花生四烯酸诱导肌球蛋白磷酸化被抑制的平滑肌细胞 迁移的信号传导途径

在应用肌球蛋白轻链激酶特异抑制剂ML-7抑制了肌球蛋白轻链磷酸化后,花生四烯酸(arachidonic acid,AA)仍可诱导兔血管平滑肌细胞（SM3）发生迁移.为了进一步阐明其信号传导途径,应用多种信号抑制剂,采用免疫印迹、Boyden小室和提取细胞膜蛋白等实验方法,对上述迁移作用的信号传导途径进行了深入的研究.结果显示,PTX（Gi蛋白抑制剂）、U73122（PLC抑制剂）、staurosporine (PKC抑制剂)、PD98059（ERK1/2抑制剂）和SB203580（p38抑制剂）分别可拮抗上述AA诱导的SM3细胞迁移作用,而SP600125（JNK抑制剂）的作用较弱.免疫印迹结果显示,AA可提高SM3细胞中PKC(ε)、ERK1/2、p38和JNK信号的磷酸化水平,呈时间依赖性, PTX或U73122可抑制上述作用;staurosporine可抑制由AA 引起的ERK1/2和JNK的磷酸化水平增强,但对p38的磷酸化水平无影响.还发现AA可促进PLCβ2的细胞膜移位, PTX可抑制其作用.上述结果表明,当肌球蛋白轻链的磷酸化被抑制后, AA可通过Gi蛋白的活化促进PLCβ2向细胞膜移位,进而通过激活PKC(ε)、ERK1/2、p38和JNK等信号转导途径而诱导SM3细胞发生迁移     

非磷酸化肌球蛋白在血小板衍生生长因子诱导的血管平滑肌细胞迁移中的作用

为了阐明非磷酸化肌球蛋白在平滑肌细胞迁移中的作用,研究探讨了非磷酸化肌球蛋白是否介导了血小板衍生生长因子(PDGF)诱导豚鼠脑基底动脉平滑肌细胞(GbaSM-4)的迁移。研究结果显示,20ng/ml以下剂量的PDGF可诱导GbaSM-4细胞发生迁移,此时肌球蛋白轻链(MLC20)磷酸化水平无变化。该迁移作用可被肌球蛋白特异性抑制剂blebbistatin所拮抗。应用RNA干扰技术抑制肌球蛋白轻链激酶表达,经免疫印迹检测经果显示,MLC20的磷酸化水平发生了显著下降;但对PDGF诱导的迁移作用无影响;在RNA干扰后blebbistatin也可抑制其迁移作用。体外ATP酶活性测定结果显示,blebbistatin对从平滑肌中提取的非磷酸化肌球蛋白的ATP酶活性有明显的抑制作用,其主要作用位点位于肌球蛋白头的头部S1。上述结果提示,非磷酸化的肌球蛋白参与了PDGF诱导的平滑肌细胞迁移。     

RhoA-ROK通路在平滑肌收缩中的作用

近年来有关平滑肌收缩的钙敏化机制研究进展迅速,一系列的证据显示这种Ca2 非依赖的调节主要是由RhoA-ROK通路介导,它主要通过磷酸化抑制肌球蛋白轻链磷酸酶(MLCP)的活性来增加肌球蛋白轻链(MLC)的磷酸化水平,从而增强平滑肌的收缩力。越来越多的研究显示RhoA-ROK通路参与了平滑肌细胞和非肌细胞的多种功能,在许多疾病如高血压、动脉粥样硬化、冠状动脉痉挛等的发生和发展中起着非常重要的作用。     

平滑肌肌球蛋白磷酸化与肌动球蛋白功能调节

在有Ｃａ２＋和钙调蛋白存在时,肌球蛋白轻链激酶催化肌球蛋白磷酸化,促使肌动蛋白激活的肌球蛋白（肌动球蛋白）Ｍｇ２＋－ＡＴＰ酶活性显著增加．然而,肌球蛋白磷酸化水平与Ｍｇ２＋－ＡＴＰ酶之间的关系是非线性的,原肌球蛋白可以进一步增加Ｍｇ２＋－ＡＴＰ酶的活性,但仍不改变它们之间的非线性关系．肌球蛋白轻链激酶的合成肽抑制剂抑制了肌球蛋白磷酸化和Ｍｇ２＋－ＡＴＰ酶活性,并导致平滑肌去膜肌纤维的等长收缩张力与速度的降低．结果提示肌球蛋白轻链激酶参与脊椎动物平滑肌收缩的调节过程,肌球蛋白轻链磷酸化作用会引起平滑肌收缩     

