Platelet-derived Growth Factor-stimulated DNA Synthesis Research Articles

Inhibition of neointimal formation by trans‐resveratrol: Role of phosphatidyl inositol 3‐kinase‐dependent Nrf2 activation in heme oxygenase‐1 induction

Neointima, defined as abnormal growth of the intimal layer of blood vessels, is believed to be a critical event in the development of vascular occlusive disease. Although resveratrol's inhibitory effects on proliferation and migration of vascular smooth muscle cells has been reported, its activity on neointimal formation is still unclear. Oral administration of trans-resveratrol significantly suppressed intimal hyperplasia in a wire-injured femoral artery mouse model. In cultured vascular smooth muscle cells, trans-resveratrol inhibited platelet-derived growth factor-stimulated DNA synthesis and cell proliferation with down-regulation of cyclin D and pRB. Moreover, platelet-derived growth factor-induced production of reactive oxygen species was inhibited by trans-resveratrol and the compound induced heme oxygenase-1 (HO-1). The anti-proliferative activity of trans-resveratrol was reversed by an HO-1 inhibitor, ZnPPIX. Subcellular fractionation and reporter gene analyses revealed that trans-resveratrol increased the level of nuclear Nrf2 and antioxidant response element reporter activity, and that these were essential for the induction of HO-1. Trans-resveratrol also enhanced the activities of phosphatidyl inositol 3-kinase and extracellular signal regulated kinase, and phosphatidyl inositol 3-kinase was required for Nrf2/antioxidant response element-dependent HO-1 induction. These data have significant implications for the elucidation of the pharmacological mechanism by which trans-resveratrol prevents vascular occlusive diseases.

Regulation of iodothyronine deiodinase and roles of thyroid hormones in human coronary artery smooth muscle cells

Thyroid hormones have been reported to have significant effects on the peripheral vascular system, including relaxation of vascular smooth muscle cells and prevention of atherosclerosis. To exert its biological activity, thyroxine (T 4) needs to be converted to 3,5,3′-triiodothyronine (T 3) by type 1 and type 2 iodothyronine deiodinases. We have previously identified type 2 iodothyronine deiodinase (D2) expression in cultured human coronary artery smooth muscle cells (hCASMCs). In the present study, we have characterized the regulation of D2 expression in hCASMCs by stable prostacyclin analogue beraprost sodium (BPS) and platelet derived growth factor (PDGF), and the roles of thyroid hormones in the functions of hCASMCs. BPS increased D2 expression, whereas PDGF suppressed BPS stimulated D2 expression without affecting cAMP production in hCASMCs. PDGF increased DNA synthesis, while BPS, T 3 or T 4 suppressed PDGF stimulated DNA synthesis in hCASMCs. Inhibition of D2 activity by 3,3′,5′-triiodothyronine (rT 3) partially restored T 4 suppression of PDGF stimulated DNA synthesis in hCASMCs. PDGF increased migration activity, whereas BPS, T 3 or T 4 suppressed PDGF stimulated migration activity of hCASMCs. These results suggest that D2 expression is increased by BPS and suppressed by PDGF in hCASMCs, and that intracellular thyroid hormone activation may be involved in the suppression of DNA synthesis and migration activity of hCASMCs.

Mixed Lineage Kinase 3 Inhibits Platelet-Derived Growth Factor-Stimulated DNA Synthesis and Matrix mRNA Expression in Mesangial Cells

Mixed lineage kinase 3 (MLK 3) is a recently described member of the MLK subfamily of Ser/Thr protein kinases that interacts with MAPK pathways. The aim of this study was to test the potential interaction of MLK 3 with signaling pathways stimulated by PDGF in rat mesangial cells. We have established a stable cell line expressing human MLK 3 in rat glomerular mesangial cells. The effects of PDGF on proliferation and matrix mRNA expression were examined. In control (vector-transfected) mesangial cells PDGF increased [³H]-thymidine incorporation significantly in a concentration-dependent manner. In mesangial cells expressing MLK 3, PDGF-induced increase in DNA synthesis was significantly reduced. PDGF also induced fibronectin and collagen I mRNA expression in control cells, the effects of which were also significantly blocked in MLK 3-transfected cells. To understand the potential interaction of MLK 3 over expression with the MAPK pathways and to examine the potential mechanism of the effects of MLK 3 over expression on proliferation and matrix expression, activation of ERK2, JNK1 and p38 were examined. ERK2 activation was increased several fold by PDGF in control cells but was attenuated significantly in MLK 3 expressing cells. PDGF did not have any effect on JNK and p38 activation, in either cell types. Using the same stable-transfected cell line, identical results were obtained on proliferation and matrix expression with sarafotoxin-s6b (endothelin receptor agonist) another potent mitogenic and sclerotic agent for mesangial cells. These results indicate an important role for MLK 3 in the regulation of growth and matrix expression in mesangial cells.

