Platelet-derived growth factor BB mediated regulation of 12-lipoxygenase in porcine aortic smooth muscle cells

Platelet-derived growth factor BB (PDGF) is a potent mitogen and chemoattractant for vascular smooth muscle cells (VSMC). In the present study, we have examined the effects of PDGF on the 12-lipoxygenase (12-LO) pathway of arachidonate metabolism in porcine aortic VSMC (PVSMC). The rationale for this is previous studies showing that LO products have growth and chemotactic effects in VSMC and that another VSMC growth factor, angiotensin II, is a potent positive regulator of 12-LO activity and expression. We observed that PDGF causes a significant increase in the formation of the 12-LO product, 12-hydroxyeicosatetraenoic acid (12-HETE) in PVSMC. In addition, PDGF also markedly increased leukocyte-type 12-LO messenger RNA and protein expression. PDGF-induced PVSMC migration was inhibited significantly by two LO blockers but not by a cyclooxygenase blocker. Furthermore, although the proliferative effects of PDGF on PVSMC were not altered by cell culture under hyperglycemic conditions (25 mM glucose, HG), the chemotactic effects of PDGF as well as those of 10% fetal calf serum were significantly greater in cells cultured in HG as compared to normal glucose conditions (5.5 mM), thus indicating a potential new mechanism for the accelerated cardiovascular disease usually observed in diabetes. These results indicate a novel mechanism for the biological effects of PDGF in leading to cardiovascular disease. © 1996 Wiley-Liss, Inc.     

Regulation of 2'',5''-oligoadenylate synthetase gene expression by interferons and platelet-derived growth factor.

In murine BALB/c 3T3 cell cultures, either beta interferon or platelet-derived growth factor (PDGF) enhanced expression of the 2',5'-oligoadenylate synthetase mRNA and protein. The time course of induction in response to beta interferon was similar to that in response to PDGF. Of several growth factors known to be present in clotted blood serum (i.e., epidermal growth factor, transforming growth factor beta, and PDGF), only PDGF enhanced expression of 2',5'-oligoadenylate synthetase. The linkage of an interferon response element-containing segment from the 5'-flanking region of a human or murine 2',-5'-oligoadenylate synthetase gene made a heterologous gene responsive to interferon. The expression of such a gene construct in transfected cells was also induced by PDGF. Induction by PDGF was inhibited by mono- or polyclonal antibodies to murine interferon, which suggested that induction by PDGF requires interferon. Both PDGF and interferon induced nuclear factors that bound to this interferon response element-containing segment in vitro.     

Platelet-derived growth factor-BB-mediated activation of Akt suppresses smooth muscle-specific gene expression through inhibition of mitogen-activated protein kinase and redistribution of serum response factor

Platelet-derived growth factor (PDGF) inhibits expression of smooth muscle (SM) genes in vascular smooth muscle cells and blocks induction by arginine vasopressin (AVP). We have previously demonstrated that suppression of SM-alpha-actin by PDGF-BB is mediated in part through a Ras-dependent pathway. This study examined the role of phosphatidylinositol 3-kinase (PI3K)y and its downstream effector, Akt, in regulating SM gene expression. PDGF caused a rapid sustained activation of Akt, whereas AVP caused only a small transient increase. PDGF selectively caused a sustained stimulation of p85/p110 alpha PI3K. In contrast, p85/110 beta PI3K activity was not altered by either PDGF or AVP, whereas both agents caused a delayed activation of Class IB p101/110 gamma PI3K. Expression of a gain-of-function PI3K or myristoylated Akt (myr-Akt) mimicked the inhibitory effect of PDGF on SM-alpha-actin and SM22 alpha expression. Pretreatment with LY 294002 reversed the inhibitory effect of PDGF. Expression of myr-Akt selectively inhibited AVP-induced activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinases, which we have shown are critical for induction of these genes. Nuclear extracts from PDGF-stimulated or myr-Akt expressing cells showed reduced serum response factor binding to SM-specific CArG elements. This was associated with appearance of serum response factor in the cytoplasm. These data indicate that activation of p85/p110 alpha/Akt mediates suppression of SM gene expression by PDGF.     