肌球蛋白轻链激酶活性片段对肌球蛋白轻链的非钙依赖性磷酸化作用特征

肌球蛋白轻链激酶 (MLCK)的活性片段 (MLCKF)能比完整的MLCK更有效地、以非钙依赖性的方式磷酸化肌球蛋白轻链 (MLC2 0 )。该片段是用胰蛋白酶水解MLCK ,再经DEAE 5 2柱层析分离而获得的 ,分子量约为 6 1kD。Western印迹已证实该MLCKF与完整的MLCK同源。MLCKF对肌球蛋白轻链的磷酸化作用及其作用特征通过甘油电泳及ScoinImage扫描软件检测 ,肌球蛋白ATP酶活性通过分光光度法检测。实验结果证实 ,MLCKF催化的MLC2 0 非钙依赖性磷酸化 (CIPM)比MLCK催化的CIPM效力高、耗能多 ,但比MLCK催化的MLC2 0 钙依赖性磷酸化 (CDPM)效力低、耗能少 ;MLCKF催化的CIPM与MLCK催化的CIPM均较MLCK催化的CDPM稳定 ,不易受温育温度、温育时间及离子浓度等变化的影响 ,且对MLCK抑制剂ML 9敏感性低。     

肌球蛋白磷酸化的研究进展

肌球蛋白是肌原纤维粗丝的组成单位,由多条重链与多条轻链组成,被视为一种分子马达。在肌肉收缩、趋化性胞质分裂、胞引作用、膜泡运输以及信号传导等生理过程中起重要作用。目前肌球蛋白磷酸化是研究的一个热点,它对细胞的迁移、收缩、胞质分裂以及其他未知功能都有着至关重要的作用。肌球蛋白磷酸化分为重链的磷酸化与轻链的磷酸化。根据国内外的最新相关研究报道,分别从肌球蛋白的结构与功能、磷酸化的作用机制、磷酸化的生物学功能以及最新研究成果等方面,对肌球蛋白的磷酸化研究进展进行阐述。     

平滑肌肌球蛋白轻链激酶及ATP结合位点突变体对ATP酶活性的调节作用

目的：探寻MLCK的非激酶活性区域对MLCK活性的影响,进一步阐明MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制。方法：利用编码MLCK全长的pColdI表达载体对其ATP结合位点进行定点突变,获得无激酶活性的MLCK突变体;应用Glycerol—PAGE鉴定肌球蛋白磷酸化水平;应用孔雀绿方法检测重组MLCK对肌球蛋白ATP酶活性的影响。结果：MLCK／△ATP（突变型）失去磷酸化肌球蛋白轻链的激酶活性;重组MLCK（野生型）和MLCK／AATP（突变型）均可以在非钙条件下激活非磷酸化肌球蛋白Mg2＋-ATP酶活性,抑制磷酸化肌球蛋白的Mg2＋．ATP酶活性,而且激活与抑制作用均随着MLCK浓度的增加而增大,但二者对肌球蛋白的ATP酶活性的作用没有显著差异（P〉0．05）。结论：平滑肌肌球蛋白轻链激酶及ATP结合位点突变体具有激活非磷酸化肌球蛋白ATP酶活性的作用。     

平滑肌肌球蛋白B的超沉淀与肌球蛋白轻链磷酸化

本文报导了牛胃肌球蛋白B(天然肌动球蛋白)的超沉淀性质。当钙离子、钙调蛋白和ATP存在时,肌球蛋白B出现超沉淀,在pH6.8和7.5处,有两个峰值。Ca~(2+)(PCa值8-4)对超沉淀影响的浓度-反应曲线呈典型的S形,表明当Ca~(2+)浓度处于微摩尔水平时产生超沉淀。伴随超沉淀发生了肌球蛋白调节轻链磷酸化。这说明肌球蛋白轻链的Ca~(2+)-CaM依赖性磷酸化可能包含在脊椎动物平滑肌收缩活动的调节机制中。     

