Troglitazone inhibits angiotensin II-induced extracellular signal-regulated kinase 1/2 nuclear translocation and activation in vascular smooth muscle cells.

The thiazolidinedione troglitazone inhibits angiotensin II-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activity in vascular smooth muscle cells. Activation of extracellular signal-regulated kinase 1/2 by angiotensin II is a multistep process involving both its phosphorylation by mitogen-activated protein kinase extracellular signal-regulated kinase kinase in the cytoplasm and a subsequent translocation to the nucleus. The cytoplasmic activation of extracellular signal-regulated kinase 1/2 in vascular smooth muscle cells proceeds through the protein kinase Czeta --> mitogen-activated protein kinase extracellular signal-regulated kinase kinase --> extracellular signal-regulated kinase pathway. Troglitazone did not affect the angiotensin II-induced activation of protein kinase Czeta or its downstream signaling kinases extracellular signal-regulated kinase 1/2 in the cytosol. In contrast, angiotensin II-induced activation of protein kinase Czeta and extracellular signal-regulated kinase 1/2 in the nucleus were both inhibited by troglitazone. Nuclear translocation of extracellular signal-regulated kinase 1/2 induced by angiotensin II was completely blocked by troglitazone. Protein kinase Czeta, however, did not translocate upon angiotensin II stimulation. Troglitazone, therefore, inhibits both angiotensin II-induced nuclear translocation of extracellular signal-regulated kinase 1/2 and the nuclear activity of its upstream signaling kinase protein kinase Czeta. Since extracellular signal-regulated kinase 1/2 nuclear translocation may be a critical signaling step for multiple growth factors that stimulate vascular smooth muscle cells proliferation and migration, troglitazone may provide a new therapeutical approach for the prevention and treatment of atherosclerosis and restenosis.     

细胞外信号调节激酶活化在慢性支气管哮喘大鼠气道平滑肌细胞增殖中的作用

本文旨在探讨细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）在慢性支气管哮喘大鼠气道平滑肌细胞（airway smooth muscle cells,ASMCs）增殖中的作用。建立慢性哮喘大鼠模型,用ERK激动剂表皮生长因子（epidermal growth factor,EGF）和抑制剂PD98059干预慢性哮喘大鼠ASMCs的培养。采用流式细胞仪、四甲基偶氮唑盐（MTT）法、^3H-thymidine（TdR）掺入法和增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）免疫组织化学法检测ASMCs增殖情况,观察ERK信号通路对ASMCs增殖的影响。RT-PCR和Western blot检测ERK mRNA和ERK1／2、磷酸化ERK1／2（p-ERK1／2）蛋白的表达。与正常对照组ASMCs比较,慢性哮喘组ASMCs的G0/G1期细胞所占比例明显减少,S＋G2／M期细胞所占比例增高;吸光度（A490）值、细胞DNA合成量和PCNA阳性表达量均明显增加,ERK mRNA、ERK1／2蛋白、P-ERK1／2蛋白的表达量以及ERK活化率显著增高。经PD98059干预之后,慢性哮喘组ASMCs的S＋G2／M期细胞所占比例、A490值、细胞DNA合成量和PCNA阳性表达量明显降低,ERK mRNA、ERK1／2蛋白、p-ERK1／2蛋白的表达量以及ERK活化率显著降低。经EGF干预后,慢性哮喘组ASMCs的S＋G2／M期细胞所占比例、A490值、细胞DNA合成量和PCNA阳性表达量进一步增高,而这一作用可以被PD98059抑制。以上结果提示,慢性哮喘大鼠ASMCs内源性增殖活性增加,ERK1／2参与其增殖活性的调控,ERK信号通路在哮喘气道重建的ASMCs增殖调控中具有重要作用。     

Extracellular signal-regulated kinase 1/2 signaling promotes oligodendrocyte myelination in vitro

