Small GTP binding protein Ras contributes to norepinephrine-induced mitogenesis of vascular smooth muscle cells(1)

Norepinephrine stimulates release of arachidonic acid from tissue lipids. Arachidonic acid metabolites generated through the lipoxygenase and cytochrome P-450 pathways but not cyclooxygenase stimulate mitogen activated protein (MAP) kinase activity and proliferation of vascular smooth muscle cells (VSMC). Moreover, norepinephrine has been shown to activate the Ras/MAP kinase pathway through generation of cytochrome P-450 metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE). The purpose of this study was to investigate the contribution of Ras in norepinephrine-induced mitogenesis in aortic VSMC. Farnesylation of Ras by farnesyl transferase is required for its full activation. Norepinephrine-induced DNA synthesis, as measured by [(3)H]-thymidine incorporation, was attenuated by inhibitors of Ras farnesyl transferase FPT III and BMS-191563. These agents also inhibited 20-HETE-stimulated [(3)H]-thymidine incorporation. In cells transiently transfected with dominant negative Ras (RasN17), norepinephrine, and 20-HETE-induced proliferation of VSMC was attenuated. Both norepinephrine and 20-HETE increased localization of Ras to plasma membrane and MAP kinase activity; FPT III attenuated these effects. These data suggest that VSMC proliferation induced by norepinephrine and 20-HETE is mediated by Ras/MAP kinase pathway.     

20-HETE inhibits the proliferation of vascular smooth muscle cells via transforming growth factor-beta

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, has been shown to modulate the growth of vascular smooth muscle cells (VSMCs). We asked whether 20-HETE modulates the proliferation of R22D cells, a clonal VSMC from neonatal rats, by releasing transforming growth factor-beta (TGF-beta). Incubation of R22D cells with 20-HETE for 24 h attenuated [(3)H]thymidine incorporation in a concentration-dependent manner without causing the release of lactate dehydrogenase. 20-HETE also inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation in R22D cells and human VSMCs. At 5 muM, 20-HETE reduced [(3)H]thymidine incorporation by 34 +/- 6%; anti-TGF-beta neutralizing antibody, but not nonspecific IgG, completely reversed the attenuated [(3)H]thymidine incorporation induced by 20-HETE. In addition, 20-HETE attenuated fetal bovine serum- and PDGF-induced expression of cyclin D1, a downstream effector of TGF-beta(1), which was reversed by anti-TGF-beta antibody. Further studies demonstrated that 20-HETE may increase TGF-beta release to a level high enough to inhibit [(3)H]thymidine incorporation without altering the steady-state mRNA level of TGF-beta. Nevertheless, pretreatment of indomethacin (a cyclooxygenase inhibitor) or paxilline (a potassium channel inhibitor) did not affect the inhibitory effect on DNA synthesis induced by 20-HETE. These results demonstrate for the first time a growth-inhibitory effect induced by 20-HETE, which may be mediated by TGF-beta.     

Phospholipase D activation by norepinephrine is mediated by 12(s)-, 15(s)-, and 20-hydroxyeicosatetraenoic acids generated by stimulation of cytosolic phospholipase a2. tyrosine phosphorylation of phospholipase d2 in response to norepinephrine

