桩蛋白的结构与功能

桩蛋白(paxillin)是近年来发现的一种信号蛋白,主要定位于黏着斑,包含LD模体、LIM结构域、SH2和SH3结合结构域,在整个分子中还散在着多种磷酸化位点,共同构成了桩蛋白的多结构域性结构。桩蛋白分子本身的酶活性尚不清楚,但很可能作为细胞内的一种接头蛋白与多种功能蛋白质结合。另外．作为黏着斑的重要组成部分,桩蛋白不仅参与了整合蛋白介导的信号转导和黏着斑的组装,在细胞黏附和迁移过程中也发挥了重要作用。     

STZ诱导的糖尿病模型小鼠肾脏的上皮-间质转分化研究

目的:探讨STZ诱导的糖尿病小鼠肾脏发生上皮-间质转分化(EMT)的情况。方法:将80只C57BL小鼠随机分为正常对照组(NC组)和糖尿病组(DM组),每组40只。DM组小鼠用1%STZ(streptozotocin,链脲佐菌素)溶液按60mg/kg体质量的剂量进行腹腔注射,每天1次,连续6天。NC组小鼠平行腹腔注射同等体积0.1mol/L的柠檬酸钠缓冲液。再将成模小鼠随机分为A、B批次,A批次用于动态观察生存率、小鼠体质量及随机血糖的监测;B批次用于在造模后第4、8、12周末观察肾组织的病理变化,并用Western blot、免疫荧光染色的方法观察肾组织中EMT标志蛋白α-SMA和E-cadherin的表达。结果:STZ诱导的糖尿病模型小鼠出现糖尿病典型症状如多饮、多尿等,血糖持续在高水平状态,体质量增长缓慢。在造模后12周末,DM组小鼠较NC组小鼠累积生存率显著降低,两组比较差异具有统计学意义(P0.001)。在造模后的第8周末,DM组小鼠肾脏出现明显的病理改变,到第12周末时,绝大部分肾小管上皮细胞被梭形的肌成纤维细胞取代,肾小球空泡,基底膜增厚。造模后的第4、8、12周末时,DM组小鼠E-cadherin表达量均显著低于NC组小鼠(P=0.004,0.026,0.004);而在第8和12周末时,DM组α-SMA表达量显著升高(P=0.009,0.015)。在第12周末,肾组织冰冻切片E-cadherin和α-SMA、免疫荧光染色结果与上述结果一致。结论:STZ诱导的糖尿病模型小鼠有较典型的糖尿病临床改变,且肾组织发生了EMT。     

极性蛋白与上皮肿瘤恶性转变

细胞极性是指细胞形态、蛋白分布以及细胞功能的不对称性,它是细胞发育、维持项一底极性、损伤修复及组织完整性等生理过程所必需的,主要是由极性蛋白调控。一旦极性蛋白之间的平衡失调,则会破坏细胞极性,诱导肿瘤发生、增殖及迁移。研究表明,极性蛋白的异常表达及错误定位均与肿瘤紧密相关。上皮细胞肿瘤发生及恶性转变过程通常伴有细胞极性丢失以及组织结构紊乱的现象,尤其是经历上皮间充质转变的上皮肿瘤细胞更易侵袭周围基质,最终引发转移。作者就目前有关极性蛋白在肿瘤方面的研究作一综述,重点阐述极性蛋白在肿瘤转移中的功能,并对相关问题进行讨论。     

波形蛋白在实验性肾间质纤维化上皮-间充质转分化中的表达及意义

目的明确肾脏纤维化中波形蛋白表达在上皮-间充质转分化观察中的意义。方法雄性SD大鼠12只,随机分成假手术组和模型组,模型组行单侧输尿管梗阻术。造模后14天处死大鼠,分别采用免疫组织化学染色和Western印迹法对梗阻侧肾组织波形蛋白和α平滑肌肌动蛋白作定性和定量检测。并通过体外实验用TGF-β1刺激诱导人近端小管上皮细胞株(HK-2)发生上皮-间充质转分化。采用间接免疫荧光法对E-钙粘蛋白和波形蛋白进行染色,采用激光共聚焦显微镜观察细胞形态改变,并用蛋白印迹法定量检测HK-2细胞波形蛋白和α平滑肌肌动蛋白表达水平。结果萎缩和扩张的肾小管上皮细胞出现波形蛋白和α平滑肌肌动蛋白表达,肾组织中波形蛋白和α平滑肌肌动蛋白表达量显著增加;正常的HK-2细胞,细胞形态为不规则圆形,TGF-β1刺激后细胞伸展成长梭形;细胞α平滑肌肌动蛋白表达水平显著增加,波形蛋白表达水平无显著变化。结论体内实验研究上皮-间充质转分化,将波形蛋白作为间充质细胞标志物具有较好的参考价值,而体外研究中,其标志作用尚存在争议。     

