modulate cellular responses, such as chemotaxis.
We have previously shown that porcine aortic endothelial cells expressing the Y934F platelet-derived growth factor (PDGF) -receptor mutant respond to PDGF-BB in a chemotaxis assay at about 100-fold lower concentration than do wild-type PDGF -receptor-expressing cells (Hansen, K., Johnell, M., Siegbahn, A., Rorsman, C., Engströ m, U., Wernstedt, C., Heldin, C.-H., and Rö nnstrand, L. (1996) EMBO J. 15, 5299 -5313). Here we show that the increased chemotaxis correlates with increased activation of phospholipase C-␥1 (PLC-␥1), measured as inositol-1,4,5-trisphosphate release. By two-dimensional phosphopeptide mapping, the increase in phosphorylation of PLC-␥1 was shown not to be selective for any site, rather a general increase in phosphorylation of PLC-␥1 was seen. Specific inhibitors of protein kinase C, bisindolylmaleimide (GF109203X), and phosphatidylinositol 3-kinase (PI3-kinase), LY294002, did not affect the activation of PLC-␥1. To assess whether increased activation of PLC-␥1 is the cause of the hyperchemotactic behavior of the Y934F mutant cell line, we constructed cell lines expressing either wildtype or a catalytically compromised version of PLC-␥1 under a tetracycline-inducible promoter. Overexpression and concomitant increased activation of wild-type PLC-␥1 in response to PDGF-BB led to a hyperchemotactic behavior of the cells, while the catalytically compromised PLC-␥1 mutant had no effect on PDGF-BBinduced chemotaxis. Furthermore, in cells expressing normal levels of PLC-␥1, chemotaxis was inhibited by LY294002. In contrast, the increase in chemotactic response seen upon overexpression of PLC-␥1 was not inhibited by the PI3-kinase inhibitor LY294002. These observations suggest the existence of two different pathways which mediate PDGF-induced chemotaxis; depending on the cellular context, the PI3-kinase pathway or the PLC-␥1 pathway may dominate.
Cerebral cavernous malformation (CCM) is a neurovascular disease that results in various neurological symptoms. Thrombi have been reported in surgically resected CCM patient biopsies; but the molecular signatures of these thrombi remain elusive. Here, we investigated the kinetics of thrombi formation in CCM and how thrombi affect the vasculature and contribute to cerebral hypoxia. We used RNA-sequencing to investigate mouse brain endothelial cells with specific Ccm3 gene deletion (Ccm3-iECKO). We found that Ccm3 deficient brain endothelial cells had a higher expression of genes related to the coagulation cascade and hypoxia when compared to wild-type brain endothelial cells. Immunofluorescent assays identified key molecular signatures of thrombi such as fibrin, von Willebrand factor, and activated platelets in Ccm3-iECKO mice and human CCM biopsies. Notably, we identified polyhedrocytes in Ccm3-iECKO mice and human CCM biopsies and report it for the first time. We also found that the parenchyma surrounding CCM lesions is hypoxic and that more thrombi correlate with higher levels of hypoxia. Lastly, we created an in vitro model to study CCM pathology and found that human brain endothelial cells deficient for CCM3, expressed elevated levels of plasminogen activator inhibitor-1 and had a redistribution of von Willebrand factor. With transcriptomics, comprehensive imaging, and an in vitro CCM preclinical model this study provides experimental evidence that genes and proteins related to the coagulation cascade affect the brain vasculature and promote neurological side effects such as hypoxia in CCM. This study supports the concept that antithrombotic therapy may be beneficial for patients with CCM.
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