Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells

Proliferation of vascular smooth muscle cells (VSMC) contributes to the pathogenesis of atherosclerosis, and glycated serum albumin (GSA, Amadori adduct of albumin) might be a mitogen for VSMC proliferation, which may further be associated with diabetic vascular complications. In this study, we investigated the involvement of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and protein kinase C (PKC), in GSA-stimulated mitogenesis, as well as the functional relationship between these factors. VSMC stimulation with GSA resulted in a marked activation of ERK. The MAPK kinase (MEK) inhibitor, PD98059, blocked GSA-stimulated MAPK activation and resulted in an inhibition of GSA-stimulated VSMC proliferation. GSA also increased PKC activity in VSMC in a dose-dependent manner. The inhibition of PKC by the PKC inhibitors, GF109203X and Rottlerin (PKCdelta specific inhibitor), as well as PKC downregulation by phorbol 12-myristate 13-acetate (PMA), inhibited GSA-induced cell proliferation and blocked ERK activation. This indicates that phorbol ester-sensitive PKC isoforms including PKCdelta are involved in MAPK activation. Thus, we show that the MAPK cascade is required for GSA-induced proliferation, and that phorbol ester-sensitive PKC isoforms contribute to cell activation and proliferation in GSA-stimulated VSMC.     

Association of insulin receptor substrate 1 (IRS-1) y895 with Grb-2 mediates the insulin signaling involved in IRS-1-deficient brown adipocyte mitogenesis

We have recently generated immortalized fetal brown adipocyte cell lines from insulin receptor substrate 1 (IRS-1) knockout mice and demonstrated an impairment in insulin-induced lipid synthesis as compared to wild-type cell lines. In this study, we investigated the consequences of IRS-1 deficiency on mitogenesis in response to insulin. The lack of IRS-1 resulted in the inability of insulin-stimulated IRS-1-deficient brown adipocytes to increase DNA synthesis and enter into S/G2/M phases of the cell cycle. These cells showed a severe impairment in activating mitogen-activated protein kinase kinase (MEK1/2) and p42-p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. IRS-1-deficient cells also lacked tyrosine phosphorylation of SHC and showed no SHC-Grb-2 association in response to insulin. The mitogenic response to insulin could be partially restored by enhancing IRS-2 tyrosine phosphorylation and its association with Grb-2 by inhibition of phosphatidylinositol 3-kinase activity through a feedback mechanism. Reconstitution of IRS-1-deficient brown adipocytes with wild-type IRS-1 restored insulin-induced IRS-1 and SHC tyrosine phosphorylation and IRS-1-Grb-2, IRS-1-SHC, and SHC-Grb-2 associations, leading to the activation of MAPK and enhancement of DNA synthesis. Reconstitution of IRS-1-deficient brown adipocytes with the IRS-1 mutant Tyr895Phe, which lacks IRS-1-Grb-2 binding, restored SHC-IRS-1 association and SHC-Grb-2 association. However, the lack of IRS-1-Grb-2 association impaired MAPK activation and DNA synthesis in insulin-stimulated mutant cells. These data provide strong evidence for an essential role of IRS-1 and its direct association with Grb-2 in the insulin signaling pathway leading to MAPK activation and mitogenesis in brown adipocytes.     

Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells.

