pp60c-src在血管平滑肌细胞内丝裂原活化蛋白激酶激活中的作用

观察ｐｐ６０ｃ－ｓｒｃ在血管紧张素Ⅱ（ＡｎｇⅡ）诱导血管平滑肌细胞（ＶＳＭＣｓ）内丝裂原活化蛋白激酶（ＭＡＰＫ）激活中的作用,以了解ＡｎｇⅡ促ＶＳＭＣｓ增殖的信号转导过程。将合成的反义ｃ－ｓｒｃ寡脱氧核苷酸（ｏｌｉｇｏｄｅｏｘｙｎｕｃｌｅ－ｏｔｉｄｅｓ,ＯＤＮｓ）以脂质体包裹转染培养的大鼠ＶＳＭＣｓ,用Ｗｅｓｔｅｒｎ印迹测得细胞裂解液中ｐｐ６０ｃ－ｓｒｃ含量明显下降,免疫沉淀方法测得ｐｐ６０ｃ－ｓ     

病原菌调节宿主细胞丝裂原活化蛋白激酶免疫防御信号的机理

丝裂原活化蛋白激酶(MAPK)家族广泛存在于高等生物中,介导多种生物学进程,在固有免疫防御中发挥重要作用,是真核细胞抵御病原菌侵染的第一道防线.越来越多的研究发现,病原菌可以利用多种方式激活或者抑制MAPK信号通路来增强其自身侵染力.简单介绍了MAPK信号通路的背景并详细总结了近几年关于病原菌如何作用于MAPK信号通路的研究工作,希望以此能够拓展对病原菌与宿主细胞作用方式的认识,深化对MAPK重要作用的了解.     

植物丝裂原活化蛋白激酶级联信号转导通路研究进展

丝裂原活化蛋白激酶(MAPK)是酵母、动物和植物等真核生物中普遍存在和高度保守的一类信号转导通路,由MAPKKK、MAPKK和MAPK等3部分组成,在应对生物非生物胁迫、激素、细胞分裂调控及植物生长发育等过程中发挥重要作用。该文对近年来国内外有关MAPK级联通路的组成、在植株体内的生物学功能以及MAPK通路的失活进行了概述,旨在为今后MAPK通路介导的信号转导机制的研究提供参考依据。     

丝裂原活化蛋白激酶和磷酸酯酶-1在心肌病和细胞凋亡中的作用

ERK、JNK和p38等丝裂原活化蛋白激酶通过生长因子、激动剂或应激反应等介导生长、分化、凋亡以及细胞间相互作用等多种过程。ERK、JNK和p38是参与心衰病理过程的主要信号元件,MKP-1是丝裂原活化蛋白激酶等的去磷酸化因子,是一种应激蛋白,在应激反应中可以抑制ERK、JNK和p38的活性,并通过凋节ERK、JNK和p38的活性,参与对心衰病理过程的调节。本文以转基因研究结果为主要线索,对丝裂原活化蛋白激酶和磷酸酯酶．1在心衰病理过程中的作用进行了综述。     

p38丝裂原活化蛋白激酶的功能与调控机制

丝裂原活化蛋白激酶家族可以在一系列细胞外刺激下调控细胞的行为。作为该家族的四个亚家族之一,p38亚族在许多生理过程中扮演着重要角色。p38信号通路可以在紫外照射、热击、高渗透压、炎症因子、生长因子等细胞外刺激时被激活,调控细胞分化、细胞周期、炎症反应等多种生理过程。文章重点讨论了p38亚族各个成员的特性、该信号通路的组成部分、调控机制以及生物学功能。另外,还分析了p38与其他信号通路的联系以及对一些生理过程的影响。     

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。     

胰岛素减轻血管平滑肌细胞钙化但增加血管平滑肌细胞的磷转运

血管钙化是糖尿病、尿毒症和衰老等疾病共同的临床病理过程。临床资料显示,80%的血管损伤和90%的冠状动脉疾病的患者伴有血管钙化。既往认为,胰岛素通过具有维持血糖平衡、抗钙化作用的“良性”的磷脂酰肌醇激酶(phosphatidylinoesitol 3 kinase,PI3K)途径和促细胞有丝分裂、增     

