Gap Junction Assembly Research Articles

Molecular mechanisms of gap junction assembly in the cochlea

Molecular mechanisms of gap junction assembly in the cochlea

Abstract 289: Improved Disease Modeling Reveals 9p21 Risk Allele Regulates Connexins to Induce Arrhythmic Phenotypes

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the non-coding 9p21 gene locus associated with increased risk of coronary artery disease (CAD) and myocardial infarction (MI). In addition, SNP correlations with sudden and arrhythmic death, even after accounting for patient and family history for CAD and MI, suggest an altered cardiac remodeling response. However, little is known about a possible cardiac phenotype as studies have largely focused on its effect on CAD and have trouble describing regulation with non-coding loci. Using induced pluripotent stem cell-derived CMs from patients that are homozygous risk/risk (R/R) and non-risk/non-risk (N/N) for 9p21 SNPs, we assessed cardiomyocyte (CM) function when cultured on hydrogels capable of mimicking the fibrotic stiffening associated with disease post-heart attack, i.e. stiffening from 10 kiloPascals (kPa) to 50 kPa. While all CMs independent of genotype beat synchronously on soft matrices, R/R CMs cultured on dynamically stiffened hydrogels exhibited asynchronous contractions versus N/N CMs in the same conditions. Dynamic stiffening reduced connexin 43 expression and gap junction assembly in R/R CMs but not N/N CMs. To eliminate patient-to-patient variability, we created an isogenic line by deleting the 9p21 locus from a R/R patient, i.e. R/R KO. R/R KO CMs maintained synchronous contractions and organized connexin 43 junctions after stiffening. The 9p21 locus suppresses the activity of the cell cycle regulator CDKN2A. p16, a protein produced by CDKN2A, prevents JNK phosphorylation (p-JNK), which in turn reduces gap junction expression in CMs and contributes to the development of arrhythmias in rabbit myocardium in response to stress. We observed that treatment with the p-JNK antagonist SP600125 after stiffening restored synchronous contractions and organized gap junction assembly to R/R CM. As a non-coding locus, 9p21 appears to repress connexin transcription, but only when the niche is stiffened as in disease. These data are the first to demonstrate that disease-specific niche remodeling can differentially affect CM function depending on SNPs within a non-coding locus.

Effects of different doses of 2,3-dimercaptosuccinic acid-modified Fe2 O3 nanoparticles on intercalated discs in engineered cardiac tissues.

Although iron oxide nanoparticles (IRONs) were applied in clinical magnetic resonance imaging in vivo and magnetic tissue engineering in vitro widely, the underlying effects of IRONs on the development of cardiomyocytes especially the intercellular junctions, intercalated discs (IDs), remain an unknown issue. Given the critical role of three-dimensional (3D) engineered cardiac tissues (ECTs) in evaluation of nanoparticles toxicology, it remained necessary to understand the effects of IRONs on IDs assembly of cardiomyocytes in 3D environment. In this study, we first reconstituted collagen/Matrigel based ECTs in vitro and prepared IRONs with 2,3-dimercaptosuccinic acid (DMSA-IRONs). We found that the internalization of DMSA-IRONs by cardiac cells in dose-dependent manner was not associated with the cell distribution in 3D environment by determination of Prussian blue staining and transmission electronic microscopy. Significantly, through determination of western blotting and immunofluorescence of connexin 43, N-cadherin, desmoplakin, and plakoglobin, DMSA-IRONs enhanced the assembly of gap junctions, decreased mechanical junctions (adherens junctions and desmosomes) of cardiac cells but not in dose-dependent manner in ECTs at seventh day. In addition, DMSA-IRONs increased the vesicles secretion of cardiac cells in ECTs apparently compared to control groups. Overall, we conclude that the internalization of DMSA-IRONs by cardiac cells in dose-dependent manner enhanced the assembly of electrochemical junctions and decreased the mechanical related microstructures. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 121-130, 2018.