Cell cycle dependent growth factor regulation of gene expression

The expression of the proto-oncogenes c-fos and c-myc is a rapid response of G0-arrested fibroblasts to serum and peptide growth factors; however, the role of the c-fos and c-myc gene products in subsequent cell cycle transit is not understood. We examined the expression of c-fos and c-myc mRNA in Balb/c 3T3 murine fibroblasts in response to platelet-derived growth factor (PDGF) and platelet-poor plasma, using arrest points associated with density dependent growth inhibition or metabolic inhibition to synchronize cells in S phase of the cell cycle. The expression of c-fos and c-myc mRNA in Balb/c 3T3 cells was differentially regulated with respect to growth factor dependence and cell cycle dependence. c-fos expression was induced in the presence of PDGF and was unaffected by plasma. The induction of c-fos expression in response to PDGF was cell cycle independent, occurring in cells transiting S phase and G2 as well as in G0 arrest. In contrast, c-myc expression was both growth factor and cell cycle dependent. In G0 arrested cells, c-myc expression was PDGF-dependent and plasma-independent, and PDGF was required for maintenance of elevated c-myc levels during G1 transit. In cells transiting S phase, c-myc mRNA was induced in response to PDGF, but was also plasma-dependent in S phase cells that had been "primed" by exposure to PDGF during S phase.     

Platelet-derived growth factor (PDGF) receptor-alpha-activated c-Jun NH2-terminal kinase-1 is critical for PDGF-induced p21WAF1/CIP1 promoter activity independent of p53

Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells. PDGF AA functions as a "competent factor" that stimulates cell cycle entry but requires additional (progression) factors in serum to transit the cell cycle beyond the G1/S checkpoint. Unlike PDGF AA, PDGF B-chain (c-sis) homodimer (PDGF BB) and its viral counterpart v-sis can serve as both competent and progression factors. PDGF BB activates alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR) and induces phenotypic transformation in NIH 3T3 cells, whereas PDGF AA activates alpha-PDGFR only and fails to induce transformation. We showed previously that alpha-PDGFR antagonizes beta-PDGFR-mediated transformation through activation of stress-activated protein kinase-1/c-Jun NH2-terminal kinase-1, whereas both alpha-PDGFR and beta-PDGFR induce mitogenic signals. These studies revealed a striking feature of PDGF signaling; the specificity and the strength of the PDGF growth signal is modulated by alpha-PDGFR-mediated simultaneous activation of growth stimulatory and inhibitory signals, whereas beta-PDGFR mainly induces a growth-promoting signal. Here we demonstrate that PDGF BB activation of beta-PDGFR alone results in more efficient cell cycle transition from G1 to S phase than PDGF BB activation of both alpha-PDGFR and beta-PDGFR. PDGF AA activation of alpha-PDGFR or PDGF BB activation of both alpha- and beta-PDGFRs up-regulates expression of p21WAF1/CIP1, an inhibitor of cell cycle-dependent kinases and a downstream mediator of the tumor suppressor gene product p53. However, beta-PDGFR activation alone fails to induce p21WAF1/CIP1 expression. We also demonstrate that alpha-PDGFR-activated JNK-1 is a critical signaling component for PDGF induction of p21WAF1/CIP1 promoter activity. The ability of PDGF/JNK-1 to induce p21WAF1/CIP1 promoter activity is independent of p53, although the overall p21WAF1/CIP1 promoter activities are greatly reduced in the absence of p53. These results provide a molecular basis for differential regulation of the cell cycle and transformation by alpha- and beta-PDGFRs.     

miR-15b induced by platelet-derived growth factor signaling is required for vascular smooth muscle cell proliferation

The platelet-derived growth factor (PDGF) signaling pathway is essential for inducing a dedifferentiated state of vascular smooth muscle cells (VSMCs). Activation of PDGF inhibits smooth muscle cell (SMC)-specific gene expression and increases the rate of proliferation and migration, leading to dedifferentiation of VSMCs. Recently, microRNAs have been shown to play a critical role in the modulation of the VSMC phenotype in response to extracellular signals. However, little is known about microRNAs regulated by PDGF in VSMCs. Herein, we identify microRNA-15b (miR-15b) as a mediator of VSMC phenotype regulation upon PDGF signaling. We demonstrate that miR-15b is induced by PDGF in pulmonary artery smooth muscle cells and is critical for PDGF-mediated repression of SMC-specific genes. In addition, we show that miR-15b promotes cell proliferation. These results indicate that PDGF signaling regulates SMC-specific gene expression and cell proliferation by modulating the expression of miR-15b to induce a dedifferentiated state in the VSMCs. [BMB Reports 2013; 46(11): 550-554]     

Inflammatory cytokines promote growth of intestinal smooth muscle cells by induced expression of PDGF‐Rβ