丝裂原活化蛋白激酶15纳米抗体的制备及其在B16-F10黑素瘤细胞生长过程中的抑制作用

丝裂原活化蛋白激酶15 (mitogen-activated protein kinase 15,MAPK15),又称ERK7或ERK8,是MAPK家族的非典型新成员。MAPK15不同程度地促进不同肿瘤细胞的增殖、迁移、自噬等细胞活动。本研究以MAPK15为靶点,筛选特异性的MAPK15纳米抗体,评估其是否能够作为免疫组织化学和Western印迹中的一抗用于其抗原表达的检测,并探究该纳米抗体在B16-F10黑色素瘤细胞中的作用。通过噬菌体展示技术从B16-F10黑色素瘤细胞纳米抗体文库中进行筛选,得到1株MAPK15特异性纳米抗体,命名为MAPK15-VHH;将该菌株构建原核表达载体,进行优化诱导表达条件时发现,0.6 nmol/L IPTG,15℃,100 r/min条件下该纳米抗体的上清表达量最高。通过竞争ELISA法检测MAPK15-VHH的亲和力,结果显示,该抗体K_D值为0.9829。通过Western印迹和免疫组织化学检测脑组织中MAPK15在蛋白质水平的表达量及分布情况,结果表明,MAPK15-VHH可与组织中的MAPK15结合,用于检测MAPK15蛋...     

丝裂原活化蛋白激酶介导脱乙酰几丁质诱导的人参细胞氧迸发与皂苷合成

脱乙酰几丁质(chitosan, CHN)可以特异性诱导人参细胞42与39 kD蛋白激酶活性, 丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)途径的抑制剂PD98059可以抑制CHN的这种诱导. 利用MAPK抗体进行免疫沉淀试验与体外激酶分析也表明CHN诱导的42 kD与39 kD蛋白为一类MAPK. PD98059还可以抑制CHN诱导的鲨烯合成酶与鲨烯环氧酶基因(gss与gse)的转录、β-香树素合成酶(β-amyrin synthase, β-AS)的累积与人参皂苷的合成. 这些结果表明, CHN诱导的MAPK对于促进人参皂苷的合成是必需的. EGTA与LaCl3可以抑制CHN诱导的42与39 kD MAPK活性, 钌红(ruthenium red, RR)可以抑制CHN诱导的39 kD MAPK活性, 而且它们又都能抑制人参皂苷的合成, 说明胞内钙离子浓度的升高对于诱导MAPK活性与人参皂苷的合成是必需的. PD98059可以抑制CHN诱导的氧迸发(包括质膜NADPH氧化酶活性与H₂O₂的产生), 但是二碘基苯(diphenylene iodonium, DPI)、二甲基硫脲(dimethylthiourea, DMTU)与2, 5-二羟基肉桂酸甲酯(2, 5-dihydroxycinnamic acid methyl ester, DHC)却不能抑制CHN诱导的MAPK活 性, 表明CHN诱导的MAPK活性可能作用于人参细胞氧迸发的上游.     

Coronary artery smooth muscle in culture: migration of heterogeneous cell populations from vessel wall

A method for establishing primary cultures of smooth muscle cells (SMCs) from the porcine coronary artery without either microdissection and/or enzymatic dispersion was developed using selective migration of cells from coronary explants in vitro. This culture method relies on the heterogeneity of cell types and differences in their migration and adherence ability to separate SMC from contaminating fibroblasts or endothelial cells. The cell type was determined by immunohistochemical staining with monoclonal antibodies to SM -actin, SM myosin, h-caldesmon and von Willebrand factor. The first wave of migration (1-7 days) consisted of a mixture of fibroblasts and SMCs. Only SMCs were present in the second wave of migration (7-14 days). Endothelial cells, which exhibited a lower capacity for migration and adherence, were restricted to the third wave of migration (14-21 days). Cells obtained from the second wave of migration exhibited the characteristic single-layered, aligned, hill-and-valley pattern of SMCs when confluent. Quiescence was attained 4-5 days after removal of serum, as established by [3H]-thymidine incorporation. Stimulation of the quiescent SMCs with 20% FBS resulted in a synchronous re-entry into the cell-cycle with S phase reached 15-18 h later. The SMCs prepared using this protocol thus exhibit the structural markers and capacity to undergo phenotypic modulation that are characteristic of SMCs in vivo. This approach to establishing primary cultures of SMCs offers the advantage of selecting for the subpopulation of cells capable of migration in response to injury or growth factor stimulation.     