Norepinephrine (NE) stimulates phospholipase D (PLD) through a Ras/MAPK pathway in rabbit vascular smooth muscle cells (VSMC). NE also activates calcium influx and calmodulin (CaM)-dependent protein kinase II-dependent cytosolic phospholipase A(2) (cPLA(2)). Arachidonic acid (AA) released by cPLA(2)-catalyzed phospholipid hydrolysis is then metabolized into hydroxyeicosatetraenoic acids (HETEs) through lipoxygenase and cytochrome P450 4A (CYP4A) pathways. HETEs, in turn, have been shown to stimulate Ras translocation and to increase MAPK activity in VSMC. This study was conducted to determine the contribution of cPLA(2)-derived AA and its metabolites (HETEs) to the activation of PLD. NE-induced PLD activation was reduced by two structurally distinct CaM antagonists, W-7 and calmidazolium, and by CaM-dependent protein kinase II inhibition. Blockade of cPLA(2) activity or protein depletion with selective cPLA(2) antisense oligonucleotides abolished NE-induced PLD activation. The increase in PLD activity elicited by NE was also blocked by inhibitors of lipoxygenases (baicalein) and CYP4A (17-octadecynoic acid), but not of cyclooxygenase (indomethacin). AA and its metabolites (12(S)-, 15(S)-, and 20-HETEs) increased PLD activity. PLD activation by AA and HETEs was reduced by inhibitors of Ras farnesyltransferase (farnesyl protein transferase III and BMS-191563) and MEK (U0126 and PD98059). These data suggest that HETEs are the mediators of cPLA(2)-dependent PLD activation by NE in VSMC. In addition to cPLA(2), PLD was also found to contribute to AA release for prostacyclin production via the phosphatidate phosphohydrolase/diacylglycerol lipase pathway. Finally, a catalytically inactive PLD(2) (but not PLD(1)) mutant inhibited NE-induced PLD activity, and PLD(2) was tyrosine-phosphorylated in response to NE by a MAPK-dependent pathway. We conclude that NE stimulates cPLA(2)-dependent PLD(2) through lipoxygenase- and CYP4A-derived HETEs via the Ras/ERK pathway by a mechanism involving tyrosine phosphorylation of PLD(2) in rabbit VSMC.     

Effects of lipoxygenase metabolites of arachidonic acid on the growth of human mononuclear marrow cells and marrow stromal cell cultures.

The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures. LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased [3H]-thymidine incorporation on marrow stromal cell cultures without affecting cell number. Only 12-HETE showed a dose-response effect on [3H]-thymidine incorporation. While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect. The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth. These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures.     

20-hydroxyeicosatetraenoic acid (20-HETE) metabolism in coronary endothelial cells

We have investigated the role of endothelial cells in the metabolism of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive mediator synthesized from arachidonic acid by cytochrome P450 omega-oxidases. Porcine coronary artery endothelial cells (PCEC) incorporated 20-[(3)H]HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. The incorporation was reduced by equimolar amounts of arachidonic, eicosapentaenoic or 8,9-epoxyeicosatrienoic acids, but some uptake persisted even when a 10-fold excess of arachidonic acid was available. The retention of 20-[(3)H]HETE increased substantially when methyl arachidonoyl fluorophosphonate, but not bromoenol lactone, was added, suggesting that a Ca(2+)-dependent cytosolic phospholipase A(2) released the 20-HETE contained in PCEC phospholipids. Addition of calcium ionophore A23187 produced a rapid release of 20-[(3)H]HETE from the PCEC, a finding that also is consistent with a Ca(2+)-dependent mobilization process. PCEC also converted 20-[(3)H]HETE to 20-carboxy-arachidonic acid (20-COOH-AA) and 18-, 16-, and 14-carbon beta-oxidation products. 20-COOH-AA produced vasodilation in porcine coronary arterioles, but 20-HETE was inactive. These results suggest that the incorporation of 20-HETE and its subsequent conversion to 20-COOH-AA in the endothelium may be important in modulating coronary vascular function.     

Evaluation of cytochrome P450 4 family as mediator of phospholipase D activation in aortic vascular smooth muscle cells