Potentiation of mitogenic activity of platelet-derived growth factor by physiological concentrations of insulin via the MAP kinase cascade in rat A10 vascular smooth muscle cells.

Hyperinsulinemia has been shown to be associated with diabetic angiopathy. Migration and proliferation of vascular smooth muscle cells (VSMC) are the processes required for the development of atherosclerosis. In this study, we attempted to determine whether insulin affects mitogenic signaling induced by plateletderived growth factor (PDGF) in a rat VSMC cell line (A10 cells). PDGF stimulated DNA synthesis which was totally dependent on Ras, because transfection of dominant negative Ras resulted in complete loss of PDGF-stimulated DNA synthesis. Initiation of DNA synthesis was preceded by activation of Raf-1, MEK and MAP kinases (Erk 1 and Erk2). Treatment of the cells with PD98059, an inhibitor of MAPK kinase (MEK) attenuated but did not abolish PDGF-stimulated DNA synthesis, suggesting that MAPK is required but not essential for DNA synthesis. PDGF also stimulated phosphorylation of protein kinase B (Akt/PKB) and p70 S6Kinase (p70S6K) in a wortmannin-sensitive manner. Rapamycin, an inhibitor of p70S6K, markedly suppressed DNA synthesis. Low concentrations of insulin (1-10 nmol/l) alone showed little mitogenic activity and no significant effect on MAPK activity. However, the presence of insulin enhanced both DNA synthesis and MAPK activation by PDGF. The enhancing effect of insulin was not seen in cells treated with PD98059. Insulin was without effect on PDGF-stimulated activations of protein kinase B (Akt/PKB) and p70S6K. We conclude that insulin, at pathophysiologically relevant concentrations, potentiates the PDGFstimulated DNA synthesis, at least in part, by potentiating activation of the MAPK cascade. These results are consistent with the notion that hyperinsulinemia is a risk factor for the development of atherosclerosis.

Identification of Nck Family Genes, Chromosomal Localization, Expression, and Signaling Specificity

Already a dozen molecules share binding to the Src homology (SH) 3 domains of human Nck, an SH3-SH3-SH3-SH2 adapter protein. We reason that there may be multiple gene members of Nck to accommodate the large binding repertoires. Here we report identification of novel human and mouse Nck genes and rename them as the Nckalpha and Nckbeta genes (including the human Nckalpha, human Nckbeta, mouse Nckalpha, and mouse Nckbeta genes). Nckalpha and Nckbeta share 68% amino acid identity, whereas the two Nckalpha and two Nckbeta across the species show 96% identity to each other. The human Nckbeta gene is mapped to 2q12, whereas the human Nckalpha gene has previously been mapped at 3q21. Antibodies specifically against Nckalpha and Nckbeta detect Nckalpha and Nckbeta with an identical molecular mass in the same cells of various origins. Ectopically expressed Nckbeta, but not its SH2 domain mutant, strongly inhibits epidermal growth factor- and platelet-derived growth factor-stimulated DNA synthesis. Consistently, epidermal growth factor receptor and platelet-derived growth factor receptor preferentially interact with Nckbeta over Nckalpha in vitro. This study indicates that Nck is a multiple gene family and that each gene may have its own signaling specificity. Because previous anti-Nck (human Nckalpha) antibodies cross-react with Nckbeta, reassessment of those studies with specific Nck genes would be necessary.

Compartmentalized Production of Ceramide at the Cell Surface

Ceramide produced by the hydrolysis of sphingomyelin is an important cellular intermediate in hormone action. Here, we present evidence that interleukin 1 beta (IL-1 beta) binding to normal human fibroblasts initiates a lipid messenger cascade that takes place in a sphingomyelin-rich plasma membrane domain with the characteristics of caveolae. Hormone binding first stimulated the appearance of diacylglycerol (DAG) in a caveolearich membrane fraction isolated from whole cells. This was immediately followed by the loss of a resident population of sphingomyelin from the fraction and the concomitant appearance of ceramide. The ceramide produced in response to IL-1 beta blocked platelet-derived growth factor-stimulated DNA synthesis. IL-1 beta stimulated the appearance of DAG in other fractions from the same cell, but this DAG was not coupled to ceramide production. This indicates that ceramide production is highly compartmentalized at the cell surface. Since caveolae are known to be involved in membrane internalization, they may be essential for the delivery of ceramide to a site of action within the cell.