The mitogenic effect of extracellular ATP on porcine aortic smooth muscle cells (SMC) was examined. Stimulation of [3H]thymidine incorporation by ATP was dose-dependent; the maximal effect was obtained at 100 microM. ATP acted synergistically with insulin, IGF-1, EGF, PDGF, and various other mitogens. Incorporation of [3H]thymidine was correlated with the fraction of [3H]thymidine-labeled nuclei and changes in cell counts. The stimulation of proliferation was also determined by measurement of cellular DNA using bisbenzamide and by following the increase of mitochondrial dehydrogenase protein. The effect of ATP was not due to hydrolysis to adenosine, which shows synergism with ATP. ATP acted as a competence factor. The mitogenic effect of ATP, but not adenosine, was further increased by lysophosphatidate, phosphatidic acid, or norepinephrine. The inhibitor of adenosine deaminase, EHNA, stimulated the effect of adenosine but not ATP. The adenosine receptor antagonist theophylline depressed adenosine-induced mitogenesis. ADP and the non-hydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate (AMP-PNP) were equally mitogenic. Thus extracellular ATP stimulated mitogenesis of SMC via P2Y purinoceptors. The mechanism of ATP acting as a mitogen in SMC was further explored. Extracellular ATP stimulated the release of [3H]arachidonic acid (AA) and prostaglandin E2 (PGE2) into the medium, and enhanced cAMP accumulation in a dose-dependent fashion similar to ATP-induced [3H]thymidine incorporation. Inhibitors of the arachidonic acid metabolism pathway, quinacrine and indomethacin, partially inhibited the mitogenic effect of ATP but not of adenosine. Pertussis toxin inhibited ATP-stimulated DNA synthesis, AA release, PGE2 formation, and cAMP accumulation. Down-regulation of protein kinase C (PKC) by long-term exposure to phorbol dibutyrate (PDBu) partially prevented stimulation of DNA synthesis and activation of the AA pathway by ATP. The PKC inhibitor, staurosporine, antagonized mitogenesis stimulated by ATP. No synergistic effect was found when PDBu and ATP were added together. Therefore, a dual mechanism, including both arachidonic acid metabolism and PKC, is involved in ATP-mediated mitogenesis in SMC. In addition, ATP acted synergistically with angiotensin II, phospholipase C, serotonin, or carbachol to stimulate DNA synthesis. Finally, the possible physiological significance of ATP as a mitogen in SMC was further studied. The effect of endothelin and heparin, which are released from endothelial cells, on ATP-dependent mitogenesis was investigated. Extracellular ATP acted synergistically with endothelin to stimulate a greater extent of [3H]thymidine incorporation than was seen with PDGF plus endothelin. Heparin, believed to have a regulatory role, partially inhibited the stimulation of DNA synthesis caused both by ATP and PDGF.(ABSTRACT TRUNCATED AT 400 WORDS)     

The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes

Ghrelin, a stomach-derived hormone, induces adiposity when administered to rodents. Because ghrelin receptor is abundantly expressed in adipose tissue, we investigated the role of ghrelin in adipocyte biology. We observed ghrelin receptor expression in 3T3-L1 preadipocytes and adipocytes. Treatment of preadipocytes with ghrelin induced cellular proliferation and differentiation to mature adipocytes, as well as basal and insulin-stimulated glucose transport, but it inhibited adipocyte apoptosis induced by serum deprivation. Exposure of 3T3-L1 cells to ghrelin caused a rapid activation of MAPKs, especially ERK1/2. Chemical inhibition of MAPK blocked the mitogenic and antiapoptotic effects of ghrelin. Ghrelin also stimulated the insulin receptor substrate-associated phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 preadipocytes and adipocytes, whereas inhibition of this pathway blocked the effects of ghrelin on cell proliferation, antiapoptosis and glucose uptake. These findings suggest that the direct effects of ghrelin on proliferation, differentiation, and apoptosis in adipocytes may play a role in regulating fat cell number. These effects may be mediated via activation of the MAPK and phosphatidylinositol 3-kinase/Akt pathways.     

Evidence for a role of MEK and MAPK during signal transduction by protein kinase C zeta.

Protein kinase C zeta (zeta PKC) is critically involved in the control of a number of cell functions, including proliferation and nuclear factor kappa B (NF-kappa B) activation. Previous studies indicate that zeta PKC is an important step downstream of Ras in the mitogenic cascade. The stimulation of Ras initiates a kinase cascade that culminates in the activation of MAP kinase (MAPK), which is required for cell growth. MAPK is activated by phosphorylation by another kinase named MAPK kinase (MEK), which is the substrate of a number of Ras-activated serine/threonine kinases such as c-Raf-1 and B-Raf. We show here that MAPK and MEK are activated in vivo by an active mutant of zeta PKC, and that a kinase-defective dominant negative mutant of zeta PKC dramatically impairs the activation of both MEK and MAPK by serum and tumour necrosis factor (TNF alpha). The stimulation of other kinases, such as stress-activated protein kinase (SAPK) or p70S6K, is shown here to be independent of zeta PKC. The importance of MEK/MAPK in the signalling mechanisms activated by zeta PKC was addressed by using the activation of a kappa B-dependent promoter as a biological read-out of zeta PKC.     