小檗碱对平滑肌肌球蛋白功能及胃肠平滑肌收缩性的影响

目的:探讨小檗碱对平滑肌肌球蛋白功能及胃肠平滑肌收缩性的影响.方法:以平滑肌肌球蛋白Mg2+-ATPase活性、肌球蛋白磷酸化以及胃与肠道平滑肌的收缩振幅为指标,考察小檗碱对平滑肌肌球蛋白Mg2+-ATPase活性和肌球蛋白磷酸化程度的影响,及其对离体小肠与胃平滑肌条收缩性的影响.结果:(1)在肌球蛋白轻链的Ca2+依赖性磷酸化反应中.小檗碱能抑制磷酸化肌球蛋白Mg2+-ATPase活性;(2)在肌球蛋白轻链的Ca2+依赖性磷酸化反应中,小檗碱可显著抑制磷酸化肌球蛋白轻链磷酸化程度;(3)小檗碱对大鼠离体小肠及胃平滑肌条收缩性均具有抑制作用.且均呈剂量依赖性.结论:小檗碱可通过抑制平滑肌肌球蛋白的功能,抑制胃肠道平滑肌的收缩性.     

d-尼古丁对血管平滑肌细胞迁移的影响

为了在分子水平上揭示吸烟导致动脉粥样硬化的机制,探讨了烟草致病的主要成分d-尼古丁对豚鼠大脑基底动脉血管平滑肌细胞GbaSM-4迁移作用的影响。应用Boyden小室实验发现,d-尼古丁具有促进GbaSM-4细胞迁移的作用。免疫荧光染色显示,在d-尼古丁作用下有GbaSM-4细胞伪足内肌动蛋白表达和分布增加的现象。为了进一步阐明d-尼古丁促进平滑肌细胞迁移作用的分子机制,应用RT-PCR方法检测到在GbaSM-4细胞内有α7型烟碱乙酰胆碱受体的表达。应用烟碱乙酰胆碱受体的特异性抑制剂甲基牛扁碱和肌肉收缩的关键酶——肌球蛋白轻链激酶（myosin light chain kinase,MLCK）抑制剂ML-9作用GbaSM-4细胞后,发现d-尼古丁对GbaSM-4细胞的诱导迁移作用被明显的抑制。采用RNA干扰技术,成功地使GbaSM-4细胞内MLCK的表达水平下调,观察到d-尼古丁对GbaSM-4细胞的诱导迁移作用也被明显的抑制。上述研究结果表明,d-尼古丁以趋化因子的作用促进血管平滑肌细胞迁移,其分子机制可能与α7型烟碱乙酰胆碱受体和MLCK等因素有关,这一发现为揭示吸烟导致动脉粥样硬化提供了实验依据。     

Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK‐mediated MLC phosphorylation

