蛋白激酶在微血管通透性中的作用

微血管内皮细胞层是一层半选择通透性屏障,可以调节血液中的液体、溶质和血浆蛋白进入组织间隙。在炎症刺激作用下,可通过旁细胞途径和跨细胞途径引起内皮通透性上升。旁细胞通路主要由内皮细胞间的紧密连接、黏附连接和细胞与外基质的黏着斑组成。炎症介质,如脂多糖和肿瘤坏死因子α可激活多种蛋白激酶。活化的蛋白激酶主要包括Rho相关的卷曲蛋白激酶、肌球蛋白轻链激酶、蛋白激酶C、酪氨酸激酶和丝裂原活化蛋白激酶等,参与引发内皮屏障生化和结构改变,旁细胞通路开放,导致通透性上升。该文对上述蛋白激酶在微血管通透性中作用机制的研究进展进行综述。     

细菌效应蛋白对宿主细胞Rho小G蛋白信号通路的调节作用

病原体细菌通过自身分泌系统分泌效应蛋白并注入宿主体内,修饰宿主的信号转导系统,破坏宿主细胞中天然免疫有关信号通路,发挥毒性作用使宿主产生疾病。吞噬作用在天然免疫系统中发挥重要作用,这个过程涉及肌动蛋白细胞骨架的重排。Rho(Ras homolog family)小G蛋白家族成员作为细胞骨架结构的重要调控蛋白可调节这一过程,其相关信号通路成为细菌效应蛋白的作用靶点。细菌效应蛋白可以模仿Rho的调节因子破坏信号通路,可以通过剪切Rho C-端的尾部结构使其从细胞膜解离并失去活性,可以直接模仿Rho发挥调控功能,可以影响Rho上游的调控事件影响其活性,也可通过对Rho进行直接的翻译后修饰使其失活,形成有利于细菌生存、繁殖、毒力释放的环境。由此导致的Rho信号通路功能紊乱使宿主产生智力缺陷、免疫功能障碍、癌症等多种疾病。     

丝裂原活化蛋白激酶激活蛋白激酶(MK)研究进展

丝裂原活化蛋白激酶(MAPK)信号通路介导多种重要的细胞生理反应.对下游蛋白激酶的磷酸化是MAPK家族成员发挥生理作用的重要方式.在MAPK的下游存在3个结构上相关的MAPK激活蛋白激酶(MAPKAPKorMK),即MK2,MK3和MK5.在被MAPK激活后,MK可将信号传递至细胞内不同靶标,从而在转录和翻译水平调节基因表达,调控细胞骨架和细胞周期,介导细胞迁移和胚胎发育.最近,在基因敲除研究的基础上,不同MK亚族成员之间的功能区分已经逐渐明晰,使我们对于MK的认识有了长足的进步.     

ROCK与动脉粥样硬化

Rho相关的卷曲螺旋型蛋白激酶（Rho—associated,coiled-coil containing protein kinase,ROCK）是ras同源家族RhoA（ras homolog family memberA）T游的靶蛋白之一,主要功能是调节肌动蛋白细胞骨架的活动,如细胞粘附、细胞运动、细胞迁移及细胞收缩。实验及临床研究表明R0cK可能与多种心血管疾病如高血压、肺动脉高压、动脉粥样硬化以及脑血管疾病有着很大相关性。此篇综述将总结近期对于RhoA／ROCK通路在调控血管功能中的关键作用并探讨其对于动脉粥样硬化相关疾病的潜在治疗价值。     

Rho小G蛋白相关激酶的结构与功能

Rho小G蛋白家族是Ras超家族成员之一,人类Rho小G蛋白包括20个成员,研究最清楚的有RhoA、Rac1和Cdc42。Rho小G蛋白参与了诸如细胞骨架调节、细胞移动、细胞增殖、细胞周期调控等重要的生物学过程。在这些生物学过程的调节中,Rho小G蛋白的下游效应蛋白质如蛋白激酶（p21-activated kinase,PAK）、ROCK（Rho-kinase）、PKN（protein kinase novel）和MRCK（myotonin-related Cdc42-binding kinase）发挥了不可或缺的作用。迄今研究发现,PAK可调节细胞骨架动力学和细胞运动,另外,PAK通过MAPK（mitogen-activated protein kinases）参与转录、细胞凋亡和幸存通路及细胞周期进程;ROCK与肌动蛋白应力纤维介导黏附复合物的形成及与细胞周期进程的调节有关;哺乳动物的PKN与RhoA/B/C相互作用介导细胞骨架调节;MRCK与细胞骨架重排、细胞核转动、微管组织中心再定位、细胞移动和癌细胞侵袭等有关。该文简要介绍Rho小G蛋白下游激酶PAK、ROCK、PKN和MRCK的结构及其在细胞骨架调节中的功能,重点总结它们在真核细胞周期调控中的作用,尤其是在癌细胞周期进程中所发挥的作用,为寻找癌症治疗的新靶点提供理论依据。     