Abstract 288: Novel Mechanisms of Non-coding Genomic Regulation Identified in Cardiac Disease-in-a-dish Models

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) at gene loci that affect cardiovascular function, and while mechanisms in protein-coding loci are obvious, those in non-coding loci are difficult to determine. 9p21 is a recently identified locus associated with increased risk of coronary artery disease (CAD) and myocardial infarction. Associations have implicated SNPs in altering smooth muscle and endothelial cell properties but have not identified adverse effects in cardiomyocytes (CMs) despite enhanced disease risk. Using induced pluripotent stem cell-derived CMs from patients that are homozygous risk/risk (R/R) and non-risk/non-risk (N/N) for 9p21 SNPs and either CAD positive or negative, we assessed CM function when cultured on hydrogels capable of mimicking the fibrotic stiffening associated with disease post-heart attack, i.e. “heart attack-in-a-dish” stiffening from 11 kiloPascals (kPa) to 50 kPa. While all CMs independent of genotype and disease beat synchronously on soft matrices, R/R CMs cultured on dynamically stiffened hydrogels exhibited asynchronous contractions and had significantly lower correlation coefficients versus N/N CMs in the same conditions. Dynamic stiffening reduced connexin 43 expression and gap junction assembly in R/R CMs but not N/N CMs. To eliminate patient-to-patient variability, we created an isogenic line by deleting the 9p21 gene locus from a R/R patient using TALEN-mediated gene editing, i.e. R/R KO. Deletion of the 9p21 locus restored synchronous contractility and organized connexin 43 junctions. As a non-coding locus, 9p21 appears to repress connexin transcription, leading to the phenotypes we observe, but only when the niche is stiffened as in disease. These data are the first to demonstrate that disease-specific niche remodeling, e.g. a “heart attack-in-a-dish” model, can differentially affect CM function depending on SNPs within a non-coding locus.

Abstract 361: Creg1 Interacts With Sec8 to Promote Cell-cell Adhesion During Cardiomyogenesis

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a 24 kD glycoprotein essential for early embryonic development. Our immunofluorescence studies revealed that CREG1 is highly expressed at myocyte junctions in both embryonic and adult hearts. To explore it role in cardiomyogenesis, we employed gain- and loss-of-function analyses demonstrating that CREG1 is required for the differentiation of mouse embryonic stem (ES) cell into cohesive myocardium-like structures. Chimeric cultures of wild-type and CREG1 knockout ES cells expressing cardiac-specific reporters showed that the cardiomyogenic effect of CREG1 is cell autonomous. Furthermore, we identified a novel interaction between CREG1 and Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Mutations of the amino acid residues D141 and P142 to alanine in CREG1 abolished its binding to Sec8. To address the role of the CREG1-Sec8 interaction in cardiomyogenesis, we rescued CREG1 knockout ES cells with wild-type and Sec8-binding mutant CREG1 and showed that CREG1 binding to Sec8 promotes cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8 and N-cadherin all localize at cell-cell adhesion sites. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. Finally, shRNA-mediated knockdown of Sec8 leads to cardiomyogenic defects similar to CREG1 knockout. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis.

CREG1 Interacts with Sec8 to Promote Cardiomyogenic Differentiation and Cell-Cell Adhesion.

Understanding the regulation of cell-cell interactions during the formation of compact myocardial structures is important for achieving true cardiac regeneration through enhancing the integration of stem cell-derived cardiomyocytes into the recipient myocardium. In this study, we found that cellular repressor of E1A-stimulated genes 1 (CREG1) is highly expressed in both embryonic and adult hearts. Gain- and loss-of-function analyses demonstrated that CREG1 is required for differentiation of mouse embryonic stem (ES) cell into cardiomyocytes and the formation of cohesive myocardium-like structures in a cell-autonomous fashion. Furthermore, CREG1 directly interacts with Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Site-directed mutagenesis and rescue of CREG1 knockout ES cells showed that CREG1 binding to Sec8 is required for cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8, and N-cadherin colocalize at intercalated discs in vivo and are enriched at cell-cell junctions in cultured cardiomyocytes. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis. Stem Cells 2016;34:2648-2660.

