间隙连接蛋白43的功能及温度响应性研究进展

在各种组织和器官中都存在允许相邻细胞的胞质区之间直接通讯的间隙连接,它们在广泛的生理过程中起关键作用。间隙连接是细胞间通道,由间隙连接蛋白组成,其中间隙连接蛋白43(Cx43)在各组织器官中广泛表达。研究发现细胞间隙连接通讯会受到冷热刺激的影响,并与Cx43表达相关。本篇综述主要介绍Cx43转录与翻译水平的调控以及它的降解途径,并对冷热刺激后Cx43表达变化的作用机制进行概述。     

癌细胞逆转过程中N-cadherin与间隙连接蛋白Cx43功能表达的关系

钙粘合蛋白 (N cadherin ,N cad)在人肺癌细胞的表达明显低于正常人肺细胞 .肺癌细胞的间隙连接通讯功能缺陷 ,连接蛋白Cx43表达抑制 .Cx43cDNA转染肺癌细胞的 4个阳性克隆其Cx43蛋白表达升高水平相近 ,但通讯功能有差别 ,与各克隆N cad的表达水平有正相关性 .N cad表达高的克隆Cx43在膜间隙连接的分布和通讯功能最明显 ,细胞分化改善 ,在裸鼠体内生长抑制 (抑制率 75 % )有显著性 .反之N cad表达低的克隆Cx43在膜间隙连接不明显 ,细胞通讯功能弱 ,恶性表型无逆转 .提示N cad与Cx43转录后表达过程的调节密切相关 ,两者介导的粘合和通讯功能有协同促进肺癌细胞逆转的作用 .     

性激素对生殖系统Cx43表达调控机制的研究进展

由连接蛋白43(connexin 43,Cx43)构成的细胞间间隙连接(gap junction,GJ)是介导细胞间直接的物质、能量交换及电、化学信号耦合的重要通路,其可保证细胞间功能活动的协调一致,在生殖系统细胞发育、分化及成熟等生理过程及功能活动中发挥非常重要的调控作用。性激素可在转录及翻译等层面调节生殖系统细胞Cx43的结构及表达,一方面通过核受体基因组机制调节Cx43基因的转录,另一方面通过非基因组快速信号传导通路机制调节Cx43的磷酸化水平,共同影响细胞间隙连接通讯(gap junction intracellular communication,GJIC),进一步干预生殖系统细胞的病理、生理过程及功能活动。近年来,性激素在心血管系统病生理状态下对连接蛋白调节的变化及机制研究较为成熟,而在子宫、卵巢等生殖器官中,对性激素通过调节连接蛋白及间隙连接进而影响细胞间信息流通的研究较少,其作用机制并不清晰。故本文结合近年来文献,综述性激素对生殖系统细胞Cx43表达及GJIC的调控机制的研究进展,旨在为今后深入地研究提供可行的思路。     

间隙连接分子Cx43相关蛋白及其功能研究进展

间隙连接是细胞间直接进行信息交流的唯一膜通道结构。Cx43是构成间隙连接中分布最广、研究最多的间隙连接分子,目前运用免疫共沉淀、免疫荧光共定位、pull-down以及酵母双杂交等多种方法研究发现了众多的Cx43相关蛋白。这些蛋白通过与Cx43相互作用在间隙连接蛋白的组装、运输、膜定位,间隙连接通道的形成以及对间隙连接通讯的调控等一系列过程中均发挥十分重要的作用。本文就目前已经研究发现的Cx43相关蛋白及其最新的功能研究进展进行综述。     

间隙连接蛋白43(Cx43)在皮肤伤口愈合中的作用

间隙连接(gap junction,GJ)是细胞膜上的通道结构,其介导的细胞间间隙连接通讯(gap junction intercellular communication,GJIC)对内环境的稳定、细胞生长调控及新陈代谢等起到重要的作用。间隙连接蛋白43(connexin43,Cx43)是哺乳动物细胞中分布最为广泛的间隙连接蛋白,越来越多的研究发现皮肤创伤后Cx43的表达会随着伤口愈合的过程发生动态变化,并影响伤口愈合的速率和质量,人为调控Cx43的表达水平会改善伤口愈合的速率和质量。主要就Cx43结构与功能、Cx43的水平对伤口愈合各阶段的影响及Cx43与慢性伤口的关系进行总结,以期为探索皮肤创伤,尤其是慢性伤口治疗新途径提供参考价值。     

