Inhibition of cell proliferation by alpha-tocopherol. Role of protein kinase C

The effect of alpha-tocopherol (vitamin E) on the proliferation of vascular smooth muscle cells (A7r5), human osteosarcoma cells (Saos-2), fibroblasts (Balb/3T3), and neuroblastoma cells (NB2A) has been studied. The proliferation of vascular smooth muscle cells was inhibited by physiologically relevant concentrations of alpha-tocopherol, neuroblastoma cells were only sensitive to higher alpha-tocopherol concentrations, and proliferation of the other cell lines was not inhibited. The inhibition of smooth muscle cell proliferation was specific for alpha-tocopherol. Trolox, phytol, and alpha-tocopherol esters had no effect. Proliferation of smooth muscle cells stimulated by platelet-derived growth factor or endothelin was completely sensitive to alpha-tocopherol. If smooth muscle cells were stimulated by fetal calf serum, proliferation was 50% inhibited by alpha-tocopherol. No effect of alpha-tocopherol was observed when proliferation of smooth muscle cells was stimulated by bombesin and lysophosphatidic acid. The possibility of an involvement of protein kinase C in the cell response to alpha-tocopherol was suggested by experiments with the isolated enzyme and supported by the 2- to 3-fold stimulation of phorbol ester binding induced by alpha-tocopherol in sensitive cells. Moreover, alpha-tocopherol also caused inhibition of protein kinase C translocation induced by phorbol esters and inhibition of the phosphorylation of its 80-kDa protein substrate in smooth muscle cells. A model is discussed by which alpha-tocopherol inhibits cell proliferation by interacting with the cytosolic protein kinase C, thus preventing its membrane translocation and activation.     

Expression of genes related to the extracellular matrix in human endothelial cells. Differential modulation by elevated glucose concentrations, phorbol esters, and cAMP.

To identify agents and mechanisms responsible for the thickened basement membranes characteristic of diabetic angiopathy we examined the effects of high glucose (30 mM) on the expression of genes related to extracellular matrix composition and turnover and investigated whether the changes induced by high glucose were mimicked and sustained by activation of protein kinase C or A. In human umbilical vein endothelial cells high glucose increased fibronectin, collagen IV, tissue plasminogen activator (tPA), and plasminogen activator-inhibitor 1 (PAI-1) mRNA levels 2-fold but did not affect type IV and interstitial collagenase expression. Acute treatment with phorbol esters resulted in increased collagen IV, tPA, PAI-1, and interstitial collagenase mRNAs; the type IV collagenase mRNA levels were instead suppressed to 50% of control. Upon longer exposure to phorbol esters (48 h) suppression of fibronectin and PAI-1 mRNAs also occurred. Intracellular elevation of cAMP led to over-expression of fibronectin and type IV collagenase and potentiated the effects of phorbol esters on collagen IV, tPA, and interstitial collagenase expression. The mRNA changes induced by high glucose occurred in the absence of protein kinase C activation or cAMP elevation. These studies indicate that events other than activation of protein kinase C or A bridge high ambient glucose to changes in endothelial cell gene expression that may contribute to diabetic angiopathy.     

Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells.

We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.     

Hormonal regulation of tissue-type plasminogen activator messenger ribonucleic acid levels in rat granulosa cells: mechanisms of induction by follicle-stimulating hormone and gonadotropin releasing hormone

FSH and GnRH both stimulate rat granulosa cells to produce tissue-type plasminogen activator (tPA). We have studied the molecular mechanisms involved in the action of these hormones by measuring tPA mRNA levels in primary cultures of rat granulosa cells. When granulosa cells were cultured in the presence of FSH or GnRH the level of tPA mRNA was increased 20- and 12-fold, respectively. The induction of tPA mRNA by FSH and GnRH was additive and the kinetics of induction differed. The effect of FSH could be mimicked by bromo-cAMP or forskolin, and was drastically enhanced by cotreatment with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. These findings are consistent with the notion that FSH mediates its effect through the protein kinase A pathway. GnRH is believed to augment phospholipid turnover in granulosa cells, leading to the activation of the protein kinase C pathway. Like GnRH, the protein kinase C activator phorbol myristate acetate also induced tPA mRNA in granulosa cells. In the presence of the protein synthesis inhibitor, cycloheximide, FSH-stimulated tPA message levels were enhanced by 30-fold, revealing superinduction of tPA mRNA levels by this pathway. In contrast the induction of tPA mRNA by GnRH was inhibited by cycloheximide indicating that the synthesis of an intermediate protein is required for the GnRH effect. Our data suggest that FSH and GnRH increase the tPA mRNA levels by two distinct pathways in cultured granulosa cells, providing a model system for studying the hormonal regulation of tPA gene expression.     

