hhLIM与actin相互作用依赖其C端LIM结构域

hhLIM是LIM蛋白家族成员之一,该蛋白质含有两个LIM结构域,在基因表达调节、细胞骨架组构及细胞肥大过程中发挥重要作用.构建hhLIM不同LIM结构域的突变体,探讨其两个LIM结构域在与actin相互结合中的作用及其可能机制.GST-pull down和hhLIM及其突变体与actin细胞定位关系的免疫荧光分析结果表明,C端的LIM结构域2是hhLIM与actin结合所必需的,该结构域中的两个Cys置换为Ser后可使hhLIM结合actin的功能完全丧失,N端的LIM结构域1突变使hhLIM结合actin的能力下降.F-actin交联实验结果显示,hhLIM通过LIM结构域2与actin直接结合并起到交联F-actin的作用.结果表明,LIM结构域2在hhLIM与actin相互作用及调节actin细胞骨架组构中起决定性作用.     

hhLIM is a novel F-actin binding protein involved in actin cytoskeleton remodeling

Human heart LIM protein (hhLIM) is a newly cloned protein. In vitro analyses showed that green fluorescent protein (GFP)-tagged hhLIM protein accumulated in the cytoplasm of C2C12 cells and colocalized with F-actin, indicating that hhLIM is an actin-binding protein in C2C12 cells. Overexpression of hhLIM-GFP in C2C12 cells significantly stabilized actin filaments and delayed depolymerization of the actin cytoskeleton induced by cytochalasin B treatment. Expression of hhLIM-GFP in C2C12 cells also induced significant changes in the organization of the actin cytoskeleton, specifically, fewer and thicker actin bundles than in control cells, suggesting that hhLIM functions as an actin-bundling protein. This hypothesis was confirmed using low-speed co-sedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of hhLIM. hhLIM has two LIM domains. To identify the essential regions and sites for association, a series of truncated mutants was constructed which showed that LIM domain 2 has the same activity as full-length hhLIM. To further characterize the binding sites, the LIM domain was functionally destructed by replacing cysteine with serine in domain 2, and results showed that the second LIM domain plays a central role in bundling of F-actin. Taken together, these data identify hhLIM as an actin-binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.     

细胞肥大过程中hhLIM的亚细胞定位变化及其对细胞骨架的影响

hhlim (humanheartlim)是从人胎心cDNA文库中筛选克隆的一个新基因 ,作为LIM家族的新成员参与心肌肥大的发生发展过程 .为了进一步研究hhLIM在心肌肥大发生过程中的作用 ,以C2C12细胞为研究对象 ,以心肌肥大强效刺激因子内皮素 1(ET 1)为诱导因素 ,探讨hhLIM与肌动蛋白的相互作用及其影响细胞骨架的分子机制 .RT PCR、Western印迹和细胞免疫荧光分析结果表明 ,心肌肥大刺激因子ET 1在诱导心肌肥大标志基因BNP和肌动蛋白表达的同时 ,使hhLIM蛋白在C2C12细胞胞核与胞质之间进行重新定位 .激光共聚焦显微镜观察结果显示 ,hhLIM与肌动蛋白在胞质中共定位 .蛋白分步提取、鉴定及hhLIM与F肌动蛋白结合与沉降实验证明 ,hhLIM多存在于细胞骨架及其相关蛋白部分 ,在体外可与F肌动蛋白共结合 .这些结果表明 ,胞质中的hhLIM作为细胞骨架相关蛋白与肌动蛋白相互作用 .进一步研究hhLIM与细胞骨架的关系时发现 ,hhLIM过表达可使C2C12细胞的骨架变成致密网状纤维并使其对细胞松弛素导致的细胞骨架解聚产生一定的抵抗作用 ,抑制hhLIM表达则使细胞骨架稀疏 ,结构模糊 .提示hhLIM参与细胞骨架组织及重构的机制与其结合并稳定F肌动蛋白有关 .     