骨组织细胞Cx43形成的间隙连接及半通道研究进展

连接子蛋43（connexin 43,Cx43）是骨组织中主要的间隙连接（gap junction）蛋白和半通道（hemichannel）蛋白,由Cx43形成的间隙连接及半通道实现了骨组织细胞间的直接通讯。连接子蛋白对骨组织的正常发育、骨重建过程的建立与平衡是非常重要的。目前研究指出,Cx43不仅参与了骨组织的力学响应过程,也参与了二磷酸盐、甲状旁腺激素等药物对骨重建的调节过程。该文以骨组织细胞内信号传递途径的关键分子Cx43为对象,就其目前的研究现状作一综述。     

缝隙连接蛋白43 研究进展

缝隙连接蛋白(Connexin,Cx)组成缝隙连接通道发挥其通讯功能,其本身也对细胞的生长、分化、凋亡、肿瘤具有调节作用。缝隙连接蛋白43(Cx43)在肺泡上皮和肺血管内皮高表达,在多种肺损伤的病理过程中起重要作用,因此成为研究热点。Cx43的表达和功能受多种因素的调节,丝裂原激活的蛋白激酶(MAPKs)是主要的调节途径。本文就Cx43在肺的病理生理过程中的作用及MAPK对其调节作用进行综述。     

间隙连接蛋白43基因在斑马鱼和金鱼中的表达模式分析

间隙连接蛋白43(connexin43,Cx43)是间隙连接蛋白家族的成员之一,在多种组织和细胞类型上广泛表达,参与体内平衡、胚胎发育、细胞分化及生长等生理活动。现以斑马鱼(Danio rerio)和金鱼(Carassius auratus)为材料,运用RT-PCR方法检测了Cx43基因在其组织和胚胎发育中的表达模式。结果显示:在斑马鱼的组织中,Cx43基因在心脏中的表达量最高,而在肾脏和卵巢的表达量较低;在不同发育时期的胚胎中,Cx43基因在体色素期和出膜期中的表达量较高,而在胚胎发育早期的表达量相对较低。在金鱼组织中,Cx43基因在心脏中的表达量较高,而在鱼鳍和眼睛的表达量较低;在不同发育时期的胚胎中,Cx43基因表达模式基本上与斑马鱼的12个时期相一致。研究表明,Cx43基因在鱼类不同组织和不同胚胎发育时期中可能行使不同的功能,但其具体的功能和机制还有待进一步研究。     

Cx43在人胚胎早期食管上皮组织中的表达

目的:探讨间隙连接蛋白Cx43在人胚胎早期食管上皮组织中的表达规律.方法:应用免疫组织化学SABC法检测第2、3、4三个月龄段人胚胎食管上皮层Cx43蛋白的表达.结果:第2～4个月龄段,食管上皮层均有Cx43蛋白阳性的细胞分布.第2个月胚龄段,Cx43蛋白在人胚胎食管上皮基底层呈强阳性表达,由基底层向管腔面,细胞阳性强度逐渐降低;第3个月胎龄段,Cx43蛋白阳性细胞在食管上皮层广泛分布;第4个月胎龄段,Cx43蛋白在食管上皮层近基底面部分细胞呈弱阳性表达.结论:Cx43蛋白在人胚胎早期食管上皮层细胞的生长发育过程中起重要的作用.     

间隙连接通讯—生长调控—癌变三者之间的关系

间隙连接普遍存在于动物细胞,是细胞群体内信号传递的主要途径,对于传播生长调节信号,调控细胞增殖有重要的作用。细胞增殖的不同状态,其间隙连接通讯功能表现不同。许多转化的细胞和肿瘤细胞的间隙连接通讯低下或无通讯。间隙连接通讯的抑制或破坏可能与细胞生长失控有关,被认为是癌变促癌阶段的重要机制。     

The role of the gap junction protein connexin43 in B lymphocyte motility and migration

The gap junction family of proteins is widely expressed in mammalian cells and form intercellular channels between adjacent cells, as well as hemichannels, for transport of molecules between the cell and the surrounding environment. In addition, gap junction proteins have recently been implicated as important for the regulation of cell adhesion and migration in a variety of cell types. The gap junction protein connexin43 (Cx43) regulates B lymphocyte adhesion, BCR- and LFA-1-mediated activation of the GTPase Rap1, and cytoskeletal rearrangements resulting in changes to cell shape and membrane spreading. We demonstrate here that the actin cytoskeleton is important for the distribution of Cx43 in the B cell plasma membrane and for other cell processes involving the cytoskeleton. Using shRNA knockdown of Cx43 in B lymphoma cells we show that Cx43 is also necessary for chemokine-mediated Rap 1 activation, motility, CXCL12-directed migration, and movement across an endothelial cell monolayer. These results demonstrate that in addition to its role in B cell spreading, Cx43 is an important regulator of B-cell motility and migration, processes essential for normal B-cell development and immune responses.     

