FHL2通过相互作用抑制分化抑制蛋白家族成员的转录抑制活性

目的：探讨FHL2与Id（分化抑制蛋白）家族蛋白之间的相互作用及FHL2对Id蛋白功能的调控效应。方法：用GST-pulldown与免疫共沉淀（CoIP）方法检测FHL2与Id家族蛋白成员之间在体内外的相互作用;用共转染与报告基因驱动的萤光素酶方法检测FHL2对Id蛋白介导转录抑制效应的调控作用。结果：FHL2与Id家族的4个蛋白均存在直接的相互作用关系,表位分析结果显示FHL2蛋白中的第2个LIM结构域在FHL2/Id相互作用中是必需的,Id蛋白N端结构域在介导FHL2/Id相互作用中是必需的,FHL2/Id相互作用不依赖于Id蛋白中的螺旋-环-螺旋结构;通过相互作用,FHL2阻止了Id蛋白对碱性螺旋-环-螺旋转录因子E47转录活性的抑制作用。结论：FHL2是一个新识别的Id蛋白广谱的相互作用因子,通过对Id蛋白功能活性的抑制效应,FHL2可能参与Id介导的多种生物学效应以及肿瘤发生与进展。     

FHL2通过相互作用抑制Id2的功能活性

分化抑制蛋白2（Id2）通过抑制碱性螺旋-环-螺旋（bHLH）类转录因子的功能活性调控多种组织细胞的分化发育,并参与人类多种肿瘤的发生与进展.Id2相互作用蛋白可能调控其翻译后的功能活性．本研究以HLH结构域缺失的Id2作为诱饵蛋白,采用酵母双杂交方法对MCF-7 cDNA文库进行筛选,识别了1个新的Id2相互作用蛋白FHL2 (属于LIM蛋白家族的一员),哺乳动物双杂交实验系统验证了Id2与FHL2之间的相互作用,同时证实,该作用不依赖于Id2中的HLH结构域;GST-pulldown、免疫共沉淀方法,进一步证实FHL2/Id2之间的相互作用;免疫荧光共定位实验结果证实,FHL2/Id2相互作用主要发生在细胞核内;共转染实验结果发现,FHL2通过相互作用阻抑了Id2对bHLH类转录因子E47的功能抑制活性．总之,本研究识别了1个新的Id2相互作用蛋白FHL2,通过直接的相互作用,FHL2抑制了Id2的功能活性,FHL2可能参与调控Id2介导的细胞分化与发育过程,并可能参与肿瘤的发生与进展．     

Spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disease of early childhood characterized by nonmalignant accumulation and multivisceral infiltration of activated T lymphocytes and histiocytes (macrophages). Cytotoxic T and natural killer (NK) cell activity is markedly reduced or absent in these patients, and mutations in a lytic granule constituent, perforin, were recently identified in a number of FHL individuals. Here, we report a comprehensive survey of 34 additional patients with FHL for mutations in the coding region of the perforin gene and the relative frequency of perforin mutations in FHL. Perforin mutations were identified in 7 of the 34 families investigated. Six children were homozygous for the mutations, and one patient was a compound heterozygote. Four novel mutations were detected: one nonsense, two missense, and one deletion of one amino acid. In four families, a previously reported mutation at codon 374, causing a premature stop codon, was identified, and, therefore, this is the most common perforin mutation identified so far in FHL patients. We found perforin mutations in 20% of all FHL patients investigated (7/34), with a somewhat higher prevalence, approximately 30% (6/20), in children whose parents originated from Turkey. No other correlation between the type of mutation and the phenotype of the patients was evident from the present study. Our combined results from mutational analysis of 34 families and linkage analysis of a subset of consanguineous families indicate that perforin mutations account for 20%-40% of the FHL cases and the FHL 1 locus on chromosome 9 for approximately 10%, whereas the major part of the FHL cases are caused by mutations in not-yet-identified genes.     