Norepinephrine (NE) stimulates phospholipase D (PLD) activity via phospholipase A2-dependent arachidonic acid release in rabbit aortic vascular smooth muscle cells (VSMC). We have previously shown that exogenous 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid generated through the cytochrome P450 (CYP) 4A pathway in vivo, stimulates PLD activity. Whether endogenous CYP4-derived arachidonic acid metabolites act as intracellular mediators of NE-induced PLD activation in VSMC is not known. In rabbit aortic VSMC, prototypical hepatic/renal CYP4A inducers such as fenofibrate and Wy 14643 inhibited both basal and NE-induced PLD activity after 48 h of exposure. The level of CYP4F, and to a lesser extent CYP4A, was also decreased by these agents. The expression levels of rabbit aortic VSMC CYP4A and CYP4F isoforms were reduced by antisense oligonucleotides treatment for 48 hours as measured by RTQ-PCR or Western blotting. This reduction in CYP4A or CYP4F levels did not change NE-induced PLD activation. The corresponding CYP4A scrambled and CYP4F sense oligonucleotides did not alter CYP levels. PLD activity was increased by ~70% after 15 min of stimulation with NE, whereas lauric acid omega-hydroxylase activity, a measure of fatty acid omega-hydroxylation, was unchanged. Inhibition of omega-hydroxylation with DDMS and HET0016, selective omega-hydroxylase inhibitors, and 20-HEDE, an antagonist of 20-HETE, increased PLD activity in a concentration-dependent manner and did not alter NE-induced PLD activation. These data suggest that PLD activation by NE is independent of the CYP4A/4F enzymes in rabbit aortic VSMC.     

Regulation of Na-K-ATPase Activity in the Proximal Tubule: Role of the Protein Kinase C Pathway and of Eicosanoids

To evaluate further the signal transduction mechanisms involved in the short-term modulation of Na-K-ATPase activity in the mammalian kidney, we examined the role of phospholipase C-protein kinase C (PLC-PKC) pathway and of various eicosanoids in this process, using microdissected rat proximal convoluted tubules. Dopamine (DA) and parathyroid hormone (either synthetic PTH_1-34 or PTH_3-34) inhibited Na-K-ATPase activity in dose-dependent manner; this effect was reproduced by PKC_530-558 fragment and blocked by the specific PKC inhibitor calphostin C, as well as by the PLC inhibitors neomycin and U-73122. Pump inhibition by DA, PTH, or arachidonic acid, and by PKC activators phorbol dibutyrate (PDBu) or dioctanoyl glycerol (DiC8) was abolished by ethoxyresorufin, an inhibitor of the cytochrome P450-dependent monooxygenase pathway, but was unaffected by indomethacin or nordihydroguaiaretic acid, inhibitors of the cyclooxygenase and lipoxygenase pathways of the arachidonic acid cascade, respectively. Furthermore, each of the three monooxygenase products tested (20-HETE, 12(R)-HETE, or 11,12-DHT) caused a dose-dependent inhibition of the pump. The effect of DA, PTH, PDBu or DiC8, as well as that of 20-HETE was not altered when sodium entry was blocked with the amiloride analog ethylisopropyl amiloride or increased with nystatin. We conclude that short-term regulation of proximal tubule Na-K-ATPase activity by dopamine and parathyroid hormone occurs via the PLC-PKC signal transduction pathway and is mediated by cytochrome P450-dependent monooxygenase products of arachidonic acid metabolism, which may interact with the pump rather than alter sodium access to it. Received: 7 January 1996/Revised: 24 April 1996     

20-HETE in neovascularization

Cytochrome P450 4A/F (CYP4A/F) converts arachidonic acid (AA) to 20-HETE by ω-hydroxylation. The contribution of 20-HETE to the regulation of myogenic response, blood pressure, and mitogenic actions has been well summarized. This review focuses on the emerging role of 20-HETE in physiological and pathological vascularization. 20-HETE has been shown to regulate vascular smooth muscle cells (VSMC) and endothelial cells (EC) by affecting their proliferation, migration, survival, and tube formation. Furthermore, the proliferation, migration, secretion of proangiogenic molecules (such as HIF-1α, VEGF, SDF-1α), and tube formation of endothelial progenitor cells (EPC) are stimulated by 20-HETE. These effects are mediated through c-Src- and EGFR-mediated downstream signaling pathways, including MAPK and PI3K/Akt pathways, eNOS uncoupling, and NOX/ROS system activation. Therefore, the CYP4A/F-20-HETE system may be a therapeutic target for the treatment of abnormal angiogenic diseases.     