Thickening of the inflamed intestinal wall involves growth of smooth muscle cells (SMC), which contributes to stricture formation. Earlier, the growth factor platelet‐derived growth factor (PDGF)‐BB was identified as a key mitogen for SMC from the rat colon (CSMC), and CSMC growth in colitis was associated with both appearance of its receptor, PDGF‐Rβ and modulation of phenotype. Here, we examined the role of inflammatory cytokines in inducing and modulating the growth response to PDGF‐BB. CSMC were enzymatically isolated from Sprague–Dawley rats, and the effect of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, transforming growth factor (TGF), IL‐17A and IL‐2 on CSMC growth and responsiveness to PDGF‐BB were assessed using proliferation assays, PCR and western blotting. Conditioned medium (CM) was obtained at 48 hrs of trinitrobenzene sulphonic acid‐induced colitis. Neither CM alone nor cytokines caused proliferation of early‐passage CSMC. However, CM from inflamed, but not control colon significantly promoted the effect of PDGF‐BB. IL‐1β, TNF‐α and IL‐17A, but not other cytokines, increased the effect of PDGF‐BB because of up‐regulation of mRNA and protein for PDGF‐Rβ without change in receptor phosphorylation. PDGF‐BB was identified in adult rat serum (RS) and RS‐induced CSMC proliferation was inhibited by imatinib, suggesting that blood‐derived PDGF‐BB is a local mitogen in vivo. In freshly isolated CSMC, CM from the inflamed colon as well as IL‐1β and TNF‐α induced the early expression of PDGF‐Rβ, while imatinib blocked subsequent RS‐induced cell proliferation. Thus, pro‐inflammatory cytokines both initiate and maintain a growth response in CSMC via PDGF‐Rβ and serum‐derived PDGF‐BB, and control of PDGF‐Rβ expression may be beneficial in chronic intestinal inflammation.     

Demonstration of stimulatory effects of platelet-derived growth factor on cultivated rat arterial smooth muscle cells : Differences between cells from young and adult animals

The effects of platelet-derived growth factor (PDGF) on DNA synthesis and proliferation in cultures of arterial smooth muscle cells obtained from young and adult rats, respectively, were measured. Addition of 10-20 ng/ml of PDGF to medium MCDB 104 induced DNA synthesis in quiescent cultures of cells from young animals to a similar extent as 10-20% whole blood serum (WBS). PDGF further stimulated proliferation of the cells in medium MCDB 104, although less markedly than 10% WBS. Antibodies against PDGF partially inhibited the growth response after stimulation with serum. This shows that PDGF is a major growth factor in serum for these cells and that PDGF can promote entrance into and passage through S phase and mitosis independent o plasma factors. Cells from adult animals were also found to respond to PDGF, although a higher concentration (25 ng/ml) was required to obtain a maximum effect. These cells, however, responded better than cells from young animals to stimulation with serum. Further, antibodies against PDGF did not inhibit the growth-stimulatory effect of serum to any appreciable extent. Thus, serum contains growth factors other than PDGF that stimulate preferentiaLly the proliferation of smooth muscle cells from adult animals.     

Calcium and growth responses of hyperresponsive airway smooth muscle to different isoforms of platelet-derived growth factor (PDGF)

Airway smooth muscle (ASM) mass is likely to be an important determinant of airway responsiveness. Highly inbred Fisher rats model innate hyperresponsiveness, and also have more ASM in vivo than control Lewis rats. Platelet derived growth factor (PDGF) is an important endogenous growth factor for ASM, and partially purified PDGF-AB causes enhanced growth of Fisher rat ASM cells, compared to Lewis cells. The aim of the present study was to determine the mitogenic effects of all three recombinant PDGF isoforms on ASM cells, and investigate the mechanisms of enhanced Fisher ASM growth responses. The potential mechanisms assessed include PDGF receptor expression and activation (tyrosine phosphorylation), and intracellular calcium (Ca2+) responses to PDGF isoforms. Fisher ASM cells had a greater mitogenic response to PDGF-AB and -AA, and a greater Ca2+ response to -BB than Lewis ASM cells. A Ca2+ response was not necessary for a mitogenic response, and the effects of PDGF isoforms on Ca2+ were not associated with their effects on growth. Therefore, we suggest that enhanced Fisher mitogenic response to PDGF-AA and -AB is not mediated by differences in Ca2+ signalling. Western analysis of the PDGF receptor indicated a similar expression of beta-PDGF receptor in ASM cells from the two rat strains, but a greater expression of alpha-PDGF receptor in Fisher cells; however, phosphorylation of the PDGF receptor following growth stimulation did not differ between strains. This suggests a role for post-receptor signals, in addition to enhanced receptor expression, in the enhanced growth response of Fisher ASM cells to PDGF-AA and -AB.     

Stimulation of platelet-derived growth factor-induced DNA synthesis by angiotensin II in rabbit vascular smooth muscle cells

In cultured rabbit vascular smooth muscle cells (VSMCs), angiotensin II by itself had little mitogenic effect even in the presence of cell-free plasma-derived serum (PDS), but markedly stimulated the platelet-derived growth factor (PDGF)-induced DNA synthesis in the presence of PDS. The maximal extent of DNA synthesis induced by PDGF plus angiotensin II was about twice that induced by PDGF alone. The stimulatory effect of angiotensin II was dose-dependent with the maximal response seen at 1 microM and was inhibited by the specific angiotensin II receptor antagonist, [Sar1, Ile8]angiotensin II. In VSMCs, both PDGF and angiotensin II induced expression of the c-fos gene in dose-dependent manners. In contrast to the synergistic effect of angiotensin II and PDGF on DNA synthesis, they induced expression of the c-fos gene in an additive manner. These results suggest that angiotensin II may act as a growth regulator for VSMCs in addition to acting as a vasoconstrictor.     