FHL2参与调控MCF-7细胞周期

目的：研究FHL2对细胞周期阻滞的影响。方法：应用pSR-GFP/Neo载体构建FHL2-siRNA干扰载体,转染MCF-7细胞,G418筛选稳定细胞系,通过流式细胞仪检测离子辐射后细胞周期的变化。结果：利用设计的FHL2-siRNA干扰载体能够干扰细胞中FHL2的表达;当离子辐射后,FHL2敲除细胞G2/M期阻滞的程度比野生型细胞显著降低。结论：FHL2参与调控MCF-7细胞离子辐射后细胞周期G2/M期阻滞。     

c-Abl调控FHL2的转录活性

目的：研究c-Abl对FHL2转录活性的调控。方法：应用免疫共沉淀和免疫印迹验证c-Abl与FHL2的相互作用及c-Abl对FHL2的磷酸化作用,并用萤光素酶报告基因研究c-Abl对FHL2转录活性的调控。结果：c-Abl与FHL2结合并磷酸化FHL2,促进FHL2的转录活性,并与FHL2一起促进FHL2下游因子骨钙蛋白的基因启动子活性。结论：c-Abl能促进FHL2的转录活性。     

Munc18‐2 is required for Syntaxin 11 Localization on the Plasma Membrane in Cytotoxic T‐Lymphocytes

Cytotoxic T‐lymphocytes (CTL) kill their targets by cytolytic granule secretion at the immunological synapse. The Sec/Munc protein, Munc18‐2, and its binding partner Syntaxin 11 (STX11) are both required for granule secretion, with mutations in either leading to the primary immunodeficiency, Familial Haemophagocytic Lymphohistiocytosis (FHL4 and 5). Understanding how Munc18‐2 and STX11 function in CTL has been hampered by not knowing the endogenous localization of these proteins. Using a novel FHL5 Munc18‐2 mutation that results in loss of protein, cytotoxicity and degranulation together with CTL from an FHL4 patient lacking STX11, enabled us to localize endogenous STX11 and Munc18‐2 in CTL. Munc18‐2 localized predominantly to cytolytic granules with low levels associated with the plasma membrane where STX11 localized. Importantly, while Munc18‐2 localization is unaffected by the absence of STX11 in FHL4 CTL, STX11 is lost from the plasma membrane in FHL5 CTL lacking Munc18‐2. These findings support a role for Munc18‐2 in chaperoning STX11 to the plasma membrane where the final fusion events involved in secretion occur.      

FHL2 蛋白在肿瘤中的转录调控及其分子生物学机制

FHL2是仅有四个半LIM结构域(FHL)蛋白家族的成员,目前在FHL蛋白家族中研究最为广泛。FHL2作为重要的衔接蛋白和支架蛋白,主要通过LIM结构域介导蛋白分子间的相互作用以实现其生物学功能。Fhl2基因在转录水平受多种肿瘤相关基因的调控,如p53,血清应答因子等。FHL2与恶性肿瘤的关系是近年来的研究热点,目前认为FHL2能够作为癌蛋白及抑癌蛋白通过不同机制广泛影响乳腺癌、胃肠道肿瘤、肝癌、前列腺癌等肿瘤的发生发展,并且在不同肿瘤中的表达具有组织特异性。本文就FHL2的结构特点、功能、转录调控及与肿瘤的关系几个方面展开综述,从而明确FHL2在不同肿瘤中所发挥的作用及其分子生物学机制将会为治疗相关肿瘤提供新的干预靶点。     

Deletion of the FHL2 gene attenuates intima‐media thickening in a partially ligated carotid artery ligated mouse model