J. Neurochem. (2012) 122, 1167-1180. ABSTRACT: Multiple extracellular factors have been implicated in orchestrating myelination of the CNS; however, less is known about the intracellular signaling cascades that regulate this process. We have previously shown that brain-derived neurotrophic factor (BDNF) promotes oligodendrocyte myelination. Here, we screened for the activation of candidate signaling pathways in in vitro myelination assays and found that extracellular signal-regulated kinase (Erk) signaling positively correlated with basal levels of oligodendrocyte myelination as well as BDNF-induced myelination in vitro. By selectively manipulating Erk1/2 activation in oligodendrocytes in vitro, we found that constitutive activation of Erk1/2 significantly increased myelination, mimicking the promyelinating effect of BDNF, and also caused myelination to occur earlier. Conversely, selective inhibition of Erk1/2 in oligodendrocytes significantly reduced the basal level of myelination and blocked the promyelinating effect of BDNF. Analysis of myelinating spinal cord and corpus callosum white matter tracts revealed that the majority of mature oligodendrocytes are co-labeled with phospho-Erk1/2, whereas phospho-Erk1/2 was rarely observed in oligodendrocyte progenitor cells. Finally, the total level of phospho-Erk1/2 correlated with myelin formation during the early postnatal period. Collectively, these data identify that Erk1/2 signaling within oligodendrocytes exerts an important and direct effect to promote myelination.     

PKC-dependent extracellular signal-regulated kinase 1/2 pathway is involved in the inhibition of Ib on AngiotensinII-induced proliferation of vascular smooth muscle cells

AngiotensinII (AngII) induces vascular smooth muscle cell (VSMC) proliferation, which plays an important role in the development and progression of hypertension. AngII-induced cellular events have been implicated, in part, in the activation of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2). In the present study, we investigated the effect of Ib, a novel nonpeptide AngII receptor type 1 (AT₁) antagonist, on the activation of PKC and ERK1/2 in VSMC proliferation induced by AngII. MTT, and [³H]thymidine incorporation assay showed that AngII-induced VSMC proliferation was inhibited significantly by Ib. The specific binding of [¹²⁵I]AngII to AT₁ receptors was blocked by Ib in a concentration-dependent manner with IC₅₀ value of 0.96 nM. PKC activity assay and Western blot analysis demonstrated that Ib significantly inhibited the activation of PKC and phosphorylation of ERK1/2 induced by AngII, respectively. Furthermore, AngII-induced ERK1/2 activation was obviously blocked by GF109203X, a PKC inhibitor. These findings suggest that the suppression of Ib on AngII-induced VSMC proliferation may be attributed to its inhibitory effect on PKC-dependent ERK1/2 pathway.     

Relation of NADH/NAD to contraction in vascular smooth muscle

The relationship of NADH/NAD to O2 consumption with respect to the different phases of contraction in vascular smooth muscle in response to a maximal depolarizing concentration of KCl was investigated. The NADH bound to cellular proteins could be distinguished from free NADH in whole tissue homogenates. Evidence suggested that the NADH was bound to pyruvate dehydrogenase and perhaps to other dehydrogenases since binding paralleled the changes in the activity of pyruvate dehydrogenase with contraction. The measured changes in NADH were attributed to that within the mitochondrial compartment since the contribution of reducing equivalents within the cytoplasmic compartment was negligible. During the phase of contraction in which force was initially being generated and at which O2 consumption was the highest, there was a net increase in NADH/NAD. After stable isometric force was maintained, at which time O2 consumption had returned to slightly above the basal pre-contraction level, there was a net decrease in NADH/NAD. Previous evidence indicates the phosphorylation potential (ATP/ADP) may decrease during this phase of contraction. It is concluded that contraction of vascular smooth muscle is accompanied by a changing pool of reducing equivalents. Factors which govern O2 consumption may change during the different phases of muscle contraction.     