Modulation of mouse neural crest cell motility by N-cadherin and connexin 43 gap junctions.

Connexin 43 (Cx43alpha1) gap junction has been shown to have an essential role in mediating functional coupling of neural crest cells and in modulating neural crest cell migration. Here, we showed that N-cadherin and wnt1 are required for efficient dye coupling but not for the expression of Cx43alpha1 gap junctions in neural crest cells. Cell motility was found to be altered in the N-cadherin-deficient neural crest cells, but the alterations were different from that elicited by Cx43alpha1 deficiency. In contrast, wnt1-deficient neural crest cells showed no discernible change in cell motility. These observations suggest that dye coupling may not be a good measure of gap junction communication relevant to motility. Alternatively, Cx43alpha1 may serve a novel function in motility. We observed that p120 catenin (p120ctn), an Armadillo protein known to modulate cell motility, is colocalized not only with N-cadherin but also with Cx43alpha1. Moreover, the subcellular distribution of p120ctn was altered with N-cadherin or Cx43alpha1 deficiency. Based on these findings, we propose a model in which Cx43alpha1 and N-cadherin may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.     

Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions.

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.     

The carboxy‐terminal tail of connexin43 gap junction protein is sufficient to mediate cytoskeleton changes in human glioma cells

Connexin43 (Cx43) is a ubiquitously expressed member of the gap junction protein family that mediates gap junction intercellular communication (GJIC) by allowing exchange of cytosolic materials. Previous studies have used Cx43 truncated at the cytoplasmic tail (C‐tail) to demonstrate that the C‐tail is essential to regulate cell growth and motility. Therefore, the aim of our study was to delineate the respective role of the truncated Cx43 and the C‐tail in mediating Cx43‐dependent signaling. A truncated Cx43 expressing the channel part of the protein (TrCx43, amino acid 1–242) and a construct encompassing only the C‐tail from amino acid 243 (243Cx43) were transduced into LN18 human glioma cells. Our results showed that the ability of Cx43 to suppress growth was independent of GJIC as assessed by dye transfer, but was dependent on the presence of a rigid extracellular matrix. We further demonstrated that the C‐tail alone is sufficient to promote motility. Surprisingly, Cx43 is also able to increase migration in the absence of the C‐tail, suggesting the presence of at least two distinct signaling mechanisms utilized by Cx43 to affect motility. Finally, we used time‐lapse imaging to examine the behavior of migrating cells and it was apparent that the C‐tail was associated with a lamellipodia‐based migration not observed in either mock or TrCx43 expressing LN18 cells. Our study shows for the first time that a free C‐tail is sufficient to induce Cx43‐dependent changes in cell morphology and that Cx43 signaling is linked to the regulation of the actin cytoskeleton. J. Cell. Biochem. 110: 589–597, 2010. © 2010 Wiley‐Liss, Inc.     

Dose-dependent differential upregulation of CCN1/Cyr61 and CCN3/NOV by the gap junction protein Connexin43 in glioma cells

Gap junctions form channels that allow exchange of materials between cells and are composed of transmembrane protein subunits called connexins. While connexins are believed to mediate cellular signaling by permitting intercellular communication to occur, there is also increasing evidence that suggest connexins may mediate growth control via a junction-independent mechanism. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes, and gliomas exhibit reduced Cx43 expression. We have previously observed that restoration of Cx43 levels in glioma cells led to increased expression of CCN3 (NOV) proteins. We now report that overexpression of Cx43 in C6-glioma cells (C6-Cx43) also upregulates the expression of CCN1 (Cyr61). Both CCN1 and CCN3 belong to the Cyr61/Connective tissue growth factor/Nephroblastoma-overexpressed (CCN) family of secretory proteins. The CCN proteins are tightly associated with the extracellular matrix and have important roles in cell proliferation and migration. CCN1 promotes growth in glioma cells, as shown by the increased proliferation rate of CCN1-overexpressing C6 cells. In addition to its effect on cell growth, CCN1 also increased the motility of glioma cells in the presence of extracellular substrates such as fibronectin. Gliomas expressing high levels of Cx43 preferentially upregulated CCN3 which resulted in reduced growth rate. CCN3 could also be observed in Cx43 gap junction plaques in confluent C6-Cx43H culture at the stationary phase of their growth. Our results suggest that the dissimilar growth characteristics between high and low Cx43 expressors may be due to differential regulation of CCN3 by varying levels of Cx43.     

Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication.

Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.     

Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes.

Recent studies using mice with genetically engineered gap junction protein connexin (Cx) genes have provided evidence that reduced gap-junctional coupling in ventricular cardiomyocytes predisposes to ventricular arrhythmia. However, the pathological processes of arrhythmogenesis due to abnormalities in gap junctions are poorly understood. We have postulated a hypothesis that dysfunction of gap junctions at the single-cell level may affect synchronization of calcium transients among cardiomyocytes. To examine this hypothesis, we developed a novel system in which gap-junctional intercellular communication in primary neonatal rat cardiomyocytes was inhibited by a mutated (Delta130-137) Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP), and calcium transients were imaged in real time while the mutated Cx43-EGFP-expressing cardiomyocytes were identified. The mutated Cx43-EGFP inhibited dye coupling not only in the liver epithelial cell line IAR 20 but also in primary neonatal rat cardiomyocytes in a dominant-negative manner, whereas wild-type Cx43-EGFP made functional gap junctions in otherwise communication-deficient HeLa cells. The mutated Cx43-EGFP induced desynchronization of calcium transients among cardiomyocytes with significantly higher frequency than wild-type Cx43-EGFP. These results suggest that dysfunction of gap-junctional intercellular communication at the single-cell level could hamper synchronous beating among cardiomyocytes as a result of desynchronization of calcium transients.     

TGF-β2 increases cell-cell communication in chondrocytes via p-Smad3 signalling

Connexin 43 (Cx43)-mediated gap junction intercellular communication (GJIC) plays a crucial role in the pathology and physiology of joint tissues. Transforming growth factor-β2 (TGF-β2), one of the potent regulatory factors in chondrocytes, plays a key role in the regulation of cell cycle and development of joint diseases. However, it is still unknown how TGF-β2 mediates GJIC in chondrocytes. The aim of this study was to explore the potential mechanism by which TGF-β2 regulates GJIC in chondrocytes. CCK-8 assays and scratch assays were performed to define the role of TGF-β2 on cell proliferation and migration. The scrape loading/dye transfer assay and scanning electron microscopy (SEM) were used to verify the effect of TGF-β2 on GJIC between chondrocytes. qPCR was performed to analyse the expression of genes in the gap junction protein family in chondrocytes. The expression of the Cx43 protein and phosphorylated Smad3 (p-Smad3) was evaluated by western blot assay. Immunofluorescence staining was used to explore p-Smad3 signalling pathway activation and Cx43 distribution. From these experiments, we found that the Cx43 protein was the most highly expressed member of the gap junction protein family in chondrocytes. We also found that TGF-β2 facilitated cell-to-cell communication in chondrocytes by upregulating Cx43 expression in chondrocytes. Finally, we found that TGF-β2 activated Smad3 signalling and promoted the nuclear aggregation of p-Smad3. Inhibition experiments by SIS3 also confirmed that TGF-β2-mediated GJIC through p-Smad3 signalling. For the first time, this study confirmed that TGF-β2 could regulate the formation of Cx43-mediated GJIC in chondrocytes via the canonical p-Smad3 signalling pathway.     

N-cadherin and Cx43alpha1 gap junctions modulates mouse neural crest cell motility via distinct pathways

Our previous studies showed an essential role for connexin 43 or alpha1 connexin (Cx43alpha1) gap junctions in the modulation of neural crest cell motility. Cx43alpha1 gap junctions and N-cadherin containing adherens junctions are expressed in migrating cardiac neural crest cells. Analysis of the N-cadherin knockout (KO) mouse model revealed that N-cadherin is essential for gap junction mediated dye coupling but not for expression of Cx43alpha1 gap junctions in neural crest cells. Time lapse videomicroscopy and motion analysis showed that the motility of N-cadherin KO neural crest cells were altered, but the motility changes differed compared to Cx43alpha1 KO neural crest cells. These observations suggest that the role of N-cadherin in cell motility is not simply mediated via the modulation of Cx43alpha1 mediated cell-cell communication. This was confirmed by a parallel analysis of wnt-1 deficient neural crest cells, which also showed a reduction in dye coupling, and yet no change in cell motility. Analysis of p120 catenin (p120ctn), an Amardillo family protein known to play a role in cell motility, showed that it is colocalized with N-cadherin and Cx43alpha1 in migrating neural crest cells. This subcellular distribution was altered in the N-cadherin and Cx43alpha1 KO neural crest cells. Given these results, we propose that N-cadherin and Cx43alpha1 may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.