Rho激酶(ROCK)在糖尿病并发症中的研究

ROCK(Rho-associated protein kinase),即Rho相关蛋白激酶,是Rho/ROCK通路的重要蛋白。ROCK与GTP(guanosine triphosphate)结合蛋白Rho相互作用,通过磷酸化激活多种下游蛋白或核因子,在机体的各项调节功能中起到重要的作用。研究表明,糖尿病患者体内ROCK异常上调,可能是导致糖尿病并发症的重要原因,最终危及患者心血管系统、泌尿系统乃至生殖系统。而对ROCK的深入研究表明,使用ROCK的拮抗剂,如法舒地尔等,可有效抑制ROCK/Rho通路,最终缓解糖尿病并发症。     

平滑肌细胞骨架结构及其信号调节途径

平滑肌细胞骨架是一个复杂的动态性网络,是细胞生命活动不可缺少的细胞结构。Rho通过活化其下游靶分子促进应力纤维的形成,其中Rho—associated coiled—coil kinase(ROCK)和Dial在该过程中起关键作用;PKC通过在细胞内不同定位的亚型使细胞骨架蛋白磷酸化,发挥其调节细胞骨架重构的作用。两条信号转导途径通过Src途径相互联系,共同参与细胞骨架动力学的调节。     

蛋白激酶C分子的成熟与活化

蛋白激酶C(Protein kinase C,PKC)是细胞内一类重要的Ser/Thr激酶,调控多种生命活动的信号转导过程,目前已发现了至少11种亚型,其结构有一定的保守性而又有所差别,导致其功能和调控的多样性。新合成的PKC一般需要经历活化茎环(Activation-loop,A-loop)、转角模体(Turn motif,TM)以及疏水模体(hydrophobic motif,HM)的程序性磷酸化过程才能成熟,获得进一步活化的功能。本文综述了近年来PKC的程序性磷酸化成熟以及活化的研究进展情况。     

p53在细胞增殖和迁移中对骨架重建的作用

p53作为最重要的抑癌因子之一,通常作为转录因子发挥肿瘤抑制作用。除转录活性外,p53及其突变型可能通过调节整合素、钙黏蛋白、Rho/ROCK信号通路等对肌动蛋白细胞骨架重建产生作用,从而影响细胞增殖和迁移。p53的这些功能在调节肌动蛋白细胞骨架重建以响应细胞外微环境和癌基因激活中起着至关重要的作用。     

ABCA1介导胆固醇外流及抗炎的信号通路研究进展

ABCA1抗动脉粥样硬化的作用主要通过以下两种途径:介导细胞内胆固醇流出和抑制炎症。载脂蛋白与ABCA1的相互作用可激活多个信号通路,包括JAK2/STAT3、蛋白激酶A(PKA)、Rho家族G蛋白CDC42和蛋白激酶C(PKC)等信号通路。ABCA1通过修饰细胞膜脂筏或直接激活信号通路而介导脂质流出和发挥抗炎功能。对这些信号通路的认识,能为动脉粥样硬化相关疾病提供新的治疗靶点。     

Ezrin/radixin/moesin proteins are phosphorylated by TNF-alpha and modulate permeability increases in human pulmonary microvascular endothelial cells