Aberrant trafficking of a Leu89Pro connexin32 mutant associated with X-linked dominant Charcot–Marie–Tooth disease

Objective: To determine the functional abnormalities of the Leu89Pro mutation in connexin32 (CX32), which we have previously reported is present within an X-linked dominant Charcot–Marie–Tooth disease family. In this family, male patients were moderately to severely affected.Methods: We performed immunofluorescence to investigate whether the Leu89Pro CX32 protein was transported to the cell membrane in HeLa and Schwann cells. First, we constructed the eukaryotic express plasmids expressing CX32 (wild-type or Leu89Pro) and enhanced green fluorescent protein by the gene recombination technology. Then the recombinant plasmids were transiently transfected into communication-incompetent HeLa cells and human Schwann cells by the lipofectamine method. Later, we double-labeled cells for both CX32 and markers of the ER (calnexin) or the Golgi (58-kDa protein) at 24 h or 48 h. The images were collected using a Leica TCS SP5 II confocal microscope.Results: The mutant CX32 protein was localized in the endoplasmic reticulum and failed to reach the cell membrane to form gap junctions.Conclusion: Our results indicated that the Leu89Pro substitution in the second transmembrane domain of CX32 disrupts the trafficking of the protein, inhibiting the assembly of CX32 gap junctions, which in turn may result in peripheral neuropathy. This functional abnormality may explain the moderate to severe phenotype seen in Leu89Pro patients, and as such represents a promising therapeutic target in the treatment of this subset of CMTX patients.

LRP6 acts as a scaffold protein in cardiac gap junction assembly.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor in the canonical Wnt/β-catenin signalling. Here, we report the scaffold function of LRP6 in gap junction formation of cardiomyocytes. Cardiac LRP6 is spatially restricted to intercalated discs and binds to gap junction protein connexin 43 (Cx43). A deficiency in LRP6 disrupts Cx43 gap junction formation and thereby impairs the cell-to-cell coupling, which is independent of Wnt/β-catenin signalling. The defect in Cx43 gap junction resulting from LRP6 reduction is attributable to the defective traffic of de novo Cx43 proteins from the endoplasmic reticulum to the Golgi apparatus, leading to the lysosomal degradation of Cx43 proteins. Accordingly, the hearts of conditional cardiac-specific Lrp6-knockout mice consistently exhibit overt reduction of Cx43 gap junction plaques without any abnormality in Wnt signalling and are predisposed to lethal arrhythmias. These findings uncover a distinct role of LRP6 as a platform for intracellular protein trafficking.

Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer.

SUMMARYBrain metastasis represents a substantial source of morbidity and mortality in various cancers, and is characterized by high resistance to chemotherapy. Here we define the role of the most abundant cell type in the brain, the astrocyte, in promoting brain metastasis. Breast and lung cancer cells express protocadherin 7 (PCDH7) to favor the assembly of carcinoma-astrocyte gap junctions composed of connexin 43 (Cx43). Once engaged with the astrocyte gap-junctional network, brain metastatic cancer cells employ these channels to transfer the second messenger cGAMP to astrocytes, activating the STING pathway and production of inflammatory cytokines IFNα and TNFα. As paracrine signals, these factors activate the STAT1 and NF-κB pathways in brain metastatic cells, which support tumour growth and chemoresistance. The orally bioavailable modulators of gap junctions meclofenamate and tonabersat break this paracrine loop, and we provide proof-of-principle for the applicability of this therapeutic strategy to treat established brain metastasis.

CELLULAR SMALL TALK.

The article discusses cellular communication. Topics include the multiple modes of information exchange cells use, such as hormone signals, research of the gap junction assembly process in cells, which contain thousands of cell communication channels, by the author and his colleagues, a type of connexin protein called Cx43, and genetic evidence found in the 1990s that connexins can participate in disease, such as the developmental disease oculodentodigital dysplasia (ODDD).

Spatiotemporal Analysis of the Role that Protein Kinase A Phosphorylation Plays In Gap Junction Assembly

Gap junction (GJ) trafficking and intercellular communication are regulated through interactions of the GJ protein, connexin, with several kinases culminating in phosphorylation on specific serines or tyrosines in the connexin C‐terminus. Connexin43 (Cx43) is an important, ubiquitous and highly phosphorylated isoform present in many vertebrate organs. Activation of Protein Kinase A (PKA) causes increased trafficking of Cx43 to the plasma membrane hypothesized to occur by direct phosphorylation on S364/S365. Here, we imaged Cx43 tagged with a PKA phosphorylation FRET biosensor (Cx43‐AKAR3). A strong Cx43‐AKAR3 FRET ratio signal was found in the Golgi apparatus and not in the GJs. Phosphorylation in the Golgi increased after stimulation with forskolin and IBMX, potent stimulators of PKA activity. However, we found no overlap of the PKA FRET ratio signal with antibody labeling for phospho‐S365 before or after PKA stimulation. Inhibition of PKA with H89 decreased but does not abrogate the effect of forskolin/IBMX stimulation. However, treatment with this inhibitor after stimulation did not result in rapid dephosphorylation. Previously published work indicated that direct PKA phosphorylation of Cx43 is low, especially as compared with PKC or MAPK. Thus, PKA activation and localization was not consistent with direct S365 phosphorylation. Most likely, PKA phosphorylates another kinase that increases Cx43 trafficking to the plasma membrane and GJ assembly.Funding: GM072881 (GS), GM043154 & DK054441 (AN), GM055632 (PD) & GM103412 (NCMIR)

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size.