Heparin and dibutyryl cAMP modulate gene expression in stimulated human saphenous vein smooth muscle cells

Summary Increased expression of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) A chain, and tissue plasminogen activator (tPA) by smooth muscle cells (SMC) has been postulated to mediate the progression of intimal hyperplasia. We tested whether heparin would suppress the expression of these genes in stimulated human saphenous vein SMC. Quiescent cultured human saphenous vein SMC were stimulated for 4 h with heat-inactivated fetal bovine serum (10% by vol) in the presence or absence of heparin (1 to 250μg/ml). Heparin (50μg/ml) attenuated the induction by serum of bFGF mRNA, tPA mRNA, and tPA secretion. Nonanticoagulant heparin also attenuated serum induction of bFGF and tPA mRNA levels. To further study the role of second messenger signaling, a more specific mode of SMC stimulation was used with thrombin (3 U/ml) in the presence or absence of dibutyryl cyclic AMP (Bu₂-cAMP; 0.5 mM). In contrast to heparin, which had no effect on PDGF expression, Bu₂-cAMP decreased the induction by thrombin of PDGF-A chain mRNA levels. In thrombin-stimulated SMC, Bu₂-cAMP significantly decreased secretion of PDGF-AA protein. Thrombin, however, caused an increase in bFGF mRNA levels which was potentiated by Bu₂-cAMP with associated potentiation by Bu₂-cAMP of intracellular bFGF protein levels. The induction of tPA mRNA and tPA secretion by thrombin was sharply blocked by Bu₂-cAMP. These results suggest that heparin reduces intimal hyperplasia at least partly via partial inhibition of SMC gene expression.     

Mechanisms of inhibition by heparin of PDGF stimulated MAP kinase activation in vascular smooth muscle cells

Heparin and heparan are potent inhibitors of vascular smooth muscle cell (VSMC) proliferation. To investigate the mechanisms by which heparin suppresses growth factor stimulated mitogenesis, the present experiments investigated the effects of heparin on platelet-derived growth factor (PDGF) stimulated signal transduction pathways. Heparin treatment substantially inhibited PDGF-BB stimulated rat VSMC growth. Western analysis showed a 30 min PDGF-BB treatment of VSMC induced the tyrosine phosphorylation of multiple protein bands; cotreatment with heparin inhibited mitogen-activated protein (MAP) kinase tyrosine phosphorylation but had little effect on PDGF receptor tyrosine phosphorylation. In-gel kinase assays demonstrated that heparin inhibited PDGF-BB stimulated MAP kinase activity at late (25 min) but not early (10 min) time points. These data indicate that heparin does not inhibit the initial signalling events after PDGF-BB binding but instead acts through an alternate mechanism to inhibit MAP kinase. To investigate if heparin directly stimulates tyrosine phosphatase-mediated suppression of MAP kinase, we treated VSMC with orthovanadate, a tyrosine phosphatase inhibitor. Heparin inhibited MAP kinase tyrosine phosphorylation after orthovanadate treatment, indicating that heparin does not suppress MAP kinase by enlistment of a tyrosine phosphatase. Experiments were performed to investigate signalling pathways upstream of MAP kinase. To determine if protein kinase C (PKC) mediates PDGF-BB, serum, and EGF stimulation of MAP kinase, we treated VSMC overnight with phorbol ester (PMA) to downregulate PKC. Abolition of conventional and novel PKC activity significantly suppressed both serum and PDGF-BB induced MAP kinase activation, indicating protein kinase C is an important mediator for these mitogens. In contrast, downregulation of these PKC isoforms had little effect on EGF stimulation of MAP kinase. As heparin inhibits PDGF and serum but not EGF stimulation of MAP kinase, these data precisely correlate heparin inhibition of MAP kinase with activation through PKC-dependent pathways. Immunoprecipitation analysis found that heparin inhibited serum, PMA, and PDGF but not EGF induced raf-1 phosphorylation. These studies demonstrate that heparin did not block PDGF-BB receptor activation, which initiates the mitogenic signalling cascade. Heparin did inhibit specific postreceptor second messenger signals, such as the late phase activation of MAP kinase, which may be mediated by suppression of PKC-dependent pathways. J. Cell. Physiol. 172:69–78, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of endothelial tissue plasminogen activator and plasminogen activator inhibitor type 1 synthesis by diacylglycerol, phorbol ester, and thrombin