20-carboxy-arachidonic acid is a dual activator of peroxisome proliferator-activated receptors alpha and gamma

20-carboxy-arachidonic acid (20-COOH-AA) is a metabolite of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid produced from arachidonic acid by cytochrome P450 (CYP) omega-oxidases. Alcohol dehydrogenases convert 20-HETE to 20-COOH-AA, and we now find that a microsomal preparation containing recombinant human CYP4F3B converts arachidonic acid to 20-HETE and 20-COOH-AA. Studies with transfected COS-7 cell expression systems indicate that 20-COOH-AA activates peroxisome proliferators-activated receptor (PPAR) alpha and PPARgamma. 20-COOH-AA was twice as potent as either 20-HETE or ciglitazone in stimulating PPARgamma-mediated luciferase expression. While 20-COOH-AA also was more potent than 20-HETE in increasing PPARalpha-mediated luciferase expression, the increase was only half as much as that produced by Wy-14643. 20-COOH-AA did not increase PPARalpha or PPARgamma expression in the transfected cells. Radiolabeled 20-COOH-AA was detected intracellularly when the COS-7 cells were incubated with either [3H]20-COOH-AA or [3H]20-HETE, and binding studies indicated that [3H]20-COOH-AA bound to the isolated ligand binding domains of PPARalpha (Kd=0.87+/-0.12 microM) and PPARgamma (Kd=1.7+/-0.5 microM). These findings suggest that 20-COOH-AA, a relatively stable metabolite of 20-HETE, might function as an endogenous dual activator of PPARalpha and PPARgamma.     

Attenuation of neonatal ischemic brain damage using a 20-HETE synthesis inhibitor

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 metabolite of arachidonic acid that that contributes to infarct size following focal cerebral ischemia. However, little is known about the role of 20-HETE in global cerebral ischemia or neonatal hypoxia-ischemia (H-I). The present study examined the effects of blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) in neonatal piglets after H-I to determine if it protects highly vulnerable striatal neurons. Administration of HET0016 after H-I improved early neurological recovery and protected neurons in putamen after 4 days of recovery. HET0016 had no significant effect on cerebral blood flow. cytochrome P450 4A immunoreactivity was detected in putamen neurons, and direct infusion of 20-HETE in the putamen increased phosphorylation of Na(+), K(+) -ATPase and NMDA receptor NR1 subunit selectively at protein kinase C-sensitive sites but not at protein kinase A-sensitive sites. HET0016 selectively inhibited the H-I induced phosphorylation at these same sites at 3 h of recovery and improved Na(+), K(+) -ATPase activity. At 3 h, HET0016 also suppressed H-I induced extracellular signal-regulated kinase 1/2 activation and protein markers of nitrosative and oxidative stress. Thus, 20-HETE can exert direct effects on key proteins involved in neuronal excitotoxicity in vivo and contributes to neurodegeneration after global cerebral ischemia in immature brain.     

Angiotensin II-induced Akt activation is mediated by metabolites of arachidonic acid generated by CaMKII-stimulated Ca2(+)-dependent phospholipase A2

Angiotensin II (ANG II) promotes vascular smooth muscle cell (VSMC) growth, stimulates Ca(2+)-calmodulin (CaM)-dependent kinase II (CaMKII), and activates cytosolic Ca(2+)-dependent phospholipase A2 (cPLA2), which releases arachidonic acid (AA). ANG II also generates H2O2 and activates Akt, which have been implicated in ANG II actions in VSMC. This study was conducted to investigate the relationship of these signaling molecules to Akt activation in rat aortic VSMC. ANG II increased Akt activity, as measured by its phosphorylation at serine-473. ANG II (200 nM)-induced Akt phosphorylation was decreased by extracellular Ca2+ depletion and calcium chelator EGTA and inhibitors of CaM [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and CaMKII [(2-[N-(2-hydroxyethyl)]-N-(4-me-thoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine)]. cPLA2 inhibitor pyrrolidine-1, antisense oligonucleotide, and retroviral small interfering RNA also attenuated ANG II-induced Akt phosphorylation. AA increased Akt phosphorylation, and AA metabolism inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) blocked ANG II- and AA-induced Akt phosphorylation (199.03 +/- 27.91% with ANG II and 110.18 +/- 22.40% with ETYA + ANG II; 405.00 +/- 86.22% with AA and 153.97 +/- 63.26% with ETYA + AA). Inhibitors of lipoxygenase (cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate) and cytochrome P-450 (ketoconazole and 17-octadecynoic acid), but not cyclooxygenase (indomethacin), attenuated ANG II- and AA-induced Akt phosphorylation. Furthermore, 5(S)-, 12(S)-, 15(S)-, and 20-hydroxyeicosatetraenoic acids and 5,6-, 11,12-, and 14,15-epoxyeicosatrienoic acids increased Akt phosphorylation. Catalase inhibited ANG II-increased H2O2 production but not Akt phosphorylation. Oleic acid, which also increased H2O2 production, did not cause Akt phosphorylation. These data suggest that ANG II-induced Akt activation in VSMC is mediated by AA metabolites, most likely generated via lipoxygenase and cytochrome P-450 consequent to AA released by CaMKII-activated cPLA2 and independent of H2O2 production.     