CaM kinase II-dependent activation of tyrosine kinases and ERK1/2 in vascular smooth muscle

In vascular smooth muscle (VSM) and manyother cells, G protein receptor-coupled activation of mitogen-activatedprotein kinases has been linked, in part, to increases in freeintracellular Ca²⁺. Previously, we demonstrated thationomycin-, angiotensin II-, and thrombin-induced activation ofextracellular signal-regulated kinase (ERK)1/2 in VSM cells wasattenuated by pretreatment with KN-93, a selective inhibitor of themultifunctional Ca²⁺/calmodulin-dependent protein kinase(CaM kinase II). In the present study, we show that theCa²⁺-dependent pathway leading to activation of ERK1/2 ispreceded by nonreceptor proline-rich tyrosine kinase (PYK2) activation and epidermal growth factor (EGF) receptor tyrosine phosphorylation andis attenuated by inhibitors of src family kinases or the EGF receptor tyrosine kinase. Furthermore, we demonstrate that pretreatment with KN-93 or a CaM kinase II inhibitor peptide inhibitsCa²⁺-dependent PYK2 activation and EGF receptor tyrosinephosphorylation in response to ionomycin, ATP, and platelet-derivedgrowth factor but has no effect on phorbol 12,13-dibutyrate- orEGF-induced responses. The results implicate CaM kinase II as anintermediate in the Ca²⁺/calmodulin-dependent activation of PYK2.

     

Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle

The potent vasodilator action of cyclic GMP-dependent protein kinase (cGK) involves decreasing the Ca(2+) sensitivity of contraction of smooth muscle via stimulation of myosin light chain phosphatase through unknown mechanisms (Wu, X., Somlyo, A. V., and Somlyo, A. P. (1996) Biochem. Biophys. Res. Commun. 220, 658-663). Myosin light chain phosphatase activity is controlled by the small GTPase RhoA and its target Rho kinase. Here we demonstrate cGMP effects mediated by cGK that inhibit RhoA-dependent Ca(2+) sensitization of contraction of blood vessels and actin cytoskeleton organization in cultured vascular myocytes. Ca(2+) sensitization and actin organization were inhibited by both 8-bromo-cGMP and sodium nitroprusside (SNP). SNP also caused translocation of activated RhoA from the membrane to the cytosol. SNP-induced actin disassembly was lost in vascular myocytes in culture after successive passages but was restored by transfection of cells with cGK I. Furthermore, cGK phosphorylated RhoA in vitro, and addition of cGK I inhibited RhoA-induced Ca(2+) sensitization in permeabilized smooth muscle. 8-Bromo-cGMP-induced actin disassembly was inhibited in vascular myocytes expressing RhoA(Ala-188), a mutant that could not be phosphorylated. Collectively, these results indicate that cGK phosphorylates and inhibits RhoA and suggest that the consequent inhibition of RhoA-induced Ca(2+) sensitization and actin cytoskeleton organization contributes to the vasodilator action of nitric oxide.     

Extracellular transglutaminase 2 activates beta-catenin signaling in calcifying vascular smooth muscle cells

Accumulation of transglutaminase 2 (TG2) is often associated with mineral deposits in vasculature. Here, we demonstrate that purified TG2 stimulated a 3-fold increase in matrix mineralization and up-regulation of osteoblastic markers in cultured primary vascular smooth muscle cells (VSMCs). Extracellular TG2 interacts with the low density lipoprotein related-protein 5 receptor and activates beta-catenin signaling in VSMCs. These results suggest that TG2 may promote vascular calcification by activating the beta-catenin signaling pathway.     

p38 MAP kinase pathway regulates angiotensin II-induced contraction of rat vascular smooth muscle

Angiotensin II (ANG II) is a multifunctional hormone that exerts potent vasoconstrictor and hypertrophic effects on vascular smooth muscle. Here, we demonstrate that the p38 mitogen-activated protein (MAP) kinase pathway is involved in ANG II-induced vascular contraction. Addition of ANG II to rat aortic smooth muscle cells (SMC) caused a rapid and transient increase of p38 activity through activation of the AT(1) receptor subtype. This response to ANG II was strongly attenuated by pretreating cells with antioxidants and diphenylene iodonium and was mimicked by exposure of cells to H(2)O(2). Stimulation of p38 by ANG II resulted in the enzymatic activation of MAP kinase-activated protein (MAPKAP) kinase-2 and the phosphorylation of heat shock protein 27 (HSP27) in aortic SMC. Pretreatment of cells with the specific p38 MAP kinase inhibitor SB-203580 completely blocked the ANG II-dependent activation of MAPKAP kinase-2 and phosphorylation of HSP27. ANG II also caused a robust activation of MAPKAP kinase-2 in the intact rat aorta. Incubation with SB-203580 significantly decreased the potency of ANG II to induce contraction of rat aortic rings and depressed the maximal hormone response. These results suggest that the p38 MAP kinase pathway selectively modulates the vasoconstrictor action of ANG II in vascular smooth muscle.     