The influence of diacylglycerols, which are physiological activators of protein kinase C, on the production of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) by human umbilical vein endothelial cells (HUVEC) was studied in order to gain insight into the regulation of fibrinolysis by these cells. 1,2-dioctanoyl-sn-glycerol (diC8) stimulated tPA production in a dose- and time-dependent manner. The tPA antigen in cell supernatants increased from 0.9 ng/10(6) cells in unstimulated cells to 12.4 ng (10(6) cells after incubation with 400 microM diC8 for 24 hours. In contrast, PAI-1 production was not influenced by diC8, whereas phorbol 12-myristate 13-acetate (PMA) or thrombin stimulated both, tPA and PAI-1 production by HUVEC. Staurosporine and H7, which are inhibitors of protein kinase C, inhibited tPA synthesis by HUVEC. The degree of inhibition was dependent on the agonist used. While diC8-induced tPA production was inhibited to more than 80% by H7 (10 microM) and staurosporine (10 nM), higher doses of inhibitors were required to inhibit thrombin- and PMA-induced tPA production. Thrombin-induced PAI-1 production was inhibited to more than 80% by H7 (10 microM) and to about 50% by staurosporine, whereas PMA-induced PAI-1 production was not inhibited by staurosporine, and only to about 50% by higher doses of H7 (30 microM). These data suggest that activation of protein kinase C is a common intracellular trigger mechanism for the induction of tPA synthesis by HUVEC. Protein kinase C is most likely also involved in the regulation of PAI-1 synthesis by HUVEC.     

1-O-alkyl-2-acetyl-sn-glycerol: a platelet-activating factor metabolite with biological activity in vascular smooth muscle cells.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a potent vasoactive ether lipid produced by activated blood cells and endothelial cells. Vascular smooth muscle cells partially convert exogenous PAF to 1-O-alkyl-2-acetyl-sn-glycerol (AAG), a biologically active diacylglycerol analogue. AAG is formed rapidly (less than 15 s) after exposure of the smooth muscle cells and does not appear to be a substrate for diacylglycerol kinase in these cells. Although most of the compound is metabolized to 1-O-alkyl-sn-glycerol, a small quantity remains as AAG for greater than or equal to 6 h. AAG inhibits phorbol ester binding, and it is as effective an activator of protein kinase C as diolein in an in vitro assay. Furthermore, AAG and PAF produce the same pattern of effects on smooth muscle cell proliferation. These observations suggest that at least some of the actions of PAF in vascular smooth muscle may be mediated through the formation of AAG, a stable, bioactive metabolite that appears to function as a diacylglycerol analogue.     

Effect of heparin on vascular smooth muscle cells. II. Specific protein synthesis

