Role of cytoskeletal elements in the recruitment of Cx43-GFP and Cx26-YFP into gap junctions

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gap junctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.     

Role of Cytoskeletal Elements in the Recruitment of Cx43-GFP and Cx26-YFP into Gap Junctions

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gapjunctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.     

Role of Cytoskeletal Elements in the Recruitment of Cx43-GFP and Cx26-YFP into Gap Junctions

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gapjunctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.     

Localisation aberrante de la connexine 43 dans le cancer du testicule

Most cells can communicate directly via gap junction channels. Gap junction intercellular communication (GJIC) participates in the control of cell proliferation. Abnormal expression of connexins (Cx), the constitutive proteins of gap junctions, has been associated with a transformed phenotype. In the seminiferous tubules, connexin Cx43 is predominantly expressed by Sertoli cell and germinal cell membranes. We studied Cx43 expression in four testicular cancers (pure seminoma). Cx43 mRNA and protein characterized by RT PCR and Western blot were found to be similar to controls (normal testes) in each case. However, immunofluorscence study of Cx43 protein indicated a cytoplasmic localization with no membrane expression, excluding the participation of Cx43 in GJIC. The significance of this aberrant localization will be discussed in relation to carcinogenesis.     

Changes of gap junctional cell-cell communication in overactive detrusor in rats

To evaluate the changes in intercellular communication through gap junctions in detrusor overactivity (DO), we studied 23 adult female Wistar rats with DO after partial outflow obstruction (DO group) and 13 sham-operated rats (control group). The two groups were compared by means of urodynamics, light and electron microscopy, expression of Cx40, Cx43, and Cx45 mRNA genes with RT-PCR, Cx43 protein with Western blot analysis, and functional intercellular communication with scrape loading dye transfer (SLDT) and fluorescence recovery after photobleaching (FRAP). The number of gap junctions and the expression of connexin mRNA and Cx43 protein were increased in DO rats, and intercellular communication through gap junctions increased after 6 wk of partial outflow obstruction as assessed with SLDT and FRAP techniques. The findings provide a theoretical rationale for using Cx43 antagonists and gap junction inhibitors in the treatment of patients with overactive detrusor secondary to partial bladder outflow obstruction.     

NBT-II carcinoma behaviour is not dependent on cell-cell communication through gap junctions

To study the mechanism(s) underlying the proliferation of heterogeneous cell populations within a solid tumour, the NBT-II rat bladder carcinoma system was used. It has been first investigated whether the different cell populations are coupled through gap junctions (GJIC). Cells overexpressing the Cx43 were generated to test for any tumour suppressive activity in vivo. To determine whether GJIC is essential for tumour proliferation and the establishment of a cooperative community effect, NBT-II cells that are incompetent for cell coupling were generated. The data report that (i) carcinoma cells expressing or not FGF-1 are coupled through GJIC in vitro and in coculture and express the gap junction protein Cx43, (ii) overexpression of Cx43 in these cells does not affect their in vitro coupling capacities and in vivo tumourigenic growth properties, (iii) inhibition of GJIC through antisense strategy has no in vivo obvious consequence on the tumour growth properties of the carcinoma, and (iv) the community effect between two carcinoma cell populations does not critically involve cell coupling through gap junctions.     

Involvement of tyrosine kinase in the hypoxia/reoxygenation-induced gap junctional intercellular communication abnormality in cultured human umbilical vein endothelial cells