VDR-mediated gene expression patterns in resting human coronary artery smooth muscle cells

Vitamin D analogs such as paricalcitol and calcitriol that activate the vitamin D receptor (VDR) provide survival benefit for Stage 5 chronic kidney disease (CKD) patients, possibly associated with a decrease in cardiovascular (CV)-related incidents. Phenotypic changes of smooth muscle cells play an important role in CV disease. The role of vitamin D analogs in modulating gene expression in smooth muscle cells is still not well understood. In this study, DNA microarray analysis of approximately 22,000 different human genes was used to characterize the VDR-mediated gene expression profile in human coronary artery smooth muscle cells (CASMC) at rest. Cells in serum free medium were treated with 0.1 microM calcitriol (1alpha,25-dihydroxyvitamin D(3)) or paricalcitol (19-nor-1alpha,25-(OH)(2)D(2)) for 30 h. A total of 181 target genes were identified, with 103 genes upregulated and 78 downregulated (>two fold changes in either drug treatment group with P < 0.01). No significant difference was observed between calcitriol and paricalcitol. Target genes fell into various categories with the top five in cellular process, cell communication, signal transduction, development, and morphogenesis. Twenty-two selected genes linked to the CV system were also impacted. Real-time RT-PCR and/or Western blotting analysis were employed to confirm the expression patterns of selected genes such as 25-hydroxyvitamin D-24-hydroxylase, Wilms' tumor gene 1, transforming growth factorbeta3, plasminogen activator inhibitor-1, thrombospondin-1 (THBS1), and thrombomodulin (TM). This study provides insight into understanding the role of VDR in regulating gene expression in resting smooth muscle cells.     

影响主动脉平滑肌细胞迁移的因素

平滑肌细胞(vascular smooth muscle cell,VSMC)的迁移对血管发育、动脉粥样硬化和术后再狭窄等起到关键性的作用。主要从激发VSMC迁移的关键炎性细胞因子、细胞间相互作用的核心成员、microRNA、细胞骨架和上述各因素的迁移信号通路这几方面来综述VSMC的迁移。     

Decorin synthesized by arterial smooth muscle cells is retained in fibrin gels and modulates fibrin contraction

Fibrin serves as a provisional extracellular matrix (ECM) for arterial smooth muscle cells (ASMC) after vascular injury, yet little is known about the effect of fibrin on ECM remodeling by these cells. To address this question, monkey ASMC were grown on fibrin gels and tissue culture (TC) plastic, and proteoglycan synthesis and accumulation were assessed by radiolabeling. Initial rates of (35)S-sulfate incorporation into proteoglycans were identical for both groups, but increased proteoglycan accumulation was observed in cultures grown for 48 h on fibrin. This increased accumulation on fibrin was due to reduced proteoglycan turnover and retention within the fibrin gel. Decorin and biglycan constituted 40 and 14% of the total proteoglycan in the fibrin gels, whereas their combined contribution was only 12% in control matrices. To explore whether the retention of decorin in fibrin had any influence on the properties of the fibrin gel, ASMC-mediated fibrin contraction assays were performed. Both de novo synthesis of decorin as well as decorin added during polymerization inhibited the ability of the cells to contract fibrin. In contrast, decorin added exogenously to mature fibrin matrices had no effect on fibrin gel contraction. This study illustrates that decorin derived from ASMC selectively accumulates in fibrin and modifies fibrin architecture and mechanical properties. Such an accumulation may influence wound healing and the thrombotic properties of this provisional pro-atherosclerotic ECM.     