Activated Gq-alpha potentiates platelet-derived growth factor-stimulated mitogenesis in confluent cell cultures.

We studied the effects of activation of the Gq-alpha signaling pathway on mitogenesis by expressing a mutant (Q209L), activated alpha-subunit of Gq (alpha q*) in NIH-3T3 cells. A clonal NIH-3T3 cell line expressing alpha q* in an inducible manner was isolated. Expression of alpha q* is induced with dexamethasone, allowing the use of non-induced cells as controls for the effects of alpha q* expression. We found that, by itself, expression of alpha q* did not increase either DNA synthesis or mitogen-activated protein (MAP) kinase activity in serum-starved cells. Because alpha q* transforms cells grown in the presence of serum (De Vivo M., Chen, J., Codina, J., and Iyengar, R. (1992) J. Biol. Chem. 267, 18263-18266), we tested whether growth factor-stimulated signaling and mitogenesis were affected by expression of alpha q*. Platelet-derived growth factor (PDGF) stimulated thymidine incorporation modestly (50%) in contact-inhibited, confluent cell cultures. In cells expressing alpha q*, PDGF stimulated DNA synthesis up to 3-fold over basal. Concomitant with the potentiation of PDGF-stimulated DNA synthesis, expression of alpha q* potentiated PDGF-stimulated p44 MAP kinase activity. PDGF was much more effective in stimulating both DNA synthesis and p44 MAP kinase activity in subconfluent cell cultures and expression of alpha q* exerted little or no effect on PDGF-stimulated effects in subconfluent cells. These data show that cooperation between signaling pathways may occur in a cell state-specific fashion. Such cooperation in part may be responsible for the triggering of complex cellular responses such as cell transformation.

Preferential inhibition of platelet-derived growth factor-stimulated DNA synthesis and protein tyrosine phosphorylation by nordihydroguaiaretic acid.

Nordihydroguaiaretic acid (NDGA), a reportedly specific lipoxygenase inhibitor, was found to selectively inhibit platelet-derived growth factor (PDGF)-stimulated DNA synthesis in Swiss 3T3 cells. Maximal inhibition of PDGF-induced [3H]thymidine incorporation (96%) was observed using 4 microM NDGA (IC50 = 1.5 microM). No effect of NDGA was observed upon DNA synthesis stimulated with either fetal bovine serum, bombesin, or epidermal growth factor (EGF) in the presence of insulin, or with the potent mitogen Pasteurella multocida toxin. The selective inhibition of PDGF-stimulated DNA synthesis by NDGA was also observed in diploid murine cells, rat, and human fibroblasts. Furthermore, 4 microM NDGA also inhibited PDGF-stimulated anchorage-independent colony growth of rat-1 cells by 76%. Using Swiss 3T3 cells, we found that PDGF-stimulated arachidonic acid mobilization and prostaglandin E2 production was abolished by NDGA in a dose-dependent manner. Inhibition of PDGF-stimulated arachidonic acid mobilization by NDGA could not, however, explain its potent inhibitory effect upon PDGF-stimulated DNA synthesis. Our results showed that NDGA also selectively inhibited PDGF receptor tyrosine phosphorylation in a dose-dependent manner in intact cells. Protein tyrosine phosphorylation stimulated by EGF or bombesin was not altered by NDGA treatment. Crucially, NDGA inhibited in vitro the tyrosine kinase activity of anti-phosphotyrosine and anti-PDGF receptor immunoprecipitates prepared from cultures stimulated with PDGF. This inhibition of receptor tyrosine phosphorylation in a cell-free system confirmed that NDGA acts directly at the level of the PDGF receptor tyrosine kinase domain. These results suggest that the potent and selective inhibitory effect of NDGA on PDGF-stimulated DNA synthesis results from its inhibitory action on tyrosine phosphorylation.

