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 .     

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.     

MLP参与心肌肥大发生过程与Calcineurin/NFAT信号通路有关

心肌肥大是心肌细胞面对多种病理刺激时的共同反应,以心肌细胞体积增大和胚胎期基因的重新表达为标志.心肌发育调控基因肌肉LIM蛋白(muscle LIM protein,MLP)的表达异常与心肌肥大有关.为研究MLP参与心肌肥大发生的分子机制,采用去氧肾上腺素（phenylephrine, PE）刺激大鼠原代培养心肌细胞,建立心肌细胞肥大模型,采用RNAi技术敲减MLP的表达,分析MLP与肥大信号通路钙调神经磷酸酶（calcineurin）/活化T细胞核因子（nuclear factor of activated T-cells, NFAT）的关系.结果显示, 原代培养的心肌细胞经一定浓度的PE刺激后细胞表面积增加,肥大标志蛋白ANP、BNP表达增高,并伴有MLP表达上调. RNAi方法敲减MLP的表达则明显抑制PE诱导的心肌细胞表面积增加和BNP表达增高,并且直接 影响NFAT的转录激活活性,提示MLP与心肌肥大的发生密切相关,并且可能是通过calcineurin/NFAT信号通路而参与心肌肥大的发生.     

A novel cardiac hypertrophic factor, neurotrophin-3, is paradoxically downregulated in cardiac hypertrophy

The neurotrophin family plays pivotal roles in the development of the nervous system. Recently, the role of the neurotrophin in non-neural tissue has been extensively investigated. Among them, neurotrophin-3 and its receptor TrkC are critical for embryonic heart development, though little is known about neurotrophin-3/TrkC function in adult heart. Moreover, the expressions of other neurotrophin and Trk families in the cardiovascular system have not been fully determined. In adult and neonatal rats, only TrkC mRNA was expressed more abundantly in heart than aorta among the neurotrophin receptors, while all neurotrophins were equally expressed in the cardiovascular system. Immunohistochemistry confirmed the protein expressions of neurotrophin-3/TrkC in rat ventricles. In primary-cultured rat cardiomyocytes, neurotrophin-3 strongly activated p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, and Jun N-terminal kinase pathways in Western blot analysis. In Northern blot analysis, neurotrophin-3 strongly increased mRNA expressions of cardiac hypertrophic markers (skeletal alpha-actin and atrial natriuretic peptide) in cardiomocytes. [(3)H]-phenylalanine uptake into cardiomyocytes, myofilament reorganization, and cardiomyocyte size were also augmented with neurotrophin-3 stimulation, indicating that neurotrophin-3 is a novel cardiac hypertrophic factor. Unexpectedly, neurotrophin-3 was downregulated in cardiac hypertrophy induced by pressure overload (in vivo), and in cardiomyocyte hypertrophy evoked by endothelin-1 stimulation (in vitro). Interestingly, the cell size and BNP mRNA expression level (markers of hypertrophy) were greater in cardiomyocytes treated with both neurotrophin-3 and endothelin-1 than in those stimulated with endothelin-1 alone. These findings demonstrate that neurotrophin-3 is a unique hypertrophic factor, which is paradoxically downregulated in cardiac hypertrophy and might counteract hypertrophic change.     

Overexpression of interleukin-15 induces skeletal muscle hypertrophy in vitro: implications for treatment of muscle wasting disorders

Interleukin-15 (IL-15) is a novel anabolic factor for skeletal muscle which inhibits muscle wasting associated with cancer (cachexia) in a rat model. To develop a cell culture system in which the mechanism of the anabolic action of IL-15 on skeletal muscle could be examined, the mouse C2 skeletal myogenic cell line was transduced with a retroviral expression vector for IL-15 and compared to sister cells transduced with a control vector. Overexpression of IL-15 induced fivefold higher levels of sarcomeric myosin heavy chain and alpha-actin accumulation in differentiated myotubes. Secreted factors from IL-15-overexpressing myogenic cells, but not from control cells, induced increased myofibrillar protein accumulation in cocultured control myotubes. IL-15 overexpression induced a hypertrophic myotube morphology similar to that described for cultured myotubes which overexpressed the well-characterized anabolic factor insulin-like growth factor-I (IGF-I). However, in contrast to IGF-I, the hypertrophic action of IL-15 on skeletal myogenic cells did not involve stimulation of skeletal myoblast proliferation or differentiation. IL-15 induced myotube hypertrophy at both low and high IGF-I concentrations. Furthermore, in contrast to IGF-I, which stimulated only protein synthesis under these culture conditions, IL-15 both stimulated protein synthesis and inhibited protein degradation in cultured skeletal myotubes. These findings indicate that IL-15 action on skeletal myogenic cells is distinct from that of IGF-I. Due to the ability of IGF-I to stimulate cell division and its association with several forms of cancer, controversy exists concerning the advisability of treating cachexia or age-associated muscle wasting with IGF-I. Administration of IL-15 or modulation of the IL-15 signaling pathway may represent an alternative strategy for maintaining skeletal muscle mass under these conditions.     

Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo

Adult skeletal muscle fibers can be categorized into fast and slow twitch subtypes based on specialized contractile and metabolic properties and on distinctive patterns of muscle gene expression. Muscle fiber-type characteristics are dependent on the frequency of motor nerve stimulation and are thought to be controlled by calcium-dependent signaling. The calcium, calmodulin-dependent protein phosphatase, calcineurin, stimulates slow fiber-specific gene promoters in cultured skeletal muscle cells, and the calcineurin inhibitor, cyclosporin A, inhibits slow fiber gene expression in vivo, suggesting a key role of calcineurin in activation of the slow muscle fiber phenotype. Calcineurin has also been shown to induce hypertrophy of cardiac muscle and to mediate the hypertrophic effects of insulin-like growth factor-1 on skeletal myocytes in vitro. To determine whether activated calcineurin was sufficient to induce slow fiber gene expression and hypertrophy in adult skeletal muscle in vivo, we created transgenic mice that expressed activated calcineurin under control of the muscle creatine kinase enhancer. These mice exhibited an increase in slow muscle fibers, but no evidence for skeletal muscle hypertrophy. These results demonstrate that calcineurin activation is sufficient to induce the slow fiber gene regulatory program in vivo and suggest that additional signals are required for skeletal muscle hypertrophy.     

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.     

The LIM proteins FHL1 and FHL3 are expressed differently in skeletal muscle

We have determined the complete mRNA sequence of FHL3 (formerly SLIM2). We have confirmed that it is a member of the family of LIM proteins that share a similar secondary protein structure, renamed as Four-and-a-Half-LIM domain (or FHL) proteins in accordance with this structure. The "half-LIM" domain is a single zinc finger domain that may represent a subfamily of LIM domains and defines this particular family of LIM proteins. The distribution of FHL mRNA expression within a variety of murine tissues is complex. Both FHL1 and FHL3 were expressed in a number of skeletal muscles while FHL2 was expressed at high levels in cardiac muscle. Localisation of FHL3 to human chromosome 1 placed this gene in the proximity of, but not overlapping with, alleles associated with muscle diseases. FHL1 and FHL3 mRNAs were reciprocally expressed in the murine C2C12 skeletal muscle cell line and this suggested that the pattern of expression was linked to key events in myogenesis.     

Identification of Serhl, a new member of the serine hydrolase family induced by passive stretch of skeletal muscle in vivo

In response to extended periods of stretch, skeletal muscle typically exhibits cell hypertrophy associated with sustained increases in mRNA and protein synthesis. Several soluble hypertrophic agonists have been identified, yet relatively little is known as to how mechanical load is converted into intracellular signals regulating gene expression or how increased cell size is maintained. In skeletal muscle, hypertrophy is generally regarded as a beneficial adaptive response to increased workload. In some cases, however, hypertrophy can be detrimental as seen in long-term cardiac hypertrophy. Skeletal muscle wasting (atrophy) is a feature of both inherited and acquired muscle disease and normal aging. Elucidating the molecular regulation of cell size is a fundamental step toward comprehending the complex molecular systems underlying muscle hypertrophy and atrophy. Subtractive hybridization between passively stretched and control murine skeletal muscle tissue identified an mRNA that undergoes increased expression in response to passive stretch. Encoded within the mRNA is an open reading frame of 311 amino acids containing a highly conserved type 1 peroxisomal targeting signal and a serine lipase active center. The sequence shows identity to a family of serine hydrolases and thus is named serine hydrolase-like (Serhl). The predicted three-dimensional structure displays a core alpha/beta-hydrolase fold and catalytic triad characteristic of several hydrolytic enzymes. Endogenous Serhl protein immunolocalizes to perinuclear vesicles as does Serhl-FLAG fusion protein transiently expressed in muscle cells in vitro. Overexpression of Serhl-FLAG has no effect on muscle cell phenotype in vitro. Serhl's expression patterns and its response to passive stretch suggest that it may play a role in normal peroxisome function and skeletal muscle growth in response to mechanical stimuli.