Endothelial cells (ECs) respond to TNF-alpha by altering their F-actin cytoskeleton and junctional permeability through mechanisms that include protein kinase C (PKC) and p38 MAPK. Ezrin, radixin, and moesin (ERM) regulate many cell processes that often require a conformational change of these proteins as a result of phosphorylation on a conserved threonine residue near the C terminus. This study tested the hypothesis that ERM proteins are phosphorylated on this critical threonine residue through TNF-alpha-induced activation of PKC and p38 and modulate permeability increases in pulmonary microvascular ECs. TNF-alpha induced ERM phosphorylation on the threonine residue that required activation of p38, PKC isoforms, and phosphatidylinositol-4-phosphate 5-kinase Ialpha, a major enzyme generating phosphatidylinositol 4,5-bisphosphate, and phosphorylated ERM were prominently localized at the EC periphery. TNF-alpha-induced ERM phosphorylation was accompanied by cytoskeletal changes, paracellular gap formation, and increased permeability to fluxes of dextran and albumin. These changes required activation of p38 and PKC and were completely prevented by inhibition of ERM protein expression using small interfering RNA. Thus, ERM proteins are phosphorylated through p38 and PKC-dependent mechanisms and modulate TNF-alpha-induced increases in endothelial permeability. Phosphorylation of ERM likely plays important roles in EC responses to TNF-alpha by modulating the F-actin cytoskeleton, adhesion molecules, and signaling events.     

Involvement of Rho/ROCK signalling in small cell lung cancer migration through human brain microvascular endothelial cells

Small cell lung cancer (SCLC) cells migration across human brain microvascular endothelial cells (HBMECs) is an essential step of brain metastases. Here we investigated signalling pathways in HBMECs contributing to the process. Inhibition of endothelial Rho kinase (ROCK) with Y27632 and overexpression of ROCK dominant-negative mutant prevented SCLC cells, NCI-H209, transendothelial migration and the concomitant changes of tight junction. Conversely, inhibition of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) had no effects. Furthermore, endothelial RhoA protein was activated during NCI-H209 cells transendothelial migration. Rho/ROCK participated in NCI-H209 cells transendothelial migration through regulating actin cytoskeleton reorganization. These results suggested that Rho/ROCK was required for SCLC cells transendothelial migration.     

Protein kinase C-related kinase and ROCK are required for thrombin-induced endothelial cell permeability downstream from Galpha12/13 and Galpha11/q

Increase in vascular permeability occurs under many physiological conditions such as wound repair, inflammation, and thrombotic reactions and is central in diverse human pathologies, including tumor-induced angiogenesis, ocular diseases, and septic shock. Thrombin is a pro-coagulant serine protease, which causes the local loss of endothelial barrier integrity thereby enabling the rapid extravasation of plasma proteins and the local formation of fibrin-containing clots. Available information suggests that thrombin induces endothelial permeability by promoting actomyosin contractility through the Rho/ROCK signaling pathway. Here we took advantage of pharmacological inhibitors, knockdown approaches, and the emerging knowledge on how permeability factors affect endothelial junctions to investigate in detail the mechanism underlying thrombin-induced endothelial permeability. We show that thrombin signals through PAR-1 and its coupled G proteins Galpha(12/13) and Galpha(11/q) to induce RhoA activation and intracellular calcium elevation, and that these events are interrelated. In turn, this leads to the stimulation of ROCK, which causes actin stress-fiber formation. However, this alone is not sufficient to account for thrombin-induced permeability. Instead, we found that protein kinase C-related kinase, a Rho-dependent serine/threonine kinase, is activated in endothelial cells upon thrombin stimulation and that its expression is required for endothelial permeability and the remodeling of cell-extracellular matrix and cell-cell adhesions. Our results demonstrate that the signal initiated by thrombin bifurcates at the level of RhoA to promote changes in the cytoskeletal architecture through ROCK, and the remodeling of focal adhesion components through protein kinase C-related kinase. Ultimately, both pathways converge to cause cell-cell junction disruption and provoke vascular leakage.     

Identification and functional implication of a Rho kinase-dependent moesin-EBP50 interaction in noradrenaline-stimulated artery

Ezrin, radixin, and moesin (ERM) proteins are known to be substrates of Rho kinase (ROCK), a key player in vascular smooth muscle regulation. Their function in arteries remains to be elucidated. The objective of the present study was to investigate ERM phosphorylation and function in rat aorta and mesenteric artery and the influence of ERM-binding phosphoprotein 50 (EBP50), a scaffold partner of ERM proteins in several cell types. In isolated arteries, ERM proteins are phosphorylated by PKC and ROCK with different kinetics after either agonist stimulation or KCl-induced depolarization. Immunoprecipitation of EBP50 in noradrenaline-stimulated arteries allowed identification of its interaction with moesin and several other proteins involved in cytoskeleton regulation. This interaction was inhibited by Y27632, a ROCK inhibitor. Moesin or EBP50 depletion after small interfering RNA transfection by reverse permeabilization in intact mesenteric arteries both potentiated the contractility in response to agonist stimulation without any effect on contractile response induced by high KCl. This effect was preserved in ionomycin-permeabilized arteries. These results indicate that, in agonist-stimulated arteries, the activation of ROCK leads to the binding of moesin to EBP50, which interacts with several components of the cytoskeleton, resulting in a decrease in the contractile response.     