S35 Bmp9 And Bmp10 Mediate Connexin Expression In Endothelial Cells: Implications For Pah And Hht

Background Germ-line mutations in the bone morphogenetic protein type-II receptor, BMPR-II, underlie 80% of heritable pulmonary arterial hypertension (PAH) cases and approximately 25% of idiopathic PAH cases. PAH may arise due to endothelial dysfunction as mice with BMPR-II deficiency exhibit increased pulmonary vascular permeability. BMP9 is an endothelial quiescence factor and is thought to maintain the integrity of the endothelium. We previously reported that BMPR-II and ALK1 are the key receptors through which BMP9 inhibits the proliferation of human pulmonary artery endothelial cells (hPAECs). We hypothesised that BMPR-II deficiency impacts on endothelial cell connectivity and may contribute to endothelial dysfunction in PAH. Methods Human pulmonary artery endothelial cells were obtained from Lonza and blood outgrowth endothelial cells (BOECs) were isolated from peripheral blood of unaffected controls or PAH patients with identified BMPR-II mutations. Cells were transfected with siRNAs targeting BMPR-II followed by stimulation with BMP9. RNA was extracted and the expression of candidate genes determined by quantitative PCR. Further siRNA studies were performed for ALK1 and endoglin siRNAs. The promotion of gap junction assembly by BMP9 and BMP10 were assessed by immunofluorescence, Western blotting and functionally using parachute assays. Results Screening of candidate BMP9-induced junctional and structural proteins highlighted a subset of endothelial connexins that are BMP9 and BMP10-responsive and dependent on BMPR-II and ALK1. BMP9 and BMP10 increased the expression of the connexins, assessed by Western blotting and immunostaining. In addition, BMP9 and BMP10 significantly increased the transfer of calcein from labelled donor cells to unlabelled acceptor cells, indicating a promotion of endothelial cell connectivity. Conclusion In addition to their roles promoting endothelial quiescence, BMP9 and BMP10 directly promote endothelial cell connectivity by increasing connexin expression and assembly. The central contributions of BMPR-II and ALK1 to this process may implicate impaired endothelial connectivity as a pathological component of PAH and HHT.

Hemichannels in neurodegenerative diseases: is there a link to pathology?

Although originally considered a structural component of gap junctions, connexin hemichannels (HCs) are now recognized as functional entities capable of influencing metabolic gradients within the CNS, allowing direct communication between the intra- and extracellular milieus. Besides connexins, HCs can also be formed by pannexins, which are not capable of gap junction assembly. Both positive and negative effects have been attributed to HC activity in the context of neurodegenerative diseases. For example, HCs can exert neuroprotective effects by promoting the uptake of neurotoxic molecules, whereas chronic HC opening can disrupt molecular gradients leading to cellular dysfunction and death. The latter scenario has been suggested for multiple neurodegenerative disorders, including Alzheimer’s disease (AD) and more recently, lysosomal storage disorders, which are the focus of this perspective. Currently available evidence suggests a complex role for HCs in neurodegenerative disorders, which sets the stage for future studies to determine whether targeting HC action may improve disease outcomes.

Cardiac to cancer: Connecting connexins to clinical opportunity

Gap junctions and their connexin components are indispensable in mediating the cellular coordination required for tissue and organ homeostasis. The critical nature of their existence mandates a connection to disease while at the same time offering therapeutic potential. Therapeutic intervention may be offered through the pharmacological and molecular disruption of the pathways involved in connexin biosynthesis, gap junction assembly, stabilization, or degradation. Chemical inhibitors aimed at closing connexin channels, peptide mimetics corresponding to short connexin sequences, and gene therapy approaches have been incredibly useful molecular tools in deciphering the complexities associated with connexin biology. Recently, therapeutic potential in targeting connexins has evolved from basic research in cell-based models to clinical opportunity in the form of human trials. Clinical promise is particularly evident with regards to targeting connexin43 in the context of wound healing. The following review is aimed at highlighting novel advances where the pharmacological manipulation of connexin biology has proven beneficial in animals or humans.