Biological properties of a hepatocyte growth factor from rat platelets

In an accompanying communication we demonstrated that about half of the potency of rat serum to stimulate DNA synthesis in cultured adult rat hepatocytes resides in a polypeptidelike substance from the platelets. A lysate of rat platelets was able to restore the potency of platelet-poor rat serum, whereas a lysate of human platelets inhibited thymidine incorporation by the hepatocytes. Moreover, addition to these cultures of either highly purified human platelet-derived growth factor (PDGF) or human platelet factor 4 (PF-4) failed to influence DNA synthesis either alone or in the presence of rat or human platelet-poor serum, which is required for expression of PDGF activity. Unlike the human platelet factors, rat platelet lysate (RPL) was moderately active by itself and was augmented equally well by platelet-poor serum from either source. At concentrations below 5%, platelet-poor serum from hypophysectomized rats was as potent as that from normal rats in augmenting RPL activity. This suggests that, unlike PDGF, which is not activated by hypophysectomized rat serum, the hepatotrophic component of RPL does not require the presence of exogenous somatomedins for activity, but interacts instead with other plasma constituents or with somatomedins produced by the hepatocytes in vitro. Rat platelets do, however, appear to contain PDGF or its rat equivalent in addition to the hepatocyte growth factor, since if they are heated to 100 degrees C for 10 min, their ability to stimulate nuclear labeling in confluent BALB/c 3T3 cells is not impaired, while their ability to stimulate DNA synthesis in rat hepatocytes is destroyed. These studies indicate that the hepatocyte growth factor from rat platelets differs from PDGF in its biological as well as physical characteristics, but that rat platelets also contain PDGF or an equivalent substance.     

Alteration of the chemotactic response of NIH/3T3 cells to PDGF by growth factors, transformation, and tumor promoters

The platelet-derived growth factor (PDGF) is a potent chemoattractant for cells that respond to PDGF as a mitogen. The chemotactic response of these cells to PDGF is inversely related to their rate of proliferation, with quiescent cells exhibiting a 25-fold greater chemotactic response than exponentially growing cells. Factors that stimulate the growth of quiescent cells (EGF, FGF, PDGF, and serum) decrease the cells' migratory response to PDGF but not to fibronectin, suggesting that the decreased migration is not due to a general paralysis of cell motility. Transformed lines of NIH/3T3 cells lose their ability to respond to PDGF as a chemoattractant but can still migrate in response to fibronectin. Similarly, after treatment of 3T3 cells with the tumor-promoter phorbol myristate acetate, which induces a transformation-like phenotype, the cells no longer respond to PDGF as a chemoattractant but retain their migratory response to fibronectin. Thus it appears that the growth state of the cells can alter their migratory response to PDGF. These data suggest that growth factors, transformation, and tumor promoters specifically alter the cells' ability to respond to the PDGF-mediated chemotactic signal. It appears that both transformation and tumor promoters accomplish this by altering PDGF-binding to the cell surface.     

Evidence that platelet derived growth factor (PDGF) action is required for mesoderm patterning in early amphibian (Xenopus laevis) embryogenesis.

Mesoderm induction is one of the major events of early vertebrate embryonic patterning. It appears to be controlled by sequential and combinatorial actions of several kinds of peptide growth factors. These include activin, fibroblast growth factor (FGF), and transforming growth factor-beta (TGF-beta), among others. In the present study, the function of platelet-derived growth factor (PDGF) in early Xenopus laevis embryogenesis was investigated. In the animal-cap assay, PDGF caused pre-ectodermal tissue to develop a mesoderm specific morphology (elongation) and to express the mesoderm marker genes, MyoD family and alpha-cardiac actin. In addition, two other genes were expressed -related serum response factor SL1 (a dorsal mesodermal marker) and myosin light chain (MLC2-heart marker). A role for PDGF in normal (in vivo) mesoderm induction is implicated because injection of PDGF receptor alpha antisense RNA into 2-cell embryos erased the animal cap's mesoderm marker expression. Those injected embryos also exhibited morphological abnormalities including incomplete gastrulation, failure of neural fold closing, and abnormal somitogenesis.     

Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing

The expression of platelet-derived growth factor (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but fibroblast-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately 20% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, fibroblasts and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell growth in vivo is dependent on factors regulating expression of the receptor.