Protein kinase Calpha-RhoA cross-talk in CCL2-induced alterations in brain endothelial permeability

Monocyte chemoattractant protein-1 (MCP-1 or CCL2) regulates blood-brain barrier permeability by inducing morphological and biochemical alterations in the tight junction (TJ) complex between brain endothelial cells. The present study used cultured brain endothelial cells to examine the signaling networks involved in the redistribution of TJ proteins (occludin, ZO-1, ZO-2, claudin-5) by CCL2. The CCL2-induced alterations in the brain endothelial barrier were associated with de novo Ser/Thr phosphorylation of occludin, ZO-1, ZO-2, and claudin-5. The phosphorylated TJ proteins were redistributed/localized in Triton X-100-soluble as well as Triton X-100-insoluble cell fractions. Two protein kinase C (PKC) isoforms, PKCalpha and PKCzeta, had a significant impact on this event. Inhibition of their activity using dominant negative mutants PKCalpha-DN and PKCzeta-DN diminished CCL2 effects on brain endothelial permeability. Previous data indicate that Rho/Rho kinase signaling is involved in CCL2 regulation of brain endothelial permeability. The interactions between the PKC and Rho/Rho kinase pathways were therefore examined. Rho, PKCalpha, and PKCzeta activities were knocked down using dominant negative mutants (T17Rho, PKCalpha-DN, and PKCzeta-DN, respectively). PKCalpha and Rho, but not PKCzeta and Rho, interacted at the level of Rho, with PKCalpha being a downstream target for Rho. Double transfection experiments using dominant negative mutants confirmed that this interaction is critical for CCL2-induced redistribution of TJ proteins. Collectively these data suggest for the first time that CCL2 induces brain endothelial hyperpermeability via Rho/PKCalpha signal pathway interactions.     

Rho signaling in Entamoeba histolytica modulates actomyosin-dependent activities stimulated during invasive behavior

Interaction of Entamoeba histolytica trophozoites with target cells and substrates activates signaling pathways in the parasite. Phosphorylation cascades triggered by phospho-inositide and adenyl-cyclase-dependent pathways modulate reorganization of the actin cytoskeleton to form structures that facilitate adhesion. In contrast, little is known about participation of Rho proteins and Rho signaling in actin rearrangements. We report here the in vivo expression of at least one Rho protein in trophozoites, whose activation induced actin reorganization and actin-myosin interaction. Antibodies to EhRhoA1 recombinant protein mainly localized Rho in the cytosol of nonactivated amoebae, but it was translocated to vesicular membranes and to some extent to the plasma membrane after treatment with lysophosphatidic acid (LPA), a specific agonist of Rho activation. Activated Rho was identified in LPA-treated trophozoites. LPA induced striking polymerization of actin into distinct dynamic structures. Disorganization of these structures by inhibition of Rho effector, Rho-kinase (ROCK), and by ML-7, an inhibitor of myosin light chain kinase dependent phosphorylation of myosin light chain, suggested that the actin structures also contained myosin. LPA stimulated concanavalin-A-mediated formation of caps, chemotaxis, invasion of extracellular matrix substrates, and erythrophagocytosis, but not binding to fibronectin. ROCK inhibition impaired LPA-stimulated functions and to some extent adhesion to fibronectin. Similar results were obtained with ML-7. These data suggest the presence and operation of Rho-signaling pathways in E. histolytica, that together with other, already described, signaling routes modulate actomyosin-dependent motile processes, particularly stimulated during invasive behavior.     