Interferons block protein kinase C-dependent but not-independent activation of Raf-1 and mitogen-activated protein kinases and mitogenesis in NIH 3T3 cells.

Interferons (IFNs) exert antiproliferative effects on many types of cells. The underlying molecular mechanism, however, is unclear. One possibility is that IFNs block growth factor-induced mitogenic signaling, which involves activation of Ras/Raf-1/MEK/mitogen-activated protein kinase. We have tested this hypothesis by using HER14 cells (NIH 3T3 cell expressing both platelet-derived growth factor [PDGF] and epidermal growth factor [EGF] receptors) as a model system. Our studies showed that IFNs (alpha/beta and gamma) blocked PDGF-and phorbol ester- but not EGF-stimulated DNA synthesis and cell proliferation. While the ligand-stimulated receptor tyrosine phosphorylation and interaction with downstream signaling molecules, such as GRB2, were not affected, IFNs specifically blocked PDGF- and phorbol ester- but not EGF-stimulated activation of Raf-1, mitogen-activated protein kinases, and tyrosine phosphorylation of an unidentified 34-kDa protein. This inhibition could be detected as early as 5 min after IFN treatments and was insensitive to cycloheximide, indicating that de novo protein synthesis is not required. The IFN-induced inhibition acted upstream of Raf-1 kinase and downstream of diacyl glycerol/phorbol ester, suggesting that protein kinase C (PKC) is the potential primary target. Consistently, downregulation of PKC by chronic phorbol myristate acetate treatment or inhibition of PKC by H7 and staurosporine blocked PDGF- and phorbol myristate acetate- but not EGF-induced signaling and DNA synthesis. Moreover, incubating cells with antisense oligodeoxyribonucleotides of PKC delta eliminated production of PKC delta protein and specifically blocked PDGF- but not EGF-stimulated mitogenesis in these cells. Thus, these studies have elucidated a major difference in the early events of EGF-and PDGF-stimulated signal transduction and, more importantly, revealed a novel mechanism by which IFNs may execute their antiproliferative function.     

Norepinephrine specifically stimulates ribonucleotide reductase subunit R2 gene expression in proliferating brown adipocytes: mediation via a cAMP/PKA pathway involving Src and Erk1/2 kinases

We have examined whether a qualitative switch occurs in the response of the ribonucleotide reductase (RNR) genes to the effect of the physiological cAMP-elevating agent norepinephrine (NE) during the development of brown adipocytes. Basal expression of the genes for both RNR subunits, R1 and R2, was high in proliferating cells, but was markedly down-regulated in parallel with adipocyte differentiation. NE stimulation, which promotes DNA synthesis and proliferation of brown preadipocytes, resulted in an increased expression of the R2 gene in proliferating cells (1.6-fold), but was without effect on R1 expression. In contrast, NE stimulation of confluent differentiating brown adipocytes reduced both R1 and R2 expression. The NE stimulation of R2 expression in preadipocytes was mimicked by forskolin and abolished by H89, demonstrating mediation via cAMP and protein kinase A (PKA). Also, inhibitors of Src and of Erk1/2 kinases markedly reduced NE-stimulated R2 expression. We conclude that adrenergic stimulation of brown adipocytes by NE specifically elevates expression of the RNR subunit R2 gene in the proliferative stage of brown adipocyte development, the mediating pathway being a cAMP/PKA cascade further involving Src and the MAP kinase Erk1/2. These results suggest that adrenergic stimulation of brown adipocyte proliferation may act at the level of gene expression of the limiting subunit for RNR activity, R2, and demonstrate a qualitative switch in the response of the R2 gene to cAMP-elevating agents as a consequence of the switch from proliferating to differentiating cell status.     