KLF5 and hhLIM cooperatively promote proliferation of vascular smooth muscle cells

Krüppel-like factor 5 (KLF5) plays an important role in cellular proliferation and differentiation. In this study, we show that adenovirus-mediated overexpression of KLF5 increased neointimal formation, while human heart LIM protein (hhLIM) decreased neointimal formation following vascular injury. Interestingly, neointimal formation was significantly increased in the animals where both hhLIM and KLF5 were introduced, suggesting that KLF5 can reverse hhLIM function in cell proliferation on the coexpression with hhLIM. These results were also confirmed the cellular level. Further mechanistic studies suggested that PDGF-BB promoted the interaction between hhLIM and KLF5 through stimulating hhLIM binding to TGF-β control element (TCE) on the cyclin E promoter in a KLF5-dependent manner. Failure of KLF5 binding to the TCE, on the knockdown of KLF5 by transfecting siRNA, not only prevented the recruitment of hhLIM to the cyclin E promoter but also affected activation of the cyclin E promoter by KLF5. These data suggest that KLF5 reverses hhLIM function from anti-proliferation to pro-proliferation through its interaction with hhLIM on the cyclin E promoter.     

Ajuba, a cytosolic LIM protein, shuttles into the nucleus and affects embryonal cell proliferation and fate decisions

Cellular adhesive events affect cell proliferation and differentiation decisions. How cell surface events mediating adhesion transduce signals to the nucleus is not well understood. After cell-cell or cell-substratum contact, cytosolic proteins are recruited to clustered adhesion receptor complexes. One such family of cytosolic proteins found at sites of cell adhesion is the Zyxin family of LIM proteins. Here we demonstrate that the family member Ajuba was recruited to the cell surface of embryonal cells, upon aggregate formation, at sites of cell-cell contact. Ajuba contained a functional nuclear export signal and shuttled into the nucleus. Importantly, accumulation of the LIM domains of Ajuba in the nucleus of P19 embryonal cells resulted in growth inhibition and spontaneous endodermal differentiation. The differentiating effect of Ajuba mapped to the third LIM domain, whereas regulation of proliferation mapped to the first and second LIM domains. Ajuba-induced endodermal differentiation of these cells correlated with the capacity to activate c-Jun kinase and required c-Jun kinase activation. These results suggest that the cytosolic LIM protein Ajuba may provide a new mechanism to transduce signals from sites of cell adhesion to the nucleus, regulating cell growth and differentiation decisions during early development.     

Nulp1基因抑制P19CL6细胞向心肌细胞分化

Nulp1基因是已克隆的一个新的bHLH转录因子亚家族成员.前期的研究表明：Nulp1蛋白作为一个转录抑制子对SRF信号途径有极强的抑制作用.为了进一步研究Nulp1基因在心肌分化中的作用,在能够高效分化为心肌细胞的P19CL6细胞系中过表达Nulp1基因,发现心肌分化标志基因的表达被抑制;用RNA干扰技术,使Nulp1基因在P19CL6细胞系中的内源表达降低,发现心肌分化标志基因的表达提高,说明Nulp1基因能够抑制P19CL6细胞系向心肌细胞的分化.     

Expression patterns of FHL/SLIM family members suggest important functional roles in skeletal muscle and cardiovascular system

LIM domain containing proteins play critical roles in animal development and cellular differentiation. Here, we describe the cloning and expression patterns of three members of the four and a half LIM domain-only protein family, FHL1, 2, and 3, from mouse. A comparison of embryonic expression patterns of these three highly-related genes indicates that they are expressed in an overlapping pattern in the developing cardiovascular system, and skeletal muscle. In adult tissues, the three genes are expressed in a predominant and overlapping manner in cardiac and skeletal muscle. Of the three genes, FHL2 appears to have the most restricted expression pattern during development, in heart, blood vessels, and skeletal muscle. Expression in heart is highest in cardiac septa and in the region adjacent to the atrio-ventricular ring, suggesting a potential role in septation or conduction system development. In the heart, FHL1expression was observed strongly in developing outflow tract, and to a lesser extent in myocardium. FHL3 displays low and ubiquitous expression during mouse development. Cardiac ventricular expression of FHL1, but not FHL2 or FHL3, was upregulated in two mouse models of cardiac hypertrophic and dilated cardiomyopathy. Taken together, these data indicate the potential importance of this FHL family in the development and maintenance of the cardiovascular system and striated muscle, and suggest that FHL1 may play a role in the development of heart disease.     