Involvement of RhoA and Rho kinase in neutrophil-stimulated endothelial hyperpermeability

Neutrophil-induced microvascular leakage is an early event in ischemic and inflammatory heart diseases. The specific signaling paradigm by which neutrophils increase microvascular permeability is not yet established. We investigated whether the small GTPase RhoA and its downstream effector Rho kinase mediate neutrophil-stimulated endothelial hyperpermeability. We assessed the effect of neutrophils on Rho activity in bovine coronary venular endothelial cells (CVEC) with a Rho-GTP pull-down assay. Permeability to FITC-albumin was evaluated using CVEC monolayers. We then tested the role of Rho kinase in the permeability response to neutrophils using two structurally distinct pharmacological inhibitors: Y-27632 and HA-1077. Furthermore, neutrophil-stimulated changes in endothelial F-actin organization were examined with fluorescence microscopy. The results show that C5a-activated neutrophils induced an increase in permeability coupled with RhoA activation in CVEC. Inhibition of Rho kinase with either Y-27632 or HA-1077 attenuated the hyperpermeability response. Rho kinase inhibition also attenuated increases in permeability stimulated by the neutrophil supernatant. In addition, activated neutrophils caused actin stress fiber formation in CVEC, which was diminished by either Y-27632 or HA-1077. These findings suggest that RhoA and Rho kinase are involved in the mediation of neutrophil-induced endothelial actin reorganization and barrier dysfunction.     

Role of Rho-kinase in reexpansion pulmonary edema in rabbits

Reexpansion of a collapsed lung increases the microvascular permeability and causes reexpansion pulmonary edema. Neutrophils and their products have been implicated in the development of this phenomenon. The small GTP-binding proteins Rho and its target Rho-kinase (ROCK) regulate endothelial permeability, although their roles in reexpansion pulmonary edema remain unclear. We studied the contribution of ROCK to pulmonary endothelial and epithelial permeability in a rabbit model of this disorder. Endothelial and epithelial permeability was assessed by measuring the tissue-to-plasma (T/P) and bronchoalveolar lavage (BAL) fluid-to-plasma (B/P) ratios with (125)I-labeled albumin. After intratracheal instillation of (125)I-albumin, epithelial permeability was also assessed from the plasma leak (PL) index, the ratio of (125)I-albumin in plasma/total amount of instilled (125)I-albumin. T/P, B/P, and PL index were significantly increased in the reexpanded lung. These increases were attenuated by pretreatment with Y-27632, a specific ROCK inhibitor. However, neutrophil influx, neutrophil elastase activity, and malondialdehyde concentrations in BAL fluid collected from the reexpanded lung were not changed by Y-27632. In endothelial monolayers, Y-27632 significantly attenuated the H(2)O(2)-induced increase in permeability and mitigated the morphological changes in the actin microfilament cytoskeleton of endothelial cells. These in vivo and in vitro observations suggest that the Rho/ROCK pathway contributes to the increase in alveolar barrier permeability associated with reexpansion pulmonary edema.     

RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration

Endothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiation-induced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15 Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial functions linked to actin cytoskeleton.     

Noise‐induced cochlear F‐actin depolymerization is mediated via ROCK2/p‐ERM signaling

Our previous work has suggested that traumatic noise activates Rho‐GTPase pathways in cochlear outer hair cells (OHCs), resulting in cell death and noise‐induced hearing loss (NIHL). In this study, we investigated Rho effectors, Rho‐associated kinases (ROCKs), and the targets of ROCKs, the ezrin‐radixin‐moesin (ERM) proteins, in the regulation of the cochlear actin cytoskeleton using adult CBA/J mice under conditions of noise‐induced temporary threshold shift (TTS) and permanent threshold shift (PTS) hearing loss, which result in changes to the F/G‐actin ratio. The levels of cochlear ROCK2 and p‐ERM decreased 1 h after either TTS‐ or PTS‐noise exposure. In contrast, ROCK2 and p‐ERM in OHCs decreased only after PTS‐, not after TTS‐noise exposure. Treatment with lysophosphatidic acid, an activator of the Rho pathway, resulted in significant reversal of the F/G‐actin ratio changes caused by noise exposure and attenuated OHC death and NIHL. Conversely, the down‐regulation of ROCK2 by pretreatment with ROCK2 siRNA reduced the expression of ROCK2 and p‐ERM in OHCs, exacerbated TTS to PTS, and worsened OHC loss. Additionally, pretreatment with siRNA against radixin, an ERM protein, aggravated TTS to PTS. Our results indicate that a ROCK2‐mediated ERM‐phosphorylation signaling cascade modulates noise‐induced hair cell loss and NIHL by targeting the cytoskeleton.