The retinal pigment epithelium (RPE) forms the outer blood–retina barrier (BRB). Most retinal diseases involve BRB breakdown, whereupon thrombin contained in serum directly contacts the RPE. Thrombin is known to promote actin stress fiber formation, an important determinant in eye diseases involving the epithelial–mesenchymal transition (EMT) and migration of RPE cells, such as proliferative vitreoretinopathy. We analyzed thrombin effect on signaling pathways leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in primary cultures of rat RPE cells, in order to support a role for thrombin in RPE transdifferentiation. MLC phosphorylation was measured by Western blot; actin cytoskeleton was visualized using immunofluorescent phalloidin, and Rho GTPase activation was assessed by ELISA. We showed that thrombin/PAR‐1 induces the time‐ and dose‐dependent phosphorylation of MLC through the activation of Rho/ROCK and myosin light chain kinase (MLCK). ROCK increased phospho‐MLC by phosphorylating MLC and by inhibiting MLC phosphatase. Thrombin effect was abolished by the ROCK inhibitor Y‐27632, whereas MLCK inhibitor ML‐7 and PLC‐β inhibitor U73122 attenuated MLC phosphorylation by ≈50%, suggesting the activation of MLCK by PLC‐β‐mediated calcium increase. Additionally, thrombin‐induced MLC phosphorylation was blocked by the inhibitory PKCζ pseudosubstrate, wortmannin, and LY294002, indicating IP₃/PKCζ involvement in the control of MLC phosphorylation. Moreover, we demonstrated that thrombin effect on MLC induces actin stress fiber formation, since this effect was prevented by inhibiting the pathways leading to MLC phosphorylation. We conclude that thrombin stimulation of MLC phosphorylation and actin stress fiber formation may be involved in thrombin‐induced RPE cell transformation subsequent to BRB dysfunction. J. Cell. Physiol. 226: 414–423, 2011. © 2010 Wiley‐Liss, Inc.     

Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells

As previously shown, constitutive activation of the small GTPase Rho and its downstream target Rho-kinase is crucial for spontaneous migration of Walker carcinosarcoma cells. We now show that after treatment of cells with either the Rho inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632, constitutive myosin light chain (MLC) phosphorylation is significantly decreased, correlating with inhibition of cell polarization and migration. Transfection with a dominant-negative Rho-kinase mutant similarly inhibits cell polarization and MLC phosphorylation. Transfection with a dominant-active Rho-kinase mutant leads to significantly increased MLC phosphorylation, membrane blebbing, and inhibition of cell polarization. This Rho-kinase-induced membrane blebbing can be inhibited by Y-27632, ML-7, and blebbistatin. Unexpectedly, overactivation of RhoA has similar effects as its inhibition. Introduction of a bacterially expressed constitutively activated mutant protein (but not of wild-type RhoA) into the cells or transfection of cells with a constitutively active RhoA mutant both inhibit polarization and decrease MLC phosphorylation. Transfection of cells with constitutively active or dominant-negative Rac both abrogate polarity, and the latter inhibits MLC phosphorylation. Our findings suggest an important role of Rac, Rho/Rho-kinase, and MLCK in controlling myosin activity in Walker carcinosarcoma cells and show that an appropriate level of RhoA, Rac, and Rho-kinase activity is required to regulate cell polarity and migration.     

Inhibition of the myosin light chain kinase prevents hypoxia-induced blood-brain barrier disruption

Increased mortality after stroke is associated with development of brain edema. The aim of the present study was to examine the contribution of endothelial myosin light chain (MLC) phosphorylation to hypoxia-induced blood-brain barrier (BBB) opening. Measurements of trans-endothelial electrical resistance (TEER) were performed to analyse BBB integrity in an in vitro co-culture model (bovine brain microvascular endothelial cells (BEC) and rat astrocytes). Brain fluid content was analysed in rats after stroke induction using a two-vein occlusion model. Dihydroethidium was used to monitor intracellular generation of reactive oxygen species (ROS) in BEC. MLC phosphorylation was detected using immunohistochemistry and immunoblot analysis. Hypoxia caused a decrease of TEER values by more than 40%, which was prevented by inhibition of the MLC-kinase (ML-7, 10 micromol/L). In addition, ML-7 significantly reduced the brain fluid content in vivo after stroke. The NAD(P)H-oxidase inhibitor apocynin (500 micromol/L) prevented the hypoxia-induced TEER decrease. Hypoxia-dependent ROS generation was completely abolished by apocynin. Furthermore, ML-7 and apocynin blocked hypoxia-dependent phosphorylation of MLC. Our data demonstrate that hypoxia causes a breakdown of the BBB in vitro and in vivo involving ROS and the contractile machinery.     

Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.

Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.     