Hydroxyeicosatetraenoic acids released through the cytochrome P-450 pathway regulate 3T6 fibroblast growth

Eicosanoids participate in the regulation of cellular proliferation. Thus, we observed that prostaglandin E(2) interaction with membrane receptors is involved in the control of 3T6 fibroblast growth induced by serum. However, our results suggested that another arachidonic acid pathway might be implicated in these events. Our results show that 3T6 fibroblasts synthesized hydroxyeicosatetraenoic acids (HETEs) such as 12-HETE through the cytochrome P-450 (CYP450) pathway. However, 3T6 fibroblasts did not produce leukotriene B(4) (LTB(4)), and lipoxygenase inhibitors and LT antagonists failed to inhibit 3T6 fibroblast growth induced by FBS. In contrast, we observed that CYP450 inhibitors such as SKF-525A, 17-octadecynoic acid, 1-aminobenzotriazole, and 6-(2-propargyloxyphenyl)hexanoic acid reduced 12(S)-HETE levels, 3T6 fibroblast growth, and DNA synthesis induced by FBS. The impairment of DNA synthesis and 3T6 fibroblast growth induced by SKF-525A were reversed by exogenous addition of HETEs. Moreover, we report that 5-HETE, 12(S)-HETE, and 15(S)-HETE are mitogenic on 3T6 fibroblast in the absence of another growth factor, and this effect was dependent on the activation of the phosphatidylinositol-3-kinase pathway. In conclusion, our results show that HETEs, probably produced by CYP450, are involved in the control of 3T6 fibroblast growth.     

Effect of arachidonic acid on activity of the apical K+ channel in the thick ascending limb of the rat kidney

We have used patch-clamp techniques to study the effects of arachidonic acid (AA) on the activity of the 70-pS K+ channel, the predominant type of the two apical K+ channels operating under physiological conditions in the thick ascending limb (TAL) of the rat kidney. Addition of 5-10 microM AA blocked the activity of the 70-pS K+ channel in both cell- attached and inside-out patches. The inhibitory effect of AA was specific, because application of 10 microM linoleic acid, oleic acid, or palmitic acid failed to mimic the effect of AA. The effect of AA could not be blocked by pretreatment of the TAL tubules with either 5 microM indomethacin (inhibitor of cyclooxygenase) or 4 microM cinnamyl- 3,4-dihydroxy-alpha-cyanocinnamate (CDC) (inhibitor of lipooxygenase). In contrast, addition of 5 microM 17-octadecynoic acid (17-ODYA), an inhibitor of P450 monooxygenases, abolised the effect of AA on the channel activity, indicating that the effect was mediated by cytochrome P450 metabolites of AA. Addition of 10 nM 20-hydroxyeicosatetraenoic acid (20-HETE), the main metabolite of the cytochrome P450 metabolic pathway in the medullary TAL, mimicked the inhibitory effect of 10 microM AA. However, addition of 100 nM 19-HETE or 17-HETE had no significant effects and 100 nM 20-carboxy AA (20-COOH) reduced the channel activity by only 20%, indicating that the inhibitory effect of 20-HETE was specific and responsible for the action of AA. Inhibition of the P450 metabolic pathway by either 5 microM 17-ODYA or 12, 12- dibromododec-11-enoic acid (DBDD) dramatically increased the channel activity by 280% in cell-attached patches. The stimulatory effect of 17- ODYA or DBDD was not observed in inside-out patches. The results strongly indicate that 20-HETE is a specific inhibitor for the 70-pS K+ channel and may play an important role in the regulation of the K+ channel activity in the TAL.     