周期蛋白D1参与香烟烟雾提取物促进人肺动脉平滑肌细胞增殖和迁移

本文旨在探讨周期蛋白D1(cyclin D1)在香烟烟雾提取物(cigarette smoke extract,CSE)所致人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells,HPASMCs)增殖和迁移中的作用。构建反义cyclin D1基因真核表达载体(pIRES2-EGFP-ascyclin D1),采用脂质体介导基因转染法将空载体(pIRES2-EGFP)和pIRES2-EGFP-ascyclin D1导入正常HPASMCs后,分别进行CSE干预。细胞随机分为6组:对照组、空载体组、反义cyclin D1组、5%CSE组、空载体+5%CSE组、反义cyclin D1+5%CSE组。用实时荧光RT-PCR和Western blot法分别检测cyclin D1mRNA和蛋白的表达,采用流式细胞术、四甲基偶氮唑盐比色法(MTT)、增殖细胞核抗原(PCNA)染色法测定细胞增殖能力,Transwell小室法检测细胞迁移能力。结果显示,反义cyclin D1基因真核表达载体pIRES2-EGFP-ascyclin D1成功构建,并成功转染入HPASMCs,转染后HPASMCs中cyclin D1的mRNA和蛋白表达水平较对照组均显著下降(P0.05)。与对照组比较,5%CSE组cyclinD1的mRNA和蛋白表达水平均明显升高(P0.05),细胞增殖和迁移能力显著增强(P0.05)。与5%CSE组比较,反义cyclinD1+5%CSE组cyclin D1mRNA和蛋白表达水平均明显下降(P0.05),细胞增殖和迁移能力显著降低(P0.05)。上述结果提示,CSE可通过上调cyclin D1表达促进HPASMCs增殖和迁移,反义cyclin D1基因真核表达载体可抑制CSE介导的HPASMCs增殖和迁移,提示cyclin D1在CSE所致HPASMCs增殖和迁移中发挥重要调控作用。     

Role of NADPH oxidase in cytomegalovirus-induced proliferation of human coronary artery smooth muscle cells

A number of infectious agents have been implicated in the development of vascular diseases such as atherosclerosis and posttransplantation arterial restenosis. Cytomegalovirus (CMV) has been reported to cause obliteration of coronary arteries by a progressive vasculopathy that involves proliferation of medial smooth muscle cells (SMC). In this study, we report that CMV enhances the serum-induced proliferation of human coronary SMC through activation of a superoxide-generating NADPH oxidase. Exposure of SMC to CMV for 2 h was associated with an 80% increase in NADPH oxidase. This increase in oxidase activity was associated with a two-fold increase in serum-induced DNA synthesis (5-bromo-2'-deoxyuridine incorporation) and significant interleukin-8 (IL-8) production by SMC. Diphenylene iodonium, an inhibitor of NADPH oxidase, significantly inhibited CMV-induced IL-8 production and promotion of serum-induced DNA synthesis. Similar effects were seen following pretreatment of SMC with N-acetyl cysteine, a potent antioxidant, suggesting that oxidative stress following CMV exposure might be responsible for triggering the proliferation of SMC. From this study, we conclude that CMV-mediated promotion of SMC growth is redox sensitive and may be mediated by NADPH oxidase.     

Arterial territory-specific phosphorylated retinoblastoma protein species and CDK2 promote differences in the vascular smooth muscle cell response to mitogens

Despite recent advances in medical procedures, cardiovascular disease remains a clinical challenge and the leading cause of mortality in the western world. The condition causes progressive smooth muscle cell (SMC) dedifferentiation, proliferation, and migration that contribute to vascular restenosis. The incidence of disease of the internal mammary artery (IMA), however, is much lower than in nearly all other arteries. The etiology of this IMA disease resistance is not well understood. Here, using paired primary IMA and coronary artery SMCs, serum stimulation, siRNA knockdowns, and verifications in porcine vessels in vivo, we investigate the molecular mechanisms that could account for this increased disease resistance of internal mammary SMCs. We show that the residue-specific phosphorylation profile of the retinoblastoma tumor suppressor protein (Rb) appears to differ significantly between IMA and coronary artery SMCs in cultured human cells. We also report that the differential profile of Rb phosphorylation may follow as a consequence of differences in the content of cyclin-dependent kinase 2 (CDK2) and the CDK4 phosphorylation inhibitor p15. Finally, we present evidence that siRNA-mediated CDK2 knockdown alters the profile of Rb phosphorylation in coronary artery SMCs, as well as the proliferative response of these cells to mitogenic stimulation. The intrinsic functional and protein composition specificity of the SMCs population in the coronary artery may contribute to the increased prevalence of restenosis and atherosclerosis in the coronary arteries as compared with the internal mammary arteries.     