糖尿病肾小管-间质纤维化进程中负性调控相关蛋白的研究进展

糖尿病肾病(DKD)是糖尿病最常见及最严重的慢性微血管并发症之一,DKD发展为慢性肾功能衰竭的最终途径是肾小球硬化和小管间质纤维化(TIF)。TIF是DKD的主要病理改变,在TIF进程中上皮间充质转化(EMT)发挥了重要作用。对于EMT这一复杂且影响因素众多的过程,虽已知有很多基因、蛋白参与,但具体的调控机制仍不清楚。迄今为止,对于在糖尿病肾病肾纤维化过程中可能起着重要作用的多种负性调节相关蛋白的表达变化及其调控机制的研究十分广泛,故对此进行综述。     

结缔组织生长因子与肾脏纤维化

结缔组织生长因子属于CNN家族,是TGF-β的下游因子,它的表达受到TGF-β强烈诱导。结缔组织生长因子介导了FN、ColⅠ、ColⅢ、ColⅣ等mRNA及蛋白的表达,在纤维化的过程中可能起着重要的作用。     

肾脏干细胞

肾脏千细胞是成体干细胞研究中最晚最新的,目前肾脏干细胞的研究也限定在发育中的胚胎肾,对胚胎肾干细胞的来源和定位的研究处于探索起步阶段。大量研究证明,胚性肾脏干细胞来源于输尿管芽诱导后的后肾间充质。胚性肾发育中主要的基因和转录因子可提供鉴定肾脏干细胞的标志分子。胚胎肾干细胞发育时,渗透压、氧压等多种因素组成的微环境提示髓质部可能是成体干细胞存在区域,肾乳头部为千细胞的壁龛。实验证明肾外组织对成体肾有修补作用。通过对胚胎肾发育和成体肾修复的细胞和分子机制进一步了解肾脏干细胞。     

肾脏带3蛋白研究进展

肾脏带3蛋白介导的HCO3－／Cl-交换在肾小管酸碱调节中起重要作用,近年对其在肾小管中的定位,结构,基因组成和功能的研究极为活跃,本对近年的研究进展作一综述。     

AGS3蛋白与G蛋白信号转导

AGS3蛋白是影响受体到G蛋白的信号转导或直接影响非受体依赖型G蛋白激活的蛋白质之一。AGS3蛋白在脑、睾丸、肝脏、肾脏、心脏、胰腺及PC-12细胞中普遍分布。它不仅具有不依赖受体的Gβγ信号转导激活物的作用,也能作为二磷酸乌苷（GDP）的解离抑制剂,并负向调节G蛋白偶联受体对G蛋白的激活。AGSl、AGS2、AGS4是AGS家族的其它几个成员,能选择性激活不同类型的G蛋白。LGN和PINS蛋白是AGS3的同系物。AGS3蛋白与信号转导的关系是目前研究的热点之一。     

The Mitogen-activated Protein (MAP) Kinases p38 and Extracellular Signal-regulated Kinase (ERK) Are Involved in Hepatocyte-mediated Phenotypic Switching in Prostate Cancer Cells

The greatest challenge for the seeding of cancer in metastatic sites is integration into the ectopic microenvironment despite the lack of an orthotopic supportive environment and presence of pro-death signals concomitant with a localized “foreign-body” inflammatory response. In this metastatic location, many carcinoma cells display a reversion of the epithelial-to-mesenchymal transition that marks dissemination in the primary tumor mass. This mesenchymal to epithelial reverting transition (MErT) is thought to help seeding and colonization by protecting against cell death. We have previously shown that hepatocyte coculture induces the re-expression of E-cadherin via abrogation of autocrine EGFR signaling pathway in prostate cancer (PCa) cells and that this confers a survival advantage. Herein, we show that hepatocytes educate PCa to undergo MErT by modulating the activity of p38 and ERK1/2. Hepatocytes inhibited p38 and ERK1/2 activity in prostate cancer cells, which allowed E-cadherin re-expression. Introduction of constitutively active MEK6 and MEK1 to DU145 cells cocultured with hepatocytes abrogated E-cadherin re-expression. At least a partial phenotypic reversion can be achieved by suppression of p38 and ERK1/2 activation in DU145 cells even in the absence of hepatocytes. Interestingly, these mitogen-activated protein kinase activities were also triggered by re-expressed E-cadherin leading to p38 and ERK1/2 activity in PCa cells; these signals provide protection to PCa cells upon challenge with chemotherapy and cell death-inducing cytokines. We propose that distinct p38/ERK pathways are related to E-cadherin levels and function downstream of E-cadherin allowing, respectively, for hepatocyte-mediated MErT and tumor cell survival in the face of death signals.     