Vascular endothelial cells (EC), communicating with one another across gap junctions, are usually made dysfunctional by hypoxia and reoxygenation (H/R); however, very limited information exists regarding the effects of H/R on the endothelial gap junctions. We investigated whether H/R interferes with endothelial gap junctional intercellular communication (GJIC). After human umbilical vein EC had grown to confluence, they were exposed to hypoxia (pO₂ < 0.1%) for 12–16 h and then returned to normal atmospheric conditions for reoxygenation. At 0-, 2-, 4-, 6-h reoxygenation, GJIC was detected by means of a fluorescence recovery after a photobleaching technique. The results demonstrated that a GJIC reduction (about 20% less than that under normoxia) was induced after 2 h of reoxygenation; after 4 h of reoxygenation, it began to recover (to about 10% less than that under normoxia); and after 6 h of reoxygenation, GJIC was restored to the normal level. Calphostin C (1 × 10⁻⁷ mol/l), a specific protein kinase C inhibitor, partially inhibited the reduction in GJIC (resulting in a level about 10% less than that under normoxia), whereas the tyrosine kinase inhibitor genistein (10 µmol/L) completely blocked the reduction in GJIC. Vanadate (1.5 mmol/l), a tyrosine phosphatase inhibitor, amplified the inhibitory effect of H/R on GJIC (to about 40% less than that under normoxia). Immunofluorescence and immunoprecipitation showed that 2-h reoxygenation significantly stimulated tyrosine protein phosphorylation, and this phosphorylation event was obviously enhanced by vanadate. The results of Western blotting showed that the gap junctional protein connexin 43 (Cx43) was phosphorylated by H/R; moreover, immunoprecipitation demonstrated that 2-h reoxygenation induced a prominent increase of tyrosine phosphorylation of Cx43 compared with that under normoxia. These data indicate that H/R induces a transient endothelial GJIC dysfunction through the activation of tyrosine kinase and phosphorylation of tyrosine residues of Cx43. J. Cell. Physiol. 180:305–313, 1999. © 1999 Wiley-Liss, Inc.     

Role of Cx43 Phosphorylation and MAP Kinase Activation in EGF Induced Enhancement of Cell Communication in Human Kidney Epithelial Cells

Epidermal growth factor (EGF) has been found to induce enhanced gap junctional intercellular communication (GJIC) in the human kidney epithelial cell line K7. This is in contrast to what is reported for other cell types, which all show decreased GJIC in response to EGF. In the present study it is shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) and EGF induce similar phosphorylation pattern of the gap junction protein connexin43 (Cx43) in K7 cells, although their effects on GJIC are opposite. Tyrosine phosphorylation of a 42 kD protein was observed to be induced concomitantly with phosphorylation of Cx43. EGF was however found to induce only serine phosphorylation of Cx43, indicating that the tyrosine kinase activity of the EGF receptor was not directly affecting the gap junction protein. The 42 kD protein phosphorylated on tyrosine was identified to be a mitogen activated protein (MAP) kinase. Both EGF and TPA was found to activate MAP kinase in these cells. Phosphorylation of Cx43 and enhancement of GJIC in response to EGF occurred with difference in time course. Phosphorylation of Cx43 was completed within 15 min, while the enhanced GJIC appeared 2-3 h later. It is therefore possible that regulation of synthesis or transport of Cx43 is responsible for the increase in GJIC, rather than direct involvement of Cx43 phosphorylation. This is in support of our previous finding that protein synthesis is necessary for EGF induced upregulation of GJIC in K7 cells.     

Human connexin26 and connexin30 form functional heteromeric and heterotypic channels

Mutations in GJB2 and GJB6, the genes that encode the human gap junction proteins connexin26 (Cx26) and connexin30 (Cx30), respectively, cause hearing loss. Cx26 and Cx30 are both expressed in the cochlea, leading to the potential formation of heteromeric hemichannels and heterotypic gap junction channels. To investigate their interactions, we expressed human Cx26 and Cx30 individually or together in HeLa cells. When they were expressed together, Cx26 and Cx30 appeared to interact directly (by their colocalization in gap junction plaques, by coimmunoprecipitation, and by fluorescence resonance energy transfer). Scrape-loading cells that express either Cx26 or Cx30 demonstrated that Cx26 homotypic channels robustly transferred both cationic and anionic tracers, whereas Cx30 homotypic channels transferred cationic but not anionic tracers. Cells expressing both Cx26 and Cx30 also transferred both cationic and anionic tracers by scrape loading, and the rate of calcein (an anionic tracer) transfer was intermediate between their homotypic counterparts by fluorescence recovery after photobleaching. Fluorescence recovery after photobleaching also showed that Cx26 and Cx30 form functional heterotypic channels, allowing the transfer of calcein, which did not pass the homotypic Cx30 channels. Electrophysiological recordings of cell pairs expressing different combinations of Cx26 and/or Cx30 demonstrated unique gating properties of cell pairs expressing both Cx26 and Cx30. These results indicate that Cx26 and Cx30 form functional heteromeric and heterotypic channels, whose biophysical properties and permeabilities are different from their homotypic counterparts. gap junctions; hearing; fluorescence resonance energy transfer; fluorescence recovery after photobleaching; immunoprecipitation; dye transfer; electrophysiology     