ANG II stimulates PKC-dependent ERK activation,DNA synthesis,and cell division in intestinal epithelial cells

PKC, a major target for the tumor-promoting phorbol esters, has been implicated in the signal transduction pathways that mediate important functions in intestinal epithelial cells, including proliferation and carcinogenesis. With the use of IEC-18 cells arrested in G0/G1, addition of phorbol esters resulted in a modest increase in [3H]thymidine incorporation and a slight shift toward the S and G2/M phases of the cell cycle, whereas the combination of EGF and phorbol 12,13-dibutyrate (PDB) synergistically stimulated DNA synthesis. To investigate the effects of receptor-mediated PKC activation on mitogenesis, we demonstrated that ANG II induced ERK activation, a response completely blocked by pretreatment with mitogen/extracellular signal-regulated kinase inhibitors or specific PKC inhibitors. Furthermore, ANG II stimulated an over threefold increase in [3H]thymidine incorporation that was corroborated by flow cytometric analysis of the cell cycle to levels comparable to that achieved by the combination of EGF and PDB. Taken together, our results indicate that receptor-mediated PKC activation, as induced by ANG II, transduces mitogenic signals leading to DNA synthesis and cell proliferation in IEC-18 cells.     

Insulin-activated protein kinase Cbeta bypasses Ras and stimulates mitogen-activated protein kinase activity and cell proliferation in muscle cells

In L6 muscle cells expressing wild-type human insulin receptors (L6hIR), insulin induced protein kinase Calpha (PKCalpha) and beta activities. The expression of kinase-deficient IR mutants abolished insulin stimulation of these PKC isoforms, indicating that receptor kinase is necessary for PKC activation by insulin. In L6hIR cells, inhibition of insulin receptor substrate 1 (IRS-1) expression caused a 90% decrease in insulin-induced PKCalpha and -beta activation and blocked insulin stimulation of mitogen-activated protein kinase (MAPK) and DNA synthesis. Blocking PKCbeta with either antisense oligonucleotide or the specific inhibitor LY379196 decreased the effects of insulin on MAPK activity and DNA synthesis by >80% but did not affect epidermal growth factor (EGF)- and serum-stimulated mitogenesis. In contrast, blocking c-Ras with lovastatin or the use of the L61,S186 dominant negative Ras mutant inhibited insulin-stimulated MAPK activity and DNA synthesis by only about 30% but completely blocked the effect of EGF. PKCbeta block did not affect Ras activity but almost completely inhibited insulin-induced Raf kinase activation and coprecipitation with PKCbeta. Finally, blocking PKCalpha expression by antisense oligonucleotide constitutively increased MAPK activity and DNA synthesis, with little effect on their insulin sensitivity. We make the following conclusions. (i) The tyrosine kinase activity of the IR is necessary for insulin activation of PKCalpha and -beta. (ii) IRS-1 phosphorylation is necessary for insulin activation of these PKCs in the L6 cells. (iii) In these cells, PKCbeta plays a unique Ras-independent role in mediating insulin but not EGF or other growth factor mitogenic signals.     

丝裂素活化蛋白激酶参与内皮素刺激的兔主动脉平滑肌细胞增生

内皮素（ｅｎｄｏｔｈｅｌｉｎ,ＥＴ）是已知的体内活性最强的缩血管物质,其缩血管作用由Ｇ蛋白偶联受体所介导。但ＥＴ强大的促血管平滑肌细胞（ＶＳＭＣ）增生效应的机理尚未完全阐明。本研究选用培养的兔胸主动脉ＶＳＭＣ,探讨丝裂素活化蛋白激酶（ＭＡＰＫ）在ＥＴ促细胞增生中的作用。结果表明：ＥＴ－１呈时间和浓度依赖性地促进细胞摄取￣３Ｈ－ＴｄＲ和激活ＭＡＰＫ,此作用可被蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ,ＰＫＣ）抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ（ＳＴＰ）,Ｈ－７和ＥＴ＿Ａ受体拮抗剂ＢＱ１２３所抑制,但不被酪氨酸激酶抑制剂ＨｅｒｂｉｍｙｃｉｎＡ（Ｈｅｒｂ）所抑制,用ＰＫＣ激动剂ＰＭＡ（Ｐｈｏｒｂｏｌｍｙｒｉｓｔａｔｅａｃｅｔａｔｅ）预处理ＶＳＭＣ,使其ＰＫＣ活性下调,可显著减弱ＥＴ－１对ＭＡＰＫ的激活能力。本结果提示：（１）ＭＡＰＫ参与ＥＴ－１所致的ＶＳＭＣ增生;（２）ＥＴ－１促细胞增生与激活ＭＡＰＫ的作用是由ＥＴ＿Ａ受体和ＰＫＣ介导的。     

Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMI-A down regulates c-myc gene expression and endothelial cell proliferation.

Imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate (NAMI-A) is a novel ruthenium-containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI-A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down-regulation of ERK phosphorylation in serum-cultured cells. In addition, NAMI-A markedly reduced serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity. NAMI-A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation. Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation.     

Bradykinin-stimulated arachidonic acid release from MDCK cells is not protein kinase C dependent

Bradykinin (BK)-induced release of arachidonic acid (AA) fromMadin-Darby canine kidney (MDCK) D1 cells was investigated. Phorbol12-myristate 13-acetate (PMA) caused a synergistic increase in BK- andA-23187-induced release of AA but alone had no effect on this release.Inhibition of protein kinase C (PKC) with bisindolmaleimide I (BIS)abolished the synergistic effects of PMA but did not affect AA releasecaused by BK or A-23187 alone. Downregulation of PKC with 100 nM PMAresulted in a reduction of AA release induced by BK or A-23187addition, which corresponded to a decrease in cytoplasmic phospholipaseA₂(cPLA₂) activity as measured incell extracts. Although Western blotting revealed no differences in cPLA₂ expression as a result ofPMA treatment, phosphorylation of the enzyme, as assessed byphosphoserine content, was significantly reduced in PKC-depleted cells.These results imply that, with PKC downregulation, subsequent BKstimulation results in aCa²⁺-dependent translocation of aless phosphorylated, less active form ofcPLA₂. Any stimulation of PKC byBK addition did not appear as a significant event in onset reponsesleading to AA release. On the other hand, inhibition of themitogen-activated protein kinase (MAPK) cascade with the MAPK kinaseinhibitor, PD-98059, significantly decreased BK-induced release of AA,a finding that, with our other results, points to the existence of aPKC-independent route for stimulation of MAPK and the propagation ofonset responses.

     

Role of protein kinase C in the translational regulation of lipoprotein lipase in adipocytes

The hypertriglyceridemia of diabetes is accompanied by decreased lipoprotein lipase (LPL) activity in adipocytes. Although the mechanism for decreased LPL is not known, elevated glucose is known to increase diacylglycerol, which activates protein kinase C (PKC). To determine whether PKC is involved in the regulation of LPL, we studied the effect of 12-O-tetradecanoyl phorbol 13-acetate (TPA) on adipocytes. LPL activity was inhibited when TPA was added to cultures of 3T3-F442A and rat primary adipocytes. The inhibitory effect of TPA on LPL activity was observed after 6 h of treatment, and was observed at a concentration of 6 nM. 100 nM TPA yielded maximal (80%) inhibition of LPL. No stimulation of LPL occurred after short term addition of TPA to cultures. To determine whether TPA treatment of adipocytes decreased LPL synthesis, cells were labeled with [35S]methionine and LPL protein was immunoprecipitated. LPL synthetic rate decreased after 6 h of TPA treatment. Western blot analysis of cell lysates indicated a decrease in LPL mass after TPA treatment. Despite this decrease in LPL synthesis, there was no change in LPL mRNA in the TPA-treated cells. Long term treatment of cells with TPA is known to down-regulate PKC. To assess the involvement of the different PKC isoforms, Western blotting was performed. TPA treatment of 3T3-F442A adipocytes decreased PKC alpha, beta, delta, and epsilon isoforms, whereas PKC lambda, theta, zeta, micro, iota, and gamma remained unchanged or decreased minimally. To directly assess the effect of PKC inhibition, PKC inhibitors (calphostin C and staurosporine) were added to cultures. The PKC inhibitors inhibited LPL activity rapidly (within 60 min). Thus, activation of PKC did not increase LPL, but inhibition of PKC resulted in decreased LPL synthesis by inhibition of translation, indicating a constitutive role of PKC in LPL gene expression.     