Heparin suppresses the proliferation of vascular smooth muscle cells both in vivo and in vitro. The mechanism of action of the antiproliferative activity of heparin is not known. We have detected differences in the synthesis of specific proteins when vascular smooth muscle cells are exposed to heparin and report here that many characteristics of these protein alterations parallel the properties of the antiproliferative activity. The induction into the culture medium of a pair of proteins of approximately 35,000 dalton mw in heparin-treated smooth muscle cell cultures and the antiproliferative effect of heparin share the following characteristics: 1) the effect is reversible, 2) the effect is specific for smooth muscle cells, 3) anticoagulant and non-anticoagulant heparin are equally effective, 4) the effect is lost with time in culture and, 5) heparin is the most potent glycosaminoglycan in producing the effect. Furthermore, heparin causes a transient suppression of a 48,000 dalton substrate-attached protein, whereas chondroitin sulfate A and C and dermatan sulfate had much less effect. Dextran sulfate was almost as effective as heparin in suppressing the synthesis of the substrate-attached protein. These proteins appear to be noncollagenous and the induced synthesis of the 35,000 dalton proteins is inhibited by actinomycin D. Although a direct relationship between these specific protein changes and the antiproliferative effect of heparin has not been proven, these protein alterations may play a crucial role in the effect of heparin on smooth muscle cell growth.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates the expression and the release of tissue plasminogen activator (tPA) in neuronal cells: involvement of tPA in the neuroprotective effect of PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) and tissue plasminogen activator (tPA) play important roles in neuronal migration and survival. However, a direct link between the neurotrophic effects of PACAP and tPA has never been investigated. In this study, we show that, in PC12 cells, PACAP induced a 9.85-fold increase in tPA gene expression through activation of the protein kinase A- and protein kinase C-dependent signaling pathways. In immature cerebellar granule neurons (CGN), PACAP stimulated tPA mRNA expression and release of proteolytically active tPA. Immunocytochemical labeling revealed the presence of tPA in the cytoplasm and processes of cultured CGN. The inhibitory effect of PACAP on CGN motility was not affected by the tPA substrate plasminogen or the tPA inhibitor plasminogen activator inhibitor-1. In contrast, plasminogen activator inhibitor-1 significantly reduced the stimulatory effect of PACAP on CGN survival. Altogether, these data indicate that tPA gene expression is activated by PACAP in both tumoral and normal neuronal cells. The present study also demonstrates that PACAP stimulates the release of tPA which promotes CGN survival by a mechanism dependent of its proteolytic activity.     

Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells [published erratum appears in J Cell Biol 1990 Mar;110(3):863]

The proliferation of arterial smooth muscle cells (SMCs) plays a critical role in the pathogenesis of arteriosclerosis. Previous studies have indicated that the glycosaminoglycan heparin specifically inhibited the growth of vascular SMCs in vivo and in culture, although the precise mechanism(s) of action have not been elucidated. In this study, we have examined the ability of specific mitogens (PDGF, EGF, heparin-binding growth factors, phorbol esters, and insulin) to stimulate SMC proliferation. Our results indicate that SMCs derived from different species and vascular sources respond differently to these growth factors. We next examined the ability of heparin to inhibit the proliferative responses to these mitogens. In calf aortic SMCs, heparin inhibits a protein kinase C-dependent pathway for mitogenesis. Detailed cell cycle analysis revealed several new features of the effects of heparin on SMCs. For example, heparin has two effects on the Go----S transition: it delays entry into S phase and also reduces the number of cells entering the cycle from Go. Using two separate experimental approaches, we found that heparin must be present during the last 4 h before S phase, suggesting a mid-to-late G1 heparin block. In addition, our data indicate that heparin-treated SMCs, while initially blocked in mid-to-late G1, slowly move back into a quiescent growth state in the continued presence of heparin. These results suggest that heparin may have multiple targets for its antiproliferative effect.     

Activation of EphB2 and its ligands promotes vascular smooth muscle cell proliferation.

EphB2 and its ligands regulate interactions between endothelial and mesenchymal cells in developing arteries. In adult arteries, the relationship between smooth muscle cells and overlying intact endothelium is responsible for maintaining the health of the vessel. Heparin inhibits vascular smooth muscle cell growth in culture and intimal hyperplasia following endothelial denudation. Using gene microarrays, we identified the tyrosine kinase receptor EphB2 as being differentially expressed in response to continuous intravenous heparin administration in the rabbit model of arterial injury. EphB2 protein levels increased in cultured bovine vascular smooth muscle cells following serum stimulation and were decreased in a dose-dependent fashion by heparin. Fc chimeras of the binding domain of the EphB2 ligands blocked the formation of the EphB2 ligand-receptor complex and reduced growth of serum-stimulated vascular smooth muscle cells in a dose-dependent fashion. Activation of the ligand by an Fc chimera to EphB2 followed a parabolic dose-response growth curve, indicating growth stimulation until the chimera begins to compete with native receptors. Co-administration of EphB2/Fc chimera with heparin shifted the dose-response curve to the right. These data indicate a possible new route of Heparin's antiproliferative effect and a role of EphB2 and its ligands in vascular smooth muscle cell proliferation.     