Expression of metalloproteinases and inhibitors in the differentiation of P19CL6 cells into cardiac myocytes

P19CL6 are a clonal derivative of P19 embryonal carcinoma cells, a euploid, multipotent mouse cell line, that differentiate efficiently into cardiac myocytes, with spontaneous beating evident within 10 days, following DMSO treatment. Using real-time quantitative RT-PCR we have profiled the expression of the complete matrix metalloproteinase and tissue inhibitor of metalloproteinase gene families during P19CL6 differentiation to cardiac myocytes. The genes subdivide into eight groups based upon their expression profile. Their expression was both qualitatively and quantitatively highly homologous to that seen during mouse heart development.     

LIM同源框基因家族与神经系统

同源框基因是指一类含有同源序列的基因,它编码的蛋白质作为转录调节因子调节细胞的发育和分化,控制基因的表达形式。ＬＩＭ同源框基因不仅含有同源框基因也含有编码ＬＩＭ结构域的保守序列。     

Inhibition of proliferation and differentiation and promotion of apoptosis by cyclin L2 in mouse embryonic carcinoma P19 cells

Cyclin L2 (CCNL2) is a novel member of the cyclin gene family. In a previous study, we demonstrated that CCNL2 expression was upregulated in ventricular septum tissues from patients with ventricular septal defect compared to healthy controls. In the present study, we established a stable CCNL2-overexpressing P19 cell line that can differentiate to myocardial cells when treated with 1% dimethyl sulfoxide (DMSO). Our data showed that stable CCNL2-overexpressing P19 cells were less differentiated after treatment with 1% DMSO and that expression of myocardial cell differentiation-related genes (such as cardiac actin, GATA4, Mef2C, Nkx2.5, and BNP) were reduced compared to vector-only transfected P19. Moreover, P19 cells overexpressing the CCNL2 gene had a reduced growth rate and a remarkably decreased S phase. We also found that these cells underwent apoptosis, as detected by two different apoptosis assays. The anti-apoptotic Bcl-2 protein was also downregulated in these cells. In addition, real-time PCR analysis revealed that expression of Wnt and β-catenin was suppressed and GSK3β was induced in the CCNL2-overexpressing P19 cells. These data suggest that overexpression of CCNL2 inhibited proliferation and differentiation of mouse embryonic carcinoma P19 cells and induced them to undergo apoptosis, possibly through the Wnt signal transduction pathway.     

<Emphasis Type="Italic">Fabp3</Emphasis> Inhibits Proliferation and Promotes Apoptosis of Embryonic Myocardial Cells

Fatty acid binding protein 3 (FABP3) is a member of a family of binding proteins. The protein is mainly expressed in cardiac and skeletal muscle cells, and it has been linked to fatty acid metabolism, trafficking, and signaling. Using suppression subtractive hybridization, we previously found that FABP3 is highly regulated in ventricular septal defect (VSD) patients and may play a significant role in the development of human VSD. We therefore aimed to identify the biological characteristics of the FABP3 gene in embryonic myocardial cells. On the basis of RT-PCR and western blotting analyses, we demonstrated that the expression levels of FABP3 mRNA and protein were up-regulated initially and then gradually decreased with P19 cell differentiation. MTT assays and cell cycle analysis showed that FABP3 inhibits P19 cell proliferation, and data from annexin V-FITC assays revealed that FABP3 can promote apoptosis of P19 cells. Further data from quantitative real-time RT-PCR revealed lower expression levels of cardiac muscle-specific molecular markers (cTnT, alpha-MHC, GATA4, and MEF2c) in FABP3-overexpressing cell lines than in the control cells during differentiation. Our results demonstrate that FABP3 may be involved in the differentiation of cardiac myocytes.