Vascular smooth muscle cells from genetically hyperlipidemic rabbit (WHHL rabbit) exhibit decreased growth response

The Watanabe Heritable Hyperlipidemic (WHHL) rabbit is a widely studied animal model for the human genetic disorder familial hypercholesterolemia, and spontaneously develops atherosclerotic disease. We studied the growth characteristics of cultured vascular smooth muscle cells (VSMC) from WHHL rabbits compared with VSMC from Japanese white rabbits. We measured cell proliferation, DNA synthesis, and c-myc proto-oncogene expression, in response to growth stimuli such as fetal bovine serum (FBS) and platelet-derived growth factor (PDGF). VSMC from Japanese white rabbits exhibited a 4-fold increase in cell numbers during a 5-day incubation period compared with those from WHHL rabbits. FBS and PDGF stimulated DNA synthesis, as measured by thymidine incorporation into VSMC, in both Japanese white rabbits and WHHL rabbits, however the response was significantly higher in the former strain. The intracellular pH value of VSMC determined using the pH-sensitive fluorescence dye 2′,7′-bis-carboxyethyl-carboxyfluorescein was significantly higher in WHHL rabbits than in Japanese white rabbits. Proto-oncogene c-myc was induced by exposure of VSMC to FBS, however there was no significant difference in c-myc mRNA levels between the two strains. These results suggest that VSMC from WHHL rabbits are not genetically growth accelerated, but show decreased growth response to growth stimuli.

The type II insulin-like growth factor receptor does not mediate deoxyribonucleic acid synthesis in human fibroblasts.

Two insulin-like growth factor (IGF) receptors, the type I and type II IGF receptors, have been described. While substantial evidence indicates that the type I receptor is involved in the regulation of cell division, it is uncertain if the type II receptor also mediates this response. Similarly, the role of the insulin receptor in mediating DNA synthesis remains controversial. To address these questions, we used a monoclonal antibody (alpha IR-3) to specifically inhibit type I IGF receptor activity and examined the effects of this inhibition on IGF- and insulin-stimulated DNA synthesis in human fibroblasts. WI-38 human embryonic lung fibroblasts have both type I and type II IGF receptors, as determined by cross-linking [125I] IGF-I and [125I]IGF-II to monolayers of these cells. In serum-free medium both IGF-I and IGF-II stimulate DNA synthesis in WI-38 fibroblasts, with half-maximal effects occurring at 1.5 +/- 0.3 (+/- SD) and 3.4 +/- 1.4 nM, respectively. At maximally effective concentrations, however, both hormones stimulate DNA synthesis to equal levels. alpha IR-3 binds to the type I, but not the type II, IGF receptor on WI-38 cells. It also inhibits IGF binding to the type I receptor on these cells. alpha IR-3 competitively inhibited both IGF-I- and IGF-II-stimulated DNA synthesis in WI-38 cells, but had no effect on either epidermal growth factor- or platelet-derived growth factor-stimulated DNA synthesis. These results indicate that in WI-38 fibroblasts the mitogenic effects of both IGF-I and IGF-II are mediated through the type I receptor and that the type II IGF receptor is not directly involved in this response. To define the role of the insulin receptor in mediating DNA synthesis we compared the effects of alpha IR-3 on insulin-stimulated DNA synthesis in a variety of human cell lines under identical experimental conditions. With WI-38 and HEL, another human embryonic lung fibroblast cell line, alpha IR-3 competitively inhibited the mitogenic effect of insulin. However, in two other fibroblast cell lines (GM498 and HES) and an osteogenic sarcoma cell line (MG63), alpha IR-3 inhibited IGF, but not insulin-stimulated DNA synthesis. These results indicate that human cell lines differ in the receptor type through which insulin stimulates DNA synthesis and that these differences are intrinsic properties of the cell lines and are not artifacts resulting from differences in experimental conditions.

Possible involvement of protein kinase C in platelet-derived growth factor-stimulated DNA synthesis in vascular smooth muscle cells

In cultured rabbit aortic vascular smooth muscle cells (VSMC, protein kinase C-activating phorbol esters such as 12- O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu) stimulated DNA synthesis in the presence of 10% cell-free plasma-derived serum. This stimulation was half that shown by PDGF. 4α-Phorbol-12,13-didecanoate, known to be inactive for protein kinase C, was without effect in stimulating DNA synthesis. Prolonged treatment of the cells with PDBu led to a marked decrease in protein kinase C. In the pDBu-treated cells, the TPA-stimulated DNA synthesis was completely abolished whereas the PDGF-stimulated DNA synthesis was decreased to about half that in the control cells. These results suggest that protein kinase C is involved in PDGF-stimulated proliferation of VSMC.