The four and a half LIM domain protein 2 (FHL2) is a member of the four and a half LIM domain (FHL) gene family, and it is associated with cholesterol‐enriched diet‐promoted atherosclerosis. However, the effect of FHL2 protein on vascular remodelling in response to hemodynamic alterations remains unclear. Here, we investigated the role of FHL2 in a model of restricted blood flow‐induced atherosclerosis. To promote neointimal hyperplasia in vivo, we subjected FHL2^+/+ and FHL2^?/? mice to partial ligation of the left carotid artery (LCA). The expression of p‐ERK and p‐AKT was decreased in FHL2^?/? mice. FHL2 bound to AKT regulated AKT phosphorylation and led to Rac1‐GTP inactivation. FHL2 silencing in human aortic smooth muscle cells down‐regulated the PDGF‐induced phosphorylation of ERK and AKT. Furthermore, FHL2 silencing reduced cytoskeleton conformational changes and caused cell cycle arrest. We concluded that FHL2 is essential for the regulation of arterial smooth muscle cell function. FHL2 modulates proliferation and migration via mitogen‐activated protein kinase (MAPK) and PI3K‐AKT signalling, leading to arterial wall thickening and thus neointimal hyperplasia.     

FHL2‐driven molecular network mediated Septin2 knockdown inducing apoptosis in mesangial cell

The apoptosis of mesangial cells (MCs) plays a critical role in the pathological progress of MesPGN. Septin2, a filamentous GTPase, is implicated in the apoptotic progress of MCs in the rat MesPGN model. However, the molecular mechanism of SEPT2 in MCs apoptosis is not clear. Here, we present the FHL2‐driven molecular network as the main mechanism of SEPT2‐mediated rat primary MCs apoptosis. First, we proved that the expression of FHL2 and Septin2 were closely related with MCs apoptosis in anti‐Thy1 nephritis model. Then, it was found that FHL2 was a new interaction protein of Septin2 and Septin2 knockdown could induce MC apoptosis by FHL2‐mediatied signal pathways including p‐ERK1 and p‐AKT. We applied label‐Free quantitative proteomics to identify the mechanism of Septin2/FHL2‐regulated apoptosis. Bioinformatics analysis revealed that FHL2‐driven molecular network composed of biological functions including glycolysis, oxidative stress, ribonucleotide metabolism, actin cytoskeleton regulation, and signaling pathway, was the main mechanism of SETP2‐mediated apoptosis. Furthermore, we showed that the effect of Septin2 knockdown on MC apoptosis could be alleviated by the overexpression of FHL2. Overall, this study illustrated the FHL2‐driven molecular network controlling SEPT2‐mediated apoptosis in MCs and their potential roles in mesangial proliferative nephritis.     

FHL2转录激活结构域的定位

LIM蛋白家族成员FHL2 (fourandhalfLIMdomainprotein)在转录调节、细胞凋亡及肿瘤的发生发展中都起着重要作用。利用GAL4转录因子中的DNA结合结构域 (DBD)和含有与DBD结合序列的荧光素酶报告基因(GAL4 LUC)构建了哺乳动物细胞转录激活系统 ,并利用该系统定位了FHL2的转录激活结构域。首先将GAL4 DBD序列以正确读框插入到pcDNA3载体的多克隆位点中 ,构建成真核表达载体pDBD ,再将野生型FHL2及其不同片段以正确读框与pDBD中GAL4 DBD序列融合 ,构建成野生型FHL2及其缺失突变体表达载体。将这些表达载体分别瞬时转染 2 93T胚胎肾细胞 ,野生型FHL2及其缺失突变体都得到了表达。利用GAL4 荧光素酶报告基因对野生型FHL2及其不同突变体的转录激活活性检测表明 ,在 2 93T胚胎肾细胞和乳腺癌MCF 7细胞中 ,野生型FHL2具有转录激活活性 ,缺失N端半个LIM结构域使FHL2转录激活活性降低 ,缺失C末端第二个LIM结构域对FHL2的转录激活功能影响不大 ,缺失C末端最后一个LIM结构域则使FHL2的转录激活功能完全丧失 ,而C末端缺失 2个LIM结构域使FHL2转录激活活性又有所恢复。这说明FHL2C末端最后一个LIM结构域对其转录激活功能是必需的 ,而C末端第二个LIM结构域可能对FHL2的转录激活功能有负调控作用 ,这种负调控作用取决于     