Effects of Cl- channel blockers on endothelin-1-induced proliferation of rat vascular smooth muscle cells

The effects of Cl- channel blockers on endothelin-1 (ET-1)-induced proliferation of rat aortic vascular smooth muscle cells (VSMC) were examined. We found ET-1 concentration-dependently increased cell count and [3H]-thymidine incorporation into VSMC, with EC50 values of 24.8 and 11.4 nM, respectively. Both nifedipine and SK&F96365 inhibited 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC with the maximal inhibitory concentrations of 1 and 10 microM, respectively. DIDS inhibited 10 nM ET-1-induced increase in cell count and [3H]-thymidine incorporation into VSMC in a concentration-dependent manner, whereas other Cl- channel blockers including IAA-94, NPPB, DPC, SITS and furosemide did not produce these effects. 3 microM DIDS reduced 10 nM ET-1-induced sustained increase in cytoplasmic Ca2+ concentration ([Ca2+]) by 52%. Pretreatment of VSMC with 1 microM nifedipine completely inhibited the DIDS effect on 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC and sustained increase in [Ca2+]i, whereas pretreatment with 10 microM SK&F96365 did not completely block these effects of DIDS. DIDS did not affect ET-1-induced Ca2+ release and 30 mM KCl-induced increase in [Ca2+]i. Our data suggest that DIDS-sensitive Cl- channels mediate VSMC proliferation induced by ET-1 by mechanisms related to membrane depolarization and Ca2+ influx through voltage-dependent Ca2+ channels.     

Ras triggers acidosis-induced activation of the extracellular-signal-regulated kinase pathway in cardiac myocytes

In cardiac myocytes, sustained (3 min) intracellular acidosis activates the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway and, through this pathway, increases sarcolemmal NHE (Na+/H+ exchanger) activity [Haworth, McCann, Snabaitis, Roberts and Avkiran (2003) J. Biol. Chem. 278, 31676-31684]. In the present study, we aimed to determine the time-dependence, pH-dependence and upstream signalling mechanisms of acidosis-induced ERK1/2 activation in ARVM (adult rat ventricular myocytes). Cultured ARVM were subjected to intracellular acidosis for up to 20 min by exposure to NH4Cl, followed by washout with a bicarbonate-free Tyrode solution containing the NHE1 inhibitor cariporide. After the desired duration of intracellular acidosis, the phosphorylation status of ERK1/2 and its downstream effector p90(RSK) (90 kDa ribosomal S6 kinase) were determined by Western blotting. This revealed a time-dependent transient phosphorylation of both ERK1/2 and p90(RSK) by intracellular acidosis (intracellular pH approximately 6.6), with maximum activation occurring at 3 min and a return to basal levels by 20 min. When the degree of intracellular acidosis was varied from approximately 6.8 to approximately 6.5, maximum ERK1/2 phosphorylation was observed at an intracellular pH of 6.64. Inhibition of MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK kinase 1/2) by pre-treatment of ARVM with U0126 or adenoviral expression of dominant-negative D208A-MEK1 protein prevented the phosphorylation of ERK1/2 by sustained intracellular acidosis, as did inhibition of Raf-1 with GW 5074 or ZM 336372. Interference with Ras signalling by the adenoviral expression of dominant-negative N17-Ras protein or with FPT III (farnesyl protein transferase inhibitor III) also prevented acidosis-induced ERK1/2 phosphorylation, whereas inhibiting G-protein signalling [by adenoviral expression of RGS4 or Lsc, the RGS domain of p115 RhoGEF (guanine nucleotide-exchange factor)] or protein kinase C (with bisindolylmaleimide I) had no effect. Our data show that, in ARVM, sustained intracellular acidosis activates ERK1/2 through proximal activation of the classical Ras/Raf/MEK pathway.     