Suppression of the transformed phenotype of breast cancer by tropomyosin-1

Gap junctional intercellular communication (GJIC) is thought to play a crucial role in cell differentiation. Small gap junction plaques are frequently associated with tight junction strands in hepatocytes, suggesting that gap junctions may be closely related to the role of tight junctions in the establishment of cell polarity. To examine the exact role of gap junctions in regulating tight junctions, we transfected connexin 32 (Cx32), Cx26, or Cx43 cDNAs into immortalized mouse hepatocytes derived from Cx32-deficient mice and examined the expression and function of the endogenous tight junction molecules. In transient wild-type Cx32 transfectants, immunocytochemistry revealed that endogenous occludin was in part localized at cell borders, where it was colocalized with Cx32, whereas neither was detected in parental cells. In Cx32 null hepatocytes transfected with Cx32 truncated at position 220 (R220stop), wild-type Cx26, or wild-type Cx43 cDNAs, occludin was not detected at cell borders. In stable wild-type Cx32 transfectants, occludin, claudin-1, and ZO-1 mRNAs and proteins were significantly increased compared to parental cells and all of the proteins were colocalized with Cx32 at cell borders. Treatment with a GJIC blocker, 18 beta-glycyrrhetinic acid, resulted in decreases of occludin and claudin-1 at cell borders in the stable transfectants. The induction of tight junction proteins in the stable transfectants was accompanied by an increase in both fence and barrier functions of tight junctions. Furthermore, in the stable transfectants, circumferencial actin filaments were also increased without a change of actin protein. These results indicate that Cx32 formation and/or Cx32-mediated intercellular communication may participate in the formation of functional tight junctions and actin organization.     

ELF magnetic field inhibits gap junctional intercellular communication and induces hyperphosphorylation of connexin43 in NIH3T3 cells.

The effects of extremely low frequency (ELF) magnetic field on gap junctional intercellular communication (GJIC), protein levels, and phosphorylation of connexin43 (Cx43) were studied in NIH3T3 cells. The suppression of GJIC by 24 h, 50 Hz, 0.8 mT ELF magnetic field, 2 h, 3 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA), or ELF combined with TPA treatment was confirmed by the fluorescence recovery after photobleaching (FRAP) analysis with a confocal microscope. The results showed that ELF or TPA exposure induced 50-60% inhibition of GJIC (P < 0.01). ELF combined with TPA enhanced the inhibition of GJIC. Western blot analysis using Cx43 specific antibodies showed obviously decreasing non phosphorylated Cx43 (P(0)) induced by ELF and/or TPA exposure. On the other hand, cells treated with ELF and/or TPA displayed a hyperphosphorylated Cx43 band (P(3)). However, there was no obvious changes in the level of Cx43 protein. The results implied that the P(3) band appeared to result from phosphorylation of P(0). But it remains possible that upon the ELF exposure P(0) is converted to P(1), P(2) or both and that P(3) is formed from P(1) or P(2) resulting in the observed hyperphosphorylation pattern. From the present study, we conclude that ELF magnetic field inhibits GJIC and the main mechanism is the hyperphosphorylation of Cx43.     

Inhibition of gap junctional intercellular communication in heptachlor- and heptachlor epoxide-treated normal human breast epithelial cells

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 μg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 μg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 μg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 μg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 μg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing ‘initiated’ cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.     

