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1.
Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly. Overall, these results demonstrate that the Cx43/beta-gal fusion protein can exert a dominant negative effect on GJC in two different cell types, and suggests that it may serve as a useful approach for probing the biological function of gap junctions.  相似文献   

2.
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。  相似文献   

3.
Loss of connexin expression and/or gap junctional communication (GJC) has been correlated with increased rates of cell growth in tumor cells compared to their normal communication-competent counterparts. Conversely, reduced rates of cell growth have been observed in tumor cells that are induced to express exogenous connexins and re-establish GJC. It is not clear how this putative growth-suppressive effect of the connexin proteins is mediated and some data has suggested that this function may be independent of GJC. In mammalian cells that express v-Src, connexin43 (Cx43) is phosphorylated on Tyr247 and Tyr265 and this results in a dramatic disruption of GJC. Cells that express a Cx43 mutant with phenylalanine mutations at these tyrosine sites form functional gap junctions that, unlike junctions formed by wild type Cx43, remain functional in cells that co-express v-Src. These cells still appear transformed; however, it is not known whether their ability to maintain GJC prevents the loss of growth restraints that confine “normal” cells, such as the inability to grow in an anchorage-independent manner or to form foci. In these studies, we have examined some of the growth properties of cells with Cx43 gap junctions that remain communication-competent in the presence of the co-expressed v-Src oncoprotein.  相似文献   

4.
Loss of connexin expression and/or gap junctional communication (GJC) has been correlated with increased rates of cell growth in tumor cells compared to their normal communication-competent counterparts. Conversely, reduced rates of cell growth have been observed in tumor cells that are induced to express exogenous connexins and re-establish GJC. It is not clear how this putative growth-suppressive effect of the connexin proteins is mediated and some data has suggested that this function may be independent of GJC. In mammalian cells that express v-Src, connexin43 (Cx43) is phosphorylated on Tyr247 and Tyr265 and this results in a dramatic disruption of GJC. Cells that express a Cx43 mutant with phenylalanine mutations at these tyrosine sites form functional gap junctions that, unlike junctions formed by wild type Cx43, remain functional in cells that co-express v-Src. These cells still appear transformed; however, it is not known whether their ability to maintain GJC prevents the loss of growth restraints that confine "normal" cells, such as the inability to grow in an anchorage-independent manner or to form foci. In these studies, we have examined some of the growth properties of cells with Cx43 gap junctions that remain communication-competent in the presence of the co-expressed v-Src oncoprotein.  相似文献   

5.
We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.  相似文献   

6.
The ability of herpes simplex virus type 1 thymidine kinase (HSV-tk)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-tk-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs via the transfer of phosphorylated GCV, the mechanism(s) of this bystander effect and the importance of gap junctions for the effect of prodrug/suicide gene therapy in primary human glioblastoma cells remains elusive. Surgical biopsies of malignant gliomas were used to establish explant primary cultures. Proliferating tumor cells were characterized immunohistochemically and found to express glial tumor markers including nestin, vimentin, glial fibrillary acidic protein (GFAP), S-100, and gap junction protein connexin 43 (Cx43). Western blot analysis revealed the presence of phosphorylated isoforms of Cx43 and Calcein/DiI fluorescent dye transfer showed evidence of efficient gap junction communication (GJC). In order to study the effect(s) of prodrug/suicide gene therapy in these cultures, human glioblastoma cell cultures were transfected with the HSVtk gene for transient or stable expression. Ganciclovir treatment of these cultures led to >90% of cells dead within 1 week. Eradication of cells could be inhibited by the addition of α-glycyrrhetinic acid (AGA), a GJC inhibitor. In parallel experiments, AGA decreased the immunodetection of phosphorylated Cx43 as analyzed by Western blot and inhibited fluorescent dye transfer. In conclusion, these observations are consistent with GJC as the mediator of the bystander effect in primary cultures of human glioblastoma cells by the transfer of phosphorylated GCV from HSVtk gene transfected cells to untransfected ones.  相似文献   