细胞外信号调节激酶1/2信号通路在慢性哮喘模型大鼠支气管平滑肌细胞迁移功能变化中的调控作用

本研究旨在探讨细胞外信号调节激酶1／2(extracellular signal-regulated kinase,ERK1/2)信号通路在慢性哮喘模型大鼠支气管平滑肌细胞(bronchial smooth muscle cells,BSMCs)迁移能力改变中的调控作用。应用卵清蛋白致敏和雾化方法制备大鼠慢性哮喘模型,体外培养大鼠BSMCs,采用免疫荧光细胞化学、Western blot和RT-PCR方法检测ERK1／2信号通路的表达,分别用平面迁移实验和跨膜迁移实验来评价BSMCs的活动和趋向迁移能力,并比较用和不用ERK1／2信号通路干预剂的差异。Western blot结果显示慢性哮喘模型大鼠BSMCs中总ERK1／2(9．13±0．87)较对照组(4．68±0．59)明显增加,磷酸化ERK1／2(p-ERK1/2)占总ERK1／2的比值(0．55±0．05)较对照组(0．48±0．04)显著提高(n=10,P<0．01)。慢性哮喘组ERK1和ERK2 mRNA的表达(1．83±0．24和1．07±0．11)较对照组(0．58±0．14和0．51±0．12)明显增高(n=10,P<0．01)。在平面迁移实验中,慢性哮喘大鼠BSMCs的迁移最远距离是对照组的(2．9±0．1)倍,在ERK1／2激动剂表皮生长因子(epidermal growth factor, EGF)刺激下增加到(5．0±0．2)倍,而在30μmol／L PD98059的作用后下降到(1．7±0．2)倍。正常对照大鼠BSMCs平面迁移能力对PD98059的反应较慢性哮喘组弱,仅在100μmol／L PD98059的作用下下降到(0．8±0．1)倍。跨膜迁移实验中,慢性哮喘大鼠BSMCs的跨膜迁移细胞是对照组的(1．9±0．1)倍,在EGF刺激下增加到(3．1±0．2)倍,而在30μmol/L PD98059作用后下降到(1.45±0.2)倍。这些结果表明慢性哮喘模型大鼠BSMCs的迁移能力明显增强,ERK1／2信号通路在该功能变化的调控中可能发挥了重要作用。     

Extracellular pyrophosphate metabolism and calcification in vascular smooth muscle

Extracellular inorganic pyrophosphate (ePP(i)) is an important endogenous inhibitor of vascular calcification, but it is not known whether systemic or local vascular PP(i) metabolism controls calcification. To determine the role of ePP(i) in vascular smooth muscle, we identified the pathways responsible for ePP(i) production and hydrolysis in rat and mouse aortas and manipulated them to demonstrate their role in the calcification of isolated aortas in culture. Rat and mouse aortas contained mRNA for ectonucleotide pyrophosphatase/phosphodiesterases (NPP1-3), the putative PP(i) transporter ANK, and tissue-nonspecific alkaline phosphatase (TNAP). Synthesis of PP(i) from ATP in aortas was blocked by β,γ-methylene-ATP, an inhibitor of NPPs. Aortas from mice lacking NPP1 (Enpp1(-/-)) did not synthesize PP(i) from ATP and exhibited increased calcification in culture. Although ANK-mediated transport of PP(i) could not be demonstrated in aortas, aortas from mutant (ank/ank) mice calcified more in culture than did aortas from normal (ANK/ANK) mice. Hydrolysis of PP(i) was reduced 25% by β,γ-methylene-ATP and 50% by inhibition of TNAP. Hydrolysis of PP(i) was increased in cells overexpressing TNAP or NPP3 but not NPP1 and was not reduced in Enpp1(-/-) aortas. Overexpression of TNAP increased calcification of cultured aortas. The results show that smooth muscle NPP1 and TNAP control vascular calcification through effects on synthesis and hydrolysis of ePP(i), indicating an important inhibitory role of locally produced PP(i). Smooth muscle ANK also affects calcification, but this may not be mediated through transport of PP(i). NPP3 is identified as an additional pyrophosphatase that could influence vascular calcification.     

Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle

Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 microM) with an increase in intracellular calcium concentration ([Ca(2+)](i)) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH(2)-cADPR (10 microM) inhibited both basal and ACh-stimulated [Ca(2+)](i) levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH(2)-cADPR decreased it. Tracheal mucosa strips, by releasing NAD(+), enhanced [Ca(2+)](i) in isolated TSMs, and this increase was abrogated by either NAD(+)-ase or 8-NH(2)-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD(+) plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.     

Tyrosine phosphorylation increases Ca2+ sensitivity of vascular smooth muscle contraction

In order to elucidate the role of tyrosine phosphorylation in vasoconstriction, we investigated the effects of inhibitors of tyrosine kinase (genistein, 30 microM) and phosphatase (sodium o-vanadate, 5 microM) on the contraction of aorta isolated from guinea pig. Genistein significantly inhibited norepinephrine-induced contraction, but it did not affect that induced by KCI. Thus, tyrosine phosphorylation may not be involved in the contractile response to KCI alone. The aortic contraction elicited by KCl was significantly augmented by sodium o-vanadate, which increased both the maximum force and pD2 values of KCl contraction. In the presence of verapamil, KCl-induced contraction was abolished even after pretreatment with sodium o-vanadate. Sodium o-vanadate also augmented Ca2+-induced contraction in the aortic strips depolarized with KCl, increasing both its maximum force and pD2 values. Neither basal 45Ca2+ uptake nor verapamil-sensitive 45Ca2+ uptake induced by KCl were affected by pretreatment with sodium o-vanadate. These results suggest that tyrosine phosphorylation is involved in the contraction of guinea-pig aorta not through transplasmalemmal Ca2+ entry but through increased Ca2+ sensitivity of the intracellular contractile pathway.     

PYK2/CAKbeta represents a redox-sensitive tyrosine kinase in vascular smooth muscle cells

In vascular smooth muscle cells (VSMCs), the focal adhesion kinase-related tyrosine kinase PYK2/CAKbeta is activated by vascular mitogens. Because reactive oxygen species (ROS) are assumed to mediate mitogenic signals by these agonists, we examined the possible link between ROS and PYK2 in cultured rat VSMCs. Here we present several lines of evidence showing that PYK2 is activated by ROS in VSMCs. The inhibitory effect of an antioxidant, N-acetyl-cysteine, on PYK2 activation by its specific agonists further suggests the pivotal role of PYK2 in vascular remodeling associated with enhanced ROS production.     

Protein kinase C in the regulation of smooth muscle contraction

The cellular and molecular mechanisms underlying smooth muscle contraction are reviewed in the light of recent studies of smooth muscle ultrastructure and of the role of polyphosphoinositide turnover and protein kinase C function in smooth muscle contraction. A new model of smooth muscle contraction is proposed that differs radically from accepted views, particularly the latch bridge hypothesis, in terms of both Ca2+ messenger function and the molecular events underlying this process. A coordinate fibrillar domain model of contraction is proposed in which the initial and sustained phases of contraction are mediated by different cellular and molecular events. The initial phase of response is mediated by a rise in [Ca2+]c and the resulting calmodulin-dependent activation of both myosin light chain kinase and the dissociation of caldesmon from the actin-caldesmon-tropomyosin-myosin fibrillar domain. These events lead to an interaction between actin and the phosphorylated light chains of myosin just as in previous models. However, this initial phase is followed by a sustained phase in which a rise in [Ca2+]sm stimulates the plasma membrane-associated, Ca2+-sensitive form of protein kinase C that results in the phosphorylation of both structural and regulatory components of the filamin-actin-desmin fibrillar domain. These events underlie the tonic phase of contraction.