Enhancement by homocysteine of plasminogen activator inhibitor-1 gene expression and secretion from vascular endothelial and smooth muscle cells

In order to elucidate the relationship between homocysteine and the fibrinolytic system, we examined the effect of homocysteine on plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (tPA) gene expression and protein secretion in cultured human vascular endothelial and smooth muscle cells in vitro. PAI-1 mRNA and secreted protein levels were both enhanced by homocysteine in a dose dependent manner, with significant stimulation of PAI-1 secretion observed at concentrations greater than 0.5 mM homocysteine. In contrast, secretion and mRNA expression of tPA were not significantly altered by homocysteine stimulation. Secretion of TGFbeta (transforming growth factor beta) and TNFalpha (tumor necrosis factor alpha), possible regulators of PAI-1 expression and secretion, were not stimulated by treatment with 1.0 mM homocysteine. These results suggests that hyperhomocysteinemia-induced atherosclerosis and/or thrombosis may be caused by homocysteine-induced stimulation of PAI-1 gene expression and secretion in the vasculatures by a mechanism independent from paracrine-autocrine activity of TGFbeta and TNFalpha.     

LRP and alphavbeta3 mediate tPA activation of smooth muscle cells

Tissue-type plasminogen activator (tPA) regulates vascular contractility through the low-density lipoprotein-related receptor (LRP), and this effect is inhibited by plasminogen activator inhibitor type 1 (PAI-1). We now report that tPA-mediated vasocontraction also requires the integrin alphavbeta3. tPA-induced contraction of rat aortic rings is inhibited by the Arg-Gly-Asp (RGD) peptide and by monoclonal anti-alphavbeta3 antibody. tPA induces the formation of a complex between LRP and alphavbeta3 in vascular smooth muscle cells. The three proteins are internalized within 10 min, causing the cells to become refractory to the readdition of tPA. LRP and alphavbeta3 return to the cell surface by 90 min, restoring cell responsiveness to tPA. PAI-1 and the PAI-1-derived hexapeptide EEIIMD abolish the vasocontractile activity of tPA and inhibit the tPA-mediated interaction between LRP and alphavbeta3. tPA induces calcium mobilization from intracellular stores in vascular smooth muscle cells, and this effect is inhibited by PAI-1, RGD, and antibodies to both LRP and alphavbeta3. These data indicate that tPA-mediated vasocontraction involves the coordinated interaction of LRP with alphavbeta3. Delineating the mechanism underlying these interactions and the nature of the signals transduced may provide new tools to regulate vascular tone and other consequences of tPA-mediated signaling.     

The mechanism of action of the immunosuppressive drug FK-506

The novel immunosuppressive drug FK-506 inhibits the induction of lymphocyte proliferation in vitro by mitogenic combinations of phorbol esters and calcium ionophores. Early events inducible by phorbol esters alone are unaffected, while changes induced by calcium ionophores alone are completely suppressed by as little as 0.1 nM FK-506. The event in lymphocyte activation inhibited by FK-506 is completed early in the response. Its completion requires the provision of a Ca2+ signal and concurrent activation of protein kinase C and is accelerated as a function of the strength of protein kinase C activation.     

Inhibition of stimulated cell responses by phorbol esters and other activators of protein kinase C: sites of action

The addition of the protein kinase C activator phorbol esters to cell suspension a few minutes prior to stimulation inhibits the agonists-induced biochemical changes and cell responses. This inhibition is prevented by protein kinase C inhibitors. Activation of protein kinase C down regulates the stimulated responses by affecting one or more of the steps in the exitation-response coupling. This includes the receptors, the quanine-nucleotide-binding protein, the activity or distribution of phospholipase C, and other steps.