上皮细胞向间充质细胞转变在肝癌进程中的作用

上皮细胞向间充质细胞转变(epithelial to mesenchymal transition,EMT)是细胞通过瞬时去分化为间充质表型,导致上皮细胞的可塑性发生变化的多步骤的生物学过程。EMT及其逆转MET多数发生在胚胎发生形态发生过程中。近期更多的证据显示EMT参与肝纤维化和肝癌进程。在肝癌转移的早期阶段,细胞由于E-钙粘蛋白的消融而丧失细胞-细胞接触抑制,迁移能力增强,得以扩散到周围或远处组织,故EMT在肝癌转移的早期阶段中起关键作用。此外,由于EMT的增强诱导剂如转移生长因子(transforming growth factor-β,TGF-β)具有协调肝纤维化及肝癌进程的作用,故肝癌进程中上皮细胞的可塑性研究显得尤为重要。在本综述中,作者将阐述EMT-MET在肝癌进程中的重要性,及EMT在肝癌进程中的作用机制。同时,概述最近在识别影响重要EMT转录因子的临床诊治方面取得的进展。     

Histone Demethylase KDM6B Promotes Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a critical event that occurs in embryonic development, tissue repair control, organ fibrosis, and carcinoma invasion and metastasis. Although significant progress has been made in understanding the molecular regulation of EMT, little is known about how chromatin is modified in EMT. Chromatin modifications through histone acetylation and methylation determine the precise control of gene expression. Recently, histone demethylases were found to play important roles in gene expression through demethylating mono-, di-, or trimethylated lysines. KDM6B (also known as JMJD3) is a histone demethylase that might activate gene expression by removing repressive histone H3 lysine 27 trimethylation marks from chromatin. Here we report that KDM6B played a permissive role in TGF-β-induced EMT in mammary epithelial cells by stimulating SNAI1 expression. KDM6B was induced by TGF-β, and the knockdown of KDM6B inhibited EMT induced by TGF-β. Conversely, overexpression of KDM6B induced the expression of mesenchymal genes and promoted EMT. Chromatin immunoprecipitation (ChIP) assays revealed that KDM6B promoted SNAI1 expression by removing histone H3 lysine trimethylation marks. Consistently, our analysis of the Oncomine database found that KDM6B expression was significantly increased in invasive breast carcinoma compared with normal breast tissues. The knockdown of KDM6B significantly inhibited breast cancer cell invasion. Collectively, our study uncovers a novel epigenetic mechanism regulating EMT and tumor cell invasion, and has important implication in targeting cancer metastasis.     

Crosstalk between Epidermal Growth Factor Receptors (EGFR) and integrins in resistance to EGFR tyrosine kinase inhibitors (TKIs) in solid tumors

Cell adhesion to the extracellular matrix (ECM) is important in a variety of physiological and pathologic processes, including development, tumor invasion, and metastasis. Integrin-mediated attachment to ECM proteins has emerged to cue events primitively important for the transformed phenotype of human cancer cells. Cross-talk between integrins and growth factor receptors takes an increasingly prominent role in defining adhesion, motility, and cell growth. This functional interaction has expanded beyond to link integrins with resistance to Tyrosine kinase inhibitors (TKIs) of Epidermal Growth Factor Receptors (EGFRs). In this regard, integrin-mediated adhesion has two separate functions one as a clear collaborator with growth factor receptor signaling and the second as a basic mechanism contributing in Epithelial to Mesenchymal Transition (EMT) which affects response to chemotherapy. This review provides an overview of these mechanisms and describes treatment options for selectively targeting and disrupting integrin interaction to EGFR for cancer therapy.     

SHP2 Positively Regulates TGFβ1-induced Epithelial-Mesenchymal Transition Modulated by Its Novel Interacting Protein Hook1

The epithelial-mesenchymal transition (EMT) is an essential process for embryogenesis. It also plays a critical role in the initiation of tumor metastasis. Src homology 2 (SH2)-domain containing protein-tyrosine phosphatase-2 (SHP2) is a ubiquitously expressed protein-tyrosine phosphatase and is mutated in many tumors. However, its functional role in tumor metastasis remains largely unknown. We found that TGFβ1-induced EMT in lung epithelial A549 cells was partially blocked when SHP2 was decreased by transfected siRNA. The constitutively active form (E76V) promoted EMT while the phosphatase-dead mutation (C459S) and the SHP2 inhibitor PHPS1 blocked EMT, which further demonstrated that the phosphatase activity of SHP2 was required for promoting TGFβ1-induced EMT. Using the protein-tyrosine phosphatase domain of SHP2 as bait, we identified a novel SHP2-interacting protein Hook1. Hook1 was down-regulated during EMT in A549 cells. Overexpression of Hook1 inhibited EMT while knockdown of Hook1 promoted EMT. Moreover, both the protein-tyrosine phosphatase domain and N-terminal SH2 domain of SHP2 directly interacted with Hook1. Down-regulation of Hook1 increased SHP2 activity. These results suggested that Hook1 was an endogenous negative regulator of SHP2 phosphatase activity. Our data showed that the protein-tyrosine phosphatase SHP2 was involved in the process of EMT and Hook1 repressed EMT by regulating the activation of SHP2. SHP2-Hook1 complex may play important roles in tumor metastases by regulating EMT in cancer cells.     