放射诱导基因FHL2在人群中表达水平的检测

目的：分析FHL2基因在健康人群个体间表达的差异,评估FHL2作为一种新的辐射生物剂量计的可行性。方法：收集20例健康人血液样本,提取白细胞RNA进行反转录,以β-actin作为内参,利用实时定量PCR方法,检测人群中FHL2基因相对表达水平,并分析其差异。结果：FHL2和β-actin基因的实时定量PCR熔解曲线均为单峰,所得到的Ct值与相应的PCR产物呈良好的线性关系;20例血液标本中FHL2表达水平之间存在较大差异,且其表达水平与性别无关。结论：公认的放射诱导基因FHL2可能不适合作为辐射生物剂量计。     

Subtypes of familial hemophagocytic lymphohistiocytosis in Japan based on genetic and functional analyses of cytotoxic T lymphocytes

Background

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare disease of infancy or early childhood. To clarify the incidence and subtypes of FHL in Japan, we performed genetic and functional analyses of cytotoxic T lymphocytes (CTLs) in Japanese patients with FHL.

Design and Methods

Among the Japanese children with hemophagocytic lymphohistiocytosis (HLH) registered at our laboratory, those with more than one of the following findings were eligible for study entry under a diagnosis of FHL: positive for known genetic mutations, a family history of HLH, and impaired CTL-mediated cytotoxicity. Mutations of the newly identified causative gene for FHL5, STXBP2, and the cytotoxicity and degranulation activity of CTLs in FHL patients, were analyzed.

Results

Among 31 FHL patients who satisfied the above criteria, PRF1 mutation was detected in 17 (FHL2) and UNC13D mutation was in 10 (FHL3). In 2 other patients, 3 novel mutations of STXBP2 gene were confirmed (FHL5). Finally, the remaining 2 were classified as having FHL with unknown genetic mutations. In all FHL patients, CTL-mediated cytotoxicity was low or deficient, and degranulation activity was also low or absent except FHL2 patients. In 2 patients with unknown genetic mutations, the cytotoxicity and degranulation activity of CTLs appeared to be deficient in one patient and moderately impaired in the other.

Conclusions

FHL can be diagnosed and classified on the basis of CTL-mediated cytotoxicity, degranulation activity, and genetic analysis. Based on the data obtained from functional analysis of CTLs, other unknown gene(s) responsible for FHL remain to be identified.     

Structural analysis of four and half LIM protein-2 in dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a cardiac disease characterized by dilated ventricle and systolic dysfunction. Most of the DCM patients are sporadic cases, but a certain population of DCM patients can be familial cases caused by mutations in genes for sarcomere/Z-disc components including titin/connectin. However, disease-causing mutations could be identified only in a part of the familial DCM patients, suggesting that there should be other disease causing genes for DCM. To explore a novel disease gene for DCM, we searched for mutations in FHL2, encoding for four and half LIM protein 2 (FHL2) in DCM patients, because FHL2 is known to associate with titin/connectin. A missense mutation, Gly48Ser, was identified in a patient with familial DCM. Functional analysis demonstrated that the FHL2 mutation affected the binding to titin/connectin. Because FHL2 protein is known to tether metabolic enzymes to titin/connectin, these observations suggest that the Gly48Ser mutation may be involved in the pathogenesis of DCM via impaired recruitment of metabolic enzymes to the sarcomere.     

PCBP2 promotes the development of glioma by regulating FHL3/TGF-β/Smad signaling pathway

The purpose of this study was to investigate the role of Poly (C)-binding protein 2 (PCBP2) and the related signaling pathway in glioma progression. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were performed to measure PCBP2 messenger RNA and protein expression in glioma tissues or cells. Cell transfection was completed using Lipofectamine 2000. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Transwell assay and flow cytometry assay were used to explore the effects of PCBP2 expression on biological behaviors of glioma cells. Western blot assay was used for the detection of pathway related proteins. Expression of PCBP2 in glioma tissues and cells were higher than that in paracancerous tissues and normal cells (both p < .01). Moreover, the elevated expression of PCBP2 was significantly correlated with tumor size (p = .001) and WHO stage (p = .010). Knockdown of PCBP2 could suppress proliferation, migration and invasion of glioma cells and promote apoptosis. Besides, the expression of transforming growth factor-β (TGF-β) pathway related proteins TGF-β1, p-Smad2 and p-Smad7 were decreased following the downregulation of PCBP2. PCBP2 also inhibited FHL3 expression by binding to FHL3-3′UTR. The inhibition of FHL3 could reverse the antitumor action caused by PCBP2 silencing. In vivo assay, PCBP2 was also found to inhibit the tumor growth of glioma. PCBP2 activates TGF-β/Smad signaling pathway by inhibiting FHL3 expression, thus promoting the development and progression of glioma.     