Down-regulation of connexin43 gap junction by serum deprivation in human endothelial cells was improved by (-)-Epigallocatechin gallate via ERK MAP kinase pathway

Intercellular communication through gap junctions (GJIC) plays an essential role in maintaining the functional integrity of vascular endothelium. Despite emerging evidence suggests that (−)-Epigallocatechin gallate (EGCG) may improve endothelial function. However, its effect on Cx43 gap junction in endothelial cells remains unexplored. Here we investigated the effect of EGCG on connexin43 (Cx43) gap junction in endothelial cells. The levels of Cx43 protein in human umbilical vein endothelial cells (HUVECs) cultured under serum-deprivation 48 h decreased about 50%, accompanied by decreased GJIC. This reduction can be reversed by treatments with EGCG. In addition, EGCG activated ERK, P38, and JNK mitogen-activated protein kinases (MAPKs), which were supposed to participate in the regulation of Cx43. A MEK inhibitor PD98059, but not SB203580 (a p38 kinase inhibitor) or SP600125 (a JNK kinase inhibitor), abolished the effects of EGCG on Cx43 expression and GJIC. Moreover, although both Akt and eNOS phosphorylation were time-dependently augmented by EGCG, neither PI3K inhibitor LY294002 nor eNOS inhibitor L-NAME blocked the effects of EGCG on Cx43 gap junctions. Thus, EGCG attenuated Cx43 down-regulation and impaired GJIC induced by serum deprivation, ERK MAPK Signal transduction pathway appears to be involved in these processes.     

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.     

Gap junctions and hemichannels in signal transmission, function and development of bone

Gap junctional intercellular communication (GJIC) mediated by connexins, in particular connexin 43 (Cx43), plays important roles in regulating signal transmission among different bone cells and thereby regulates development, differentiation, modeling and remodeling of the bone. GJIC regulates osteoblast formation, differentiation, survival and apoptosis. Osteoclast formation and resorptive ability are also reported to be modulated by GJIC. Furthermore, osteocytes utilize GJIC to coordinate bone remodeling in response to anabolic factors and mechanical loading. Apart from gap junctions, connexins also form hemichannels, which are localized on the cell surface and function independently of the gap junction channels. Both these channels mediate the transfer of molecules smaller than 1.2kDa including small ions, metabolites, ATP, prostaglandin and IP(3). The biological importance of the communication mediated by connexin-forming channels in bone development is revealed by the low bone mass and osteoblast dysfunction in the Cx43-null mice and the skeletal malformations observed in occulodentodigital dysplasia (ODDD) caused by mutations in the Cx43 gene. The current review summarizes the role of gap junctions and hemichannels in regulating signaling, function and development of bone cells. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

The connexin 43 C-terminus: A tail of many tales

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.     

Lipopolysaccharide-induced inhibition of connexin43 gap junction communication in astrocytes is mediated by downregulation of caveolin-3

Astrocytes play a crucial role in maintaining the homeostasis of the brain. Changes to gap junctional intercellular communication (GJIC) in astrocytes and excessive inflammation may trigger brain damage and neurodegenerative diseases. In this study, we investigated the effect of lipopolysaccharide (LPS) on connexin43 (Cx43) gap junctions in rat primary astrocytes. Following LPS treatment, dose- and time-dependent inhibition of Cx43 expression was seen. Moreover, LPS induced a reduction in Cx43 immunoreactivity at cell–cell contacts and significantly inhibited GJIC, as revealed by the fluorescent dye scrape loading assay. Toll-like receptor 4 (TLR4) protein expression was increased 2–3-fold following LPS treatment. To study the pathways underlying these LPS-induced effects, we examined downstream effectors of TLR4 signaling and found that LPS induced a significant increase in phosphorylated extracellular signal-regulated kinase (pERK) levels up to 6 h, followed by signal attenuation and downregulation of caveolin-3 expression. Interestingly, LPS treatment also induced a dramatic increase in inducible nitric oxide synthase (iNOS) levels at 6 h, which were sustained up to 18–24 h. The LPS-induced downregulation of Cx43 and caveolin-3 was prevented by co-treatment of astrocytes with the iNOS cofactor inhibitor 1400W, but not the ERK inhibitor PD98059. Specific knockdown of caveolin-3 using siRNA had a significant inhibitory effect on GJIC and resulted in a downregulation of Cx43. Our results suggest that long-term LPS treatment of astrocytes leads to inhibition of Cx43 gap junction communication by the activation of iNOS and downregulation of caveolin-3 via a TLR4-mediated signaling pathway.