Lens fiber cell elongation and differentiation is associated with a robust increase in myosin light chain phosphorylation in the developing mouse

Myosin II, a molecular motor, plays a critical role in cell migration, cell shape changes, cell adhesion, and cytokinesis. To understand the role of myosin II in lens fiber cell elongation and differentiation, we determined the distribution pattern of nonmuscle myosin IIA, IIB, and phosphorylated regulatory myosin light chain-2 (phospho-MLC) in frozen sections of the developing mouse lens by immunofluorescence analysis. While myosin IIA was distributed uniformly throughout the differentiating lens, including the epithelium and fibers, myosin IIB was localized predominantly to the epithelium and the posterior tips of the lens fibers. In contrast, immunostaining with a di-phospho-MLC antibody localized intensely and precisely to the elongating and differentiating primary and secondary lens fibers, co-localizing with actin filaments. An in situ analysis of Rho GTPase activation revealed that Rho-GTP was distributed uniformly throughout the embryonic lens, including epithelium and fibers. Inhibition of myosin light chain kinase (MLCK) activity by ML-7 in organ cultured mouse lenses led to development of nuclear lens opacity in association with abnormal fiber cell organization. Taken together, these data reveal a distinct spatial distribution pattern of myosin II isoforms in the developing lens and a robust activation of MLC phosphorylation in the differentiating lens fibers. Moreover, the regulation of MLC phosphorylation by MLCK appears to be critical for crystallin organization and for maintenance of lens transparency and lens membrane function.     

Process Formation in Astrocytes: Modulation of Cytoskeletal Proteins

Studies on primary astrocytes cultured in vitro have shown that process formation involves changes in cytoskeletal proteins and release of tension on the substratum. Actin filament reorganization has previously been found to be the major cytoskeletal change occurring during process formation. These changes are relatively rapid with breakdown of the actin web and release of contacts occur within 15 min. of cyclic AMP treatment. The former is regulated by myosin light chain (MLC) and actin depolymerizing factor (ADF), with MLC involved in the initial release of contractile tension and ADF in both initial and longer term actin breakdown. Our results show that the dephosphorylation of MLC is due to the phosphorylation and inactivation of myosin light chain kinase (MLCK) in response to cyclic AMP. To further study the mechanisms underlying the process formation in astrocytes we used endothelin-1 (ET-1), a vasopeptide which has been shown to inhibit process formation in astrocytes and sodium fluoride which is a general phosphatase inhibitor. We observe an increase in phosphorylation of MLC on inhibition of process formation. To study the role of adhesion in process formation we used suspension cultures of astrocytes. Our results with the astrocytes in suspension suggest that the process formation in astrocytes is adhesion dependent and the changes in ADF and MLC occur only when there is process formation.     

Intracellular signal transduction for migration and actin remodeling in vascular smooth muscle cells after sphingosylphosphorylcholine stimulation

Molecular mechanisms underlying migration of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (SPC) were analyzed in light of the hypothesis that remodeling of the actin cytoskeleton should be involved. After SPC stimulation, mitogen-activated protein kinases (MAPKs), including p38 MAPK (p38) and p42/44 MAPK (p42/44), were found to be phosphorylated. Migration of cells toward SPC was reduced in the presence of SB-203580, an inhibitor of p38, but not PD-98059, an inhibitor of p42/44. Pertussis toxin (PTX), a Gi protein inhibitor, induced an inhibitory effect on p38 phosphorylation and VSMC migration. Myosin light chain (MLC) phosphorylation occurred after SPC stimulation with or without pretreatment with SB-203580 or PTX. The MLC kinase inhibitor ML-7 and the Rho kinase inhibitor Y-27632 inhibited MLC phosphorylation but only partially inhibited SPC-directed migration. Complete inhibition was achieved with the addition of SB-203580. After SPC stimulation, the actin cytoskeleton formed thick bundles of actin filaments around the periphery of cells, and the cells were surrounded by elongated filopodia, i.e., magunapodia. The peripheral actin bundles consisted of alpha- and beta-actin, but magunapodia consisted exclusively of beta-actin. Such a remodeling of actin was reversed by addition of SB-203580 and PTX, but not ML-7 or Y-27632. Taken together, our biochemical and morphological data confirmed the regulation of actin remodeling and suggest that VSMCs migrate toward SPC, not only by an MLC phosphorylation-dependent pathway, but also by an MLC phosphorylation-independent pathway.