Histone Deacetylase 3 Unconventional Splicing Mediates Endothelial-to-mesenchymal Transition through Transforming Growth Factor β2

Histone deacetylase 3 (HDAC3) plays a critical role in the maintenance of endothelial integrity and other physiological processes. In this study, we demonstrated that HDAC3 undergoes unconventional splicing during stem cell differentiation. Four different splicing variants have been identified, designated as HD3α, -β, -γ, and -δ, respectively. HD3α was confirmed in stem cell differentiation by specific antibody against the sequences from intron 12. Immunofluorescence staining indicated that the HD3α isoform co-localized with CD31-positive or α-smooth muscle actin-positive cells at different developmental stages of mouse embryos. Overexpression of HD3α reprogrammed human aortic endothelial cells into mesenchymal cells featuring an endothelial-to-mesenchymal transition (EndMT) phenotype. HD3α directly interacts with HDAC3 and Akt1 and selectively activates transforming growth factor β2 (TGFβ2) secretion and cleavage. TGFβ2 functioned as an autocrine and/or paracrine EndMT factor. The HD3α-induced EndMT was both PI3K/Akt- and TGFβ2-dependent. This study provides the first evidence of the role of HDAC3 splicing in the maintenance of endothelial integrity.     

MicroRNAs in the Imprinted DLK1-DIO3 Region Repress the Epithelial-to-Mesenchymal Transition by Targeting the TWIST1 Protein Signaling Network

Development of metastatic disease accounts for the vast majority of cancer-related deaths. Nevertheless, few treatments exist that are designed to specifically inhibit processes that drive tumor metastasis. The imprinted DLK1-DIO3 region contains tumor-suppressing miRNAs, but their identity and function remain indeterminate. In this study we identify seven miRNAs in the imprinted DLK1-DIO3 region that function cooperatively to repress the epithelial-to-mesenchymal transition, a critical step that drives tumor metastasis, as well as proliferation of carcinoma cells. These seven miRNAs (miRs 300, 382, 494, 495, 539, 543, and 544) repress a signaling network comprising TWIST1, BMI1, ZEB1/2, and miR-200 family miRNAs and silencing of the cluster, which occurs via hypermethylation of upstream CpG islands in human ductal carcinomas, confers morphological, molecular, and function changes consistent with an epithelial-to-mesenchymal transition. Moreover, ectopic expression of miR-544 independently inhibited proliferation of numerous tumor cell lines by inducing the ATM cell cycle checkpoint pathway. These results establish the DLKI-DIO3 miRNA cluster as a critical checkpoint regulating tumor growth and metastasis and implicate epigenetic modification of the cluster in driving tumor progression. These results also suggest that promoter methylation status and miRNA expression levels represent new diagnostic tools and therapeutic targets to predict and inhibit, respectively, tumor metastasis in carcinoma patients.     

雌激素对A549细胞系EMT过程的影响

目的:探讨雌激素对A549细胞系EMT标志物表达量的影响。方法:用不同浓度的雌激素刺激A549细胞系,并用q-RT-PCR和Western-blot实验检测各组细胞中EMT标志物表达量的变化,用Transwell实验检测不同浓度雌激素对细胞迁移能力的影响,计算各组间有无统计学差异。结果:当雌激素浓度为1×10-9 mol/L、1×10-8 mol/L、1×10-7 mol/L时,Vimentin的m RNA表达量分别为:2.14±0.55、4.72±0.63、2.21±0.47,显著高于空白对照组,组间有统计学差异,E-cadherin的m RNA表达量分别为:0.64±0.15、0.46±0.11、0.59±0.13,显著低于空白对照组,组间有统计学差异,蛋白表达量也有同样趋势;细胞迁移数分别为58.63±7.33、80.12±9.32、61.89±8.22,组间有统计学差异。当雌激素浓度为1×10-8 mol/L时,Vimentin的表达量最高,E-cadherin的表达量最低,细胞迁移数最高。结论:适宜浓度雌激素可以促进Vimentin的表达,抑制E-cadherin的表达,提高细胞迁移能力,当雌激素浓度为1×10-8 mol/L时,促进Vimentin表达、抑制E-cadherin表达和促进细胞迁移的作用最显著。由此认为,雌激素对A549细胞系发生EMT过程有促进作用。