Statins suppress oxidized low density lipoprotein-induced macrophage proliferation by inactivation of the small G protein-p38 MAPK pathway

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) ameliorate atherosclerotic diseases. Macrophages play an important role in the development and subsequent stability of atherosclerotic plaques. We reported previously that oxidized low density lipoprotein (Ox-LDL) induced macrophage proliferation through the secretion of granulocyte/macrophage colony-stimulating factor (GM-CSF) and the consequent activation of p38 MAPK. The present study was designed to elucidate the mechanism of the inhibitory effect of statins on macrophage proliferation. Mouse peritoneal macrophages were used in our study. Cerivastatin and simvastatin each inhibited Ox-LDL-induced [(3)H]thymidine incorporation into macrophages. Statins did not inhibit Ox-LDL-induced GM-CSF production, but inhibited GM-CSF-induced p38 MAPK activation. Farnesyl transferase inhibitor and geranylgeranyl transferase inhibitor inhibited GM-CSF-induced macrophage proliferation, and farnesyl pyrophosphate and geranylgeranyl pyrophosphate prevented the effect of statins. GM-CSF-induced p38 MAPK phosphorylation was also inhibited by farnesyl transferase inhibitor or geranylgeranyl transferase inhibitor, and farnesyl pyrophosphate and geranylgeranyl pyrophosphate prevented the suppression of GM-CSF-induced p38 MAPK phosphorylation by statins. Furthermore, we found that statin significantly inhibited the membrane translocation of the small G protein family members Ras and Rho. GM-CSF-induced p38 MAPK activation and macrophage proliferation was partially inhibited by overexpression of dominant negative Ras and completely by that of RhoA. In conclusion, statins inhibited GM-CSF-induced Ras- or RhoA-p38 MAPK signal cascades, thereby suppressing Ox-LDL-induced macrophage proliferation. The significant inhibition of macrophage proliferation by statins may also explain, at least in part, their anti-atherogenic action.     

Farnesyl protein transferase inhibition interferes with activation of MAP kinase family members in human peripheral blood monocytes

BACKGROUND: Farnesyl protein transferase inhibitors have emerged as promising novel agents for combating cancerous disease. Nevertheless, the importance for farnesyl protein transferase enzymatic activity for cellular physiology of untransformed cells remains poorly investigated. MATERIALS AND METHODS: Peripheral blood monocytes, isolated from the blood of eight healthy volunteers, were treated with a farnesyl protein transferase inhibitor (FTI 744,832) or vehicle control for 16 hr. Subsequently cells were challenged with different concentrations of lipopolysaccharide (LPS), colony stimulating factor-1 (CSF-1), or phorbol esters for 10 min, after which the activation state of p42/p44 MAP kinase, p38 MAP kinase, and Jun-N-terminal kinase was investigated using Western blotting and phosphospecific antibodies. RESULTS: We observed that farnesyl protein transferase inhibition abrogated activation of p38 MAP kinase by LPS, CSF-1, and phorbol esters. Also the activation of Jun-N-terminal kinase by LPS was not seen after farnesyl protein transferase inhibition. Finally, stimulation of p42/p44 MAP kinase with CSF-1 was strongly reduced by farnesyl protein transferase inhibition, whereas activation of p42/p44 MAP kinase by phorbol ester was only slightly effected. CONCLUSIONS: Farnesyl protein transferase enzymatic activity is required for proper activation of all major members of the MAP kinase family. The observation that activation the p38 MAP kinase and Jun-N-terminal kinase is sensitive to farnesyl protein transferase inhibition raises the possibility that, in addition to cancerous disease, farnesyl protein transferase inhibitors may be useful compounds in combating inflammatory disease.     

Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca(2+), PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.