Specific Cx43 phosphorylation events regulate gap junction turnover in vivo

Gap junctions, composed of proteins from the connexin gene family, are highly dynamic structures that are regulated by kinase-mediated signaling pathways and interactions with other proteins. Phosphorylation of Connexin43 (Cx43) at different sites controls gap junction assembly, gap junction size and gap junction turnover. Here we present a model describing how Akt, mitogen activated protein kinase (MAPK) and src kinase coordinate to regulate rapid turnover of gap junctions. Specifically, Akt phosphorylates Cx43 at S373 eliminating interaction with zona occludens-1 (ZO-1) allowing gap junctions to enlarge. Then MAPK and src phosphorylate Cx43 to initiate turnover. We integrate published data with new data to test and refine this model. Finally, we propose that differential coordination of kinase activation and Cx43 phosphorylation controls the specific routes of disassembly, e.g., annular junction formation or gap junctions can potentially "unzip" and be internalized/endocytosed into the cell that produced each connexin.

Gap junctions

In vertebrates and invertebrates, signaling among neurons is most commonly mediated by chemical synapses. At these synapses neurotransmitter released by presynaptic neurons is detected by receptors on the postsynaptic neurons, leading to an influx of ions through the receptors themselves or through channels activated by intracellular signaling downstream of the receptors. But neurons can communicate with each other in a more direct way, by passing signals composed of small molecules and ions through pores called gap junctions. Gap junctions that transmit electrical signals are called electrical synapses. Unlike most chemical synapses, electrical synapses interact through axon-to-axon or dendrite-to-dendrite contacts. Found throughout the nervous system, they are probably best known for linking the relatively few inhibitory, GABAergic, neurons into large, effective networks within vertebrate brains. They are particularly important early in development before the formation of most chemical synapses, but recent work shows gap junctions play important roles in the adult nervous system, too. Gap junctions are sometimes thought to be mere passageways between cells. But, as recent work shows, their properties can be complex and surprising. Gap junctions help generate, propagate, and regulate neural oscillations, can filter electrical signals, and can be modulated in a variety of ways. Here we discuss recent work highlighting the diversity and importance of gap junctions throughout the nervous system.

Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis

ObjectiveThis study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels.MethodsFrozen cartilage samples were used for immunofluorescence...

Context dependent reversion of tumor phenotype by connexin-43 expression in MDA-MB231 cells and MCF-7 cells: Role of β-catenin/connexin43 association

Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of β-catenin, α-catenin and ZO-2, it decreased nuclear levels of β-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, β-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, β-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering β-catenin away from nucleus.

The Canonical WNT2 Pathway and FSH Interact to Regulate Gap Junction Assembly in Mouse Granulosa Cells1

WNTs are extracellular signaling molecules that exert their actions through receptors of the frizzled (FZD) family. Previous work indicated that WNT2 regulates cell proliferation in mouse granulosa cells acting through CTNNB1 (beta-catenin), a key component in canonical WNT signaling. In other cells, WNT signaling has been shown to regulate expression of connexin43 (CX43), a gap junction protein, as well as gap junction assembly. Since previous work demonstrated that CX43 is also essential in ovarian follicle development, the objective of this study was to determine if WNT2 regulates CX43 expression and/or gap-junctional intercellular communication (GJIC) in granulosa cells. WNT2 knockdown via siRNA markedly reduced CX43 expression and GJIC. CX43 expression, the extent of CX43-containing gap junction membrane, and GJIC were also reduced by CTNNB1 transient knockdown. CTNNB1 is mainly localized to the membranes between granulosa cells but disappeared from this location after WNT2 knockdown. Furthermore, CTNNB1 knockdown interfered with the ability of follicle-stimulating hormone (FSH) to promote the mobilization of CX43 into gap junctions. We propose that the WNT2/CTNNB1 pathway regulates CX43 expression and GJIC in granulosa cells by modulating CTNNB1 stability and localization in adherens junctions, and that this is essential for FSH stimulation of GJIC.