The LIM-only protein FHL2 interacts with beta-catenin and promotes differentiation of mouse myoblasts

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified beta-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of beta-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts beta-catenin-mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell-specific manner. After injection into Xenopus embryos, FHL2 inhibited the beta-catenin-induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with beta-catenin.     

Deficiency of the LIM-Only Protein FHL2 Reduces Intestinal Tumorigenesis in Apc Mutant Mice

Background

The four and a half LIM-only protein 2 (FHL2) is capable of shuttling between focal adhesion and nucleus where it signals through direct interaction with a number of proteins including β-catenin. Although FHL2 activation has been found in various human cancers, evidence of its functional contribution to carcinogenesis has been lacking.

Methodology/Principal Findings

Here we have investigated the role of FHL2 in intestinal tumorigenesis in which activation of the Wnt pathway by mutations in the adenomatous polyposis coli gene (Apc) or in β-catenin constitutes the primary transforming event. In this murine model, introduction of a biallelic deletion of FHL2 into mutant Apc^Δ14/+ mice substantially reduces the number of intestinal adenomas but not tumor growth, suggesting a role of FHL2 in the initial steps of tumorigenesis. In the lesions, Wnt signalling is not affected by FHL2 deficiency, remaining constitutively active. Nevertheless, loss of FHL2 activity is associated with increased epithelial cell migration in intestinal epithelium, which might allow to eliminate more efficiently deleterious cells and reduce the risk of tumorigenesis. This finding may provide a mechanistic basis for tumor suppression by FHL2 deficiency. In human colorectal carcinoma but not in low-grade dysplasia, we detected up-regulation and enhanced nuclear localization of FHL2, indicating the activation of FHL2 during the development of malignancy.

Conclusions/Significance

Our data demonstrate that FHL2 represents a critical factor in intestinal tumorigenesis.     

Familial Hemophagocytic Lymphohistiocytosis May Present during Adulthood: Clinical and Genetic Features of a Small Series

Familial Hemophagocytic lymphohistiocytosis (FHL) is a rare immune deficiency with defective cytotoxic function. The age at onset is usually young and the natural course is rapidly fatal if untreated. A later onset of the disease has been sporadically reported even in adolescents and adults. We report the results of our retrospective data collection of all cases diagnosed with FHL at an age of 18 years or older and enrolled in the Italian Registry of HLH. All cases were diagnosed with FHL based on evidence of genetic defect in one FHL-related gene. A total of 11 patients were diagnosed with FHL. They were 9 males and 2 females, from 10 unrelated families; their age ranged between 18 and 43 years (median, 23 years). Family history was unremarkable in eight families at the time of the diagnosis. Their genetic diagnoses are: FHL2 (n = 6), FHL3 (n = 2), FHL5 (n = 1), XLP1 (n = 2). Clinical, molecular and functional data are described. These data confirm that FHL may present beyond the pediatric age and up to the fifth decade. FHL2 due to perforin defect is the most frequently reported subtype. Adult specialists should consider FHL in the differential diagnosis of patients with cytopenia and liver or central nervous system disorders, especially when a lymphoproliferative disease is suspected but eventually not confirmed. FHL may turn to be fatal within a short time course even in adults. This risk, together with the continuous improvement in the transplant technique, especially in the area of transplant from matched unrelated donor, resulting in reduced treatment related mortality, might suggest a wider use of SCT in this population. Current diagnostic approach allows prompt identification of patients by flow-cytometry screening, then confirmed by the genetic study, and treatment with chemo-immunotherapy followed by stem cell transplantation.