Effect of the beta(3)-adrenergic agonist Cl316,243 on functional differentiation of white and brown adipocytes in primary cell culture

We investigated the effect of the specific beta(3)-adrenergic receptor agonist CL 316,243 (CL) on proliferation and functional differentiation of the Siberian hamster (Phodopus sungorus) white and brown preadipocytes in primary cell culture. Proliferation of both white and brown preadipocytes was stimulated by a general beta-adrenergic agonist (isoproterenol) but not by CL. Lipolysis of differentiated white and brown adipocytes was stimulated similarly by CL with maximum effect at 10 nM. Thermogenic properties of cells were assessed by immunodetection of UCP-1, the brown adipocyte specific uncoupling protein, and measurement of cytochrome c oxidase (COx) activity as an index of mitochondrial capacity. UCP-1 content was largely increased by CL in BAT but not in WAT cultures. Basal UCP-2 mRNA levels were similar in WAT and BAT cultures and increased by both CL and isoproterenol. COx activity of BAT cultures was twice as high as that of WAT cultures but in neither cell culture system could it be increased by beta-adrenergic stimulation. We suggest (i) that white and brown preadipocyte proliferation is increased in vitro via beta1 or beta(2), but not beta(3)-adrenergic pathways, (ii) that white and brown preadipocytes represent different cell types, and (iii) that in vitro beta-adrenergic stimulation it is not sufficient to induce complete thermogenic adaptation of brown adipocytes.     

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.     

Induction of plasminogen activator activity by phorbol ester in transformed fetal bovine aortic endothelial cells. Possible independence from protein kinase C

12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol (diC8) activate protein kinase C (PKC) in transformed fetal bovine aortic endothelial GM 7373 cells. Both molecules cause a similar increase in membrane-associated PKC activity and in the phosphorylation of a PKC-specific endogenous 87-kDa substrate in intact cells. Even though both TPA and diC8 exert a mitogenic activity in GM 7373 cells, only TPA induces also an increase in cell-associated plasminogen activator (PA) activity. Down-regulation of PKC which follows TPA-pretreatment completely abolishes the mitogenic activity of diC8 and the mitogenic and PA-inducing activity of TPA. However, both the PKC inhibitor H-7 and the down-regulation of PKC which follows a prolonged stimulation with diC8 do not abolish the PA-inducing activity of TPA. The PA-inducing activity of TPA is instead inhibited in cultures incubated in the presence of 1 mM EGTA or in a calcium-free medium. The data indicate that TPA and diC8 induce a different pattern of cellular activation in GM 7373 cells and that the PA-inducing activity of TPA might not be mediated by PKC.     

Tissue factor pathway inhibitor-2 is a novel mitogen for vascular smooth muscle cells

A mitogen for growth-arrested cultured bovine aortic smooth muscle cells was purified to homogeneity from the supernatant of cultured human umbilical vein endothelial cells by heparin affinity chromatography and reverse-phase high performance liquid chromatography. This mitogen was revealed to be tissue factor pathway inhibitor-2 (TFPI-2), which is a Kunitz-type serine protease inhibitor. TFPI-2 was expressed in baby hamster kidney cells using a mammalian expression vector. Recombinant TFPI-2 (rTFPI-2) stimulated DNA synthesis and cell proliferation in a dose-dependent manner (1-500 nM). rTFPI-2 activated mitogen-activated protein kinase (MAPK) activity and stimulated early proto-oncogene c-fos mRNA expression in smooth muscle cells. MAPK, c-fos expression and the mitogenic activity were inhibited by a specific inhibitor of MAPK kinase, PD098059. Thus, the mitogenic function of rTFPI-2 is considered to be mediated through MAPK pathway. TFPI has been reported to exhibit antiproliferative action after vascular smooth muscle injury in addition to the ability to inhibit activation of the extrinsic coagulation cascade. However, structurally similar TFPI-2 was found to have a mitogenic activity for the smooth muscle cell.