7.
Our previous studies showed an essential role for connexin 43 or α1 connexin (Cx43α1) gap junctions in the modulation of neural crest cell motility. Cx43α1 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 Cx43α1 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 Cx43α1 KO neural crest cells. These observations suggest that the role of N-cadherin in cell motility is not simply mediated via the modulation of Cx43α1 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 p 120 catenin (p 120ctn), an Amardillo family protein known to play a role in cell motility, showed that it is colocalized with N-cadherin and Cx43α1 in migrating neural crest cells. This subcellular distribution was altered in the N-cadherin and Cx43α1 KO neural crest cells. Given these results, we propose that N-cadherin and Cx43α1 may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.  相似文献   

8.
It is not clear how the v-Src oncoprotein disrupts gap junctional communication (GJC) established by connexin43 (Cx43) in mammalian cells. In this study, an experimental system was established to stably express v-Src and wild type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout (KO) mouse cell line. When co-expressed with v-Src, the levels of phosphotyrosine (pTyr) from Y247F, Y265F, and Y247F/Y265F Cx43 mutants were reduced to approximately 57%, 10%, and 2% of the level of pTyr from wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. These data also implied that phosphorylation of Cx43 at Y265 was required for efficient phosphorylation of Cx43 at Y247. Most importantly, our measurements of GJC demonstrated that, in contrast to the wt Cx43 gap junction channels, the Y247F, Y265F, and Y247F/Y265F Cx43 channels were resistant to the disruption by v-Src. In conclusion, our studies support a model for processive phosphorylation of Cx43 on tyrosine, at the Y265 site followed by the Y247 site, in mediating the disruption of GJC induced by v-Src in mammalian cells.  相似文献   

9.
Connexins are chordate gap junction channel proteins that, by enabling direct communication between the cytosols of adjacent cells, create a unique cell signalling network. Gap junctional intercellular communication (GJIC) has important roles in controlling cell growth and differentiation and in tissue development and homeostasis. Moreover, several non-canonical connexin functions unrelated to GJIC have been discovered. Of the 21 members of the human connexin family, connexin 43 (Cx43) is the most widely expressed and studied. The long cytosolic C-terminus (CT) of Cx43 is subject to extensive post-translational modifications that modulate its intracellular trafficking and gap junction channel gating. Moreover, the Cx43 CT contains multiple domains involved in protein interactions that permit crosstalk between Cx43 and cytoskeletal and regulatory proteins. These domains endow Cx43 with the capacity to affect cell growth and differentiation independently of GJIC. Here, we review the current understanding of the regulation and unique functions of the Cx43 CT, both as an essential component of full-length Cx43 and as an independent signalling hub. We highlight the complex regulatory and signalling networks controlled by the Cx43 CT, including the extensive protein interactome that underlies both gap junction channel-dependent and -independent functions. We discuss these data in relation to the recent discovery of the direct translation of specific truncated forms of Cx43. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.  相似文献   

10.
Enzyme prodrug therapy using neural progenitor cells (NPCs) as delivery vehicles has been applied in animal models of gliomas and relies on gap junction communication (GJC) between delivery and target cells. This study investigated the effects of histone deacetylase (HDAC) inhibitors on GJC for the purpose of facilitating transfer of therapeutic molecules from recombinant NPCs. We studied a novel immortalized midbrain cell line, NGC-407 of embryonic human origin having neural precursor characteristics, as a potential delivery vehicle. The expression of gap junction protein connexin 43 (Cx43) was analyzed by western blot and immunocytochemistry. While Cx43 levels were decreased in untreated differentiating NGC-407 cells, the HDAC inhibitor 4-phenylbutyrate (4-PB) increased Cx43 expression along with increased membranous deposition in both proliferating and differentiating cells. Simultaneously, Ser 279/282-phosphorylated form of Cx43 was declined in both culture conditions by 4-PB. The 4-PB effect in NGC-407 cells was verified by using HNSC.100 human neural progenitors and Trichostatin A. Improved functional GJC is of imperative importance for therapeutic strategies involving intercellular transport of low molecular-weight compounds. We show here an enhancement by 4-PB, of the functional GJC among NGC-407 cells, as well as between NGC-407 and human glioma cells, as indicated by increased fluorescent dye transfer.  相似文献   

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