Urokinase plasminogen activator induces human smooth muscle cell migration and proliferation via distinct receptor-dependent and proteolysis-dependent mechanisms

In order to define the relative contribution of the proteolytic domain and the receptor-binding domain of urokinase plasminogen activator (uPA) toward its mitogenic properties we studied the effects of different uPA isoforms on migration and proliferation of human aortic smooth muscle cells (hSMC). The isoforms tested included native human glycosylated uPA, and two recombinant uPA forms, namely a recombinant uPA with wild type structure (r-uPA), and a uPA-mutant in which the first 24 N-terminal amino acid residues of the receptor binding domain were replaced by 13 foreign amino acid residues (r-uPAmut). Cell migration was evaluated using a micro-Boyden chamber assay, and cell proliferation assessed by measurement of [3H]-thymidine incorporation into DNA. Competition binding studies on hSMC using 125I-r-uPA as ligand demonstrated that r-uPA and r-uPAmut exhibited equivalent displacement profiles. However, migration of hSMC was promoted by r-uPA and not by r-uPAmut. r-uPA-induced migration occurred at concentrations (half-maximally effective concentration of 2 nM) approximating the Kd for uPA-uPAR binding (1 nM). r-uPA-induced migration was not affected by the plasmin inhibitor aprotinin. In contrast to their differential chemotactic properties, uPA, r-uPA and r-uPAmut, which possess similar proteolytic activities, all stimulated [3H]-thymidine incorporation in hSMC. Since the [3H]-thymidine incorporation response to each isoform occurred at concentrations (> 50 nM) much higher than necessary for uPAR saturation by ligand (1 nM), this mitogenic response may be independent of binding to uPAR. [3H]-thymidine incorporation responses to r-uPA and -uPAmut were sensitive to the plasmin inhibitor aprotinin, and uPA stimulated DNA synthesis was inhibited by plasminogen activator inhibitor. We conclude that hSMC migration in response to uPA depends upon on its binding to uPAR, whereas uPA-stimulated DNA synthesis in these cells requires proteolysis and plasmin generation.     

Changes in the balance of phosphoinositide 3-kinase/protein kinase B (Akt) and the mitogen-activated protein kinases (ERK/p38MAPK) determine a phenotype of visceral and vascular smooth muscle cells.

The molecular mechanisms behind phenotypic modulation of smooth muscle cells (SMCs) remain unclear. In our recent paper, we reported the establishment of novel culture system of gizzard SMCs (Hayashi, K., H. Saga, Y. Chimori, K. Kimura, Y. Yamanaka, and K. Sobue. 1998. J. Biol. Chem. 273: 28860-28867), in which insulin-like growth factor-I (IGF-I) was the most potent for maintaining the differentiated SMC phenotype, and IGF-I triggered the phosphoinositide 3-kinase (PI3-K) and protein kinase B (PKB(Akt)) pathway. Here, we investigated the signaling pathways involved in de-differentiation of gizzard SMCs induced by PDGF-BB, bFGF, and EGF. In contrast to the IGF-I-triggered pathway, PDGF-BB, bFGF, and EGF coordinately activated ERK and p38MAPK pathways. Further, the forced expression of active forms of MEK1 and MKK6, which are the upstream kinases of ERK and p38MAPK, respectively, induced de-differentiation even when SMCs were stimulated with IGF-I. Among three growth factors, PDGF-BB only triggered the PI3-K/PKB(Akt) pathway in addition to the ERK and p38MAPK pathways. When the ERK and p38MAPK pathways were simultaneously blocked by their specific inhibitors or an active form of either PI3-K or PKB(Akt) was transfected, PDGF-BB in turn initiated to maintain the differentiated SMC phenotype. We applied these findings to vascular SMCs, and demonstrated the possibility that the same signaling pathways might be involved in regulating the vascular SMC phenotype. These results suggest that changes in the balance between the PI3-K/PKB(Akt) pathway and the ERK and p38MAPK pathways would determine phenotypes of visceral and vascular SMCs. We further reported that SMCs cotransfected with active forms of MEK1 and MKK6 secreted a nondialyzable, heat-labile protein factor(s) which induced de-differentiation of surrounding normal SMCs.