Functional Gap Junctional Intercellular Communication Research Articles

TGF-β3 enhances cell-to-cell communication in chondrocytes via the ALK5/p-Smad3 axis

Gap junctional intercellular communication (GJIC) is indispensable for the maintenance of physiological balance in articular cartilage. Transforming growth factor-β3 (TGF-β3), an important growth factor of TGF-β superfamily, is well recognized to play a unique regulatory role in cartilage development and diseases. However, the role of TGF-β3 in GJIC in adult chondrocytes remains elusive. This work aims to investigate the effect of TGF-β3 on gap-junction mediated intercellular communication in chondrocytes. We first showed that TGF-β3 could enhance the synaptic connections between chondrocytes by scanning electron microscopy (SEM) and promote the cell-to-cell communication in living chondrocytes by scrape loading/dye transfer assay. We then confirmed that TGF-β3 enhanced cell-to-cell communication via up-regulation of connexin 43 (Cx43). We next found that TGF-β3-enhanced GJIC required the participation of TGF-beta type I receptor ALK5 and depended on the activation of p-Smad3 signalling. Finally, through inhibitor experiments of SB525334 and SIS3, we demonstrated that TGF-β3-induced functional GJIC in chondrocytes via the axis of ALK5/p-Smad3 signalling. Taking together, these results demonstrate a strong correlation between TGF-β3 and GJIC in chondrocytes, which provides a new perspective on the importance of TGF-β3 on cartilage physiology and pathobiology.

Gap Junction Protein, Connexin 43 is a Key Regulator of Metastasis in Syngeneic Ovarian Cancer Mouse Model

Ovarian cancer is the 7th most common cancer of women and the most lethal gynecological cancer that demands rigorous treatments involving increased radiation cycles and higher drug concentration that triggers toxicity and eventually mortality of patients. Understanding of the disease's progression warrants effective therapeutic inventions. Epithelial Ovarian Cancer cell lines ID8 derived from C57Bl/6 mice and ID8 transfected with Vascular Endothelial Growth Factor‐A (ID8‐VEGF) were used in vitro to generate a Syngeneic Ovarian Cancer Mouse Model. Vascular Endothelial Growth Factor‐A (VEGF‐A/VEGF) is the critical regulator of tumor angiogenesis that supports the transport of nutrients, hormones and O2 to the tumor cells and removal of waste products and CO2. It regulates vascular permeability, actin reorganization, cell migration, enhances tumor growth and progression, which may be associated with aberrant cellular metabolism due to altered gap junction intercellular communication (GJIC) contributing to the tumorigenesis. The transmembrane protein Connexin forms hexameric hemichannels known as connexons and docks with another connexon of adjacent cells to form a functional GJIC. Connexin 43 (Cx43) is one of the most ubiquitously expressed connexins and its C‐terminus interacts with various molecules to regulate many biological functions, including modulation of hemichannel activity, apoptotic signaling, cell cycle regulation and, cytoskeletal rearrangements. Our study aims to understand the effect of VEGF expression on the spatial expression of Cx43 and its nexus with other cellular factors contributing to consequent GJIC formation in the ovarian cancer cells as well as in the mouse model. Our findings suggest that upregulation of VEGF in ID8‐VEGF cell lines is associated with accelerated tumor formation in syngeneic mouse models. A series of biochemical and cellular studies revealed that Sodium Phenylbutyrate(4PBA) treatment induced differential upregulation of Cx43 trafficking to the cell surface, rearrangement of cytoskeletal proteins, cell migration, and the formation of GJIC in ID8 and ID8‐VEGF cells. 4PBA treatment on ID8‐VEGF cells significantly reduced the rate of tumor formation. These differential cellular activities denote that VEGF may directly or indirectly interact with cytoskeletal proteins that regulate Cx43 trafficking and its GJIC formation. Overall, The increased capacity of GJIC formation post‐4PBA treatment is associated with reduction in cell migration in vitro (ID8 and ID8‐VEGF) and tumor formation in vivo (ID8‐VEGF). Interactome studies combining RNASeq and IPA are designed to understand the nexus between several cellular pathways, including oxidative stress in the regulation of Cx43 in the context of VEGF, to dissect the cellular and molecular mechanism of metastasis.

PDGF-AA promotes gap junction intercellular communication in chondrocytes via the PI3K/Akt pathway

ABSTRACT Background Gap junction intercellular communication (GJIC) plays an important role in cell growth, development and homeostasis. Connexin 43 (Cx43) is an important half-channel protein responsible for gap junction formation. Platelet-derived growth factor AA (PDGF-AA) regulates the proliferation, migration, metabolism, apoptosis and cell cycle of chondrocytes. However, the role of PDGF-AA in gap junction intercellular communication in chondrocytes is not fully understood. In the current study, we performed experiments to explore the effect of PDGF-AA on GJIC and its underlying biomechanical mechanism. Methods qPCR was performed to determine the expression of PDGF, PDGFR and connexin family genes in chondrocytes and/or cartilage. A scrape loading/dye transfer assay was used to determine GJIC. Western blot analysis was applied to detect the expression of Cx43 and PI3K/Akt signaling pathway proteins. Immunofluorescence staining was utilized to examine protein distribution. Scanning electron microscopy was used to delineate the morphology of chondrocytes. Results Expression of PDGF-A mRNA was highest among the PDGF family in chondrocytes and cartilage tissues. PDGF-AA promoted functional GJIC formation in chondrocytes by upregulating the expression of Cx43. Enhanced functional GJIC formation in chondrocytes induced by PDGF-AA occurred through the activation of PI3K/Akt signaling and its nuclear accumulation. Conclusion For the first time, this study provides evidence demonstrating the role of PDGF-AA in cell-to-cell communication in chondrocytes through mediating Cx43 expression.

The Concept of "Cancer Stem Cells" in the Context of Classic Carcinogenesis Hypotheses and Experimental Findings.

In this Commentary, the operational definition of cancer stem cells or cancer initiating cells includes the ability of certain cells, found in a heterogeneous mixture of cells within a tumor, which are able to sustain growth of that tumor. However, that concept of cancer stem cells does not resolve the age-old controversy of two opposing hypotheses of the origin of the cancer, namely the stem cell hypothesis versus the de-differentiation or re-programming hypothesis. Moreover, this cancer stem concept has to take into account classic experimental observations, techniques, and concepts, such as the multi-stage, multi-mechanism process of carcinogenesis; roles of mutagenic, cytotoxic and epigenetic mechanisms; the important differences between errors of DNA repair and errors of DNA replication in forming mutations; biomarkers of known characteristics of normal adult organ-specific stem cells and of cancer stem cells; and the characteristics of epigenetic mechanisms involved in the carcinogenic process. In addition, vague and misleading terms, such as carcinogens, immortal and normal cells have to be clarified in the context of current scientific facts. The ultimate integration of all of these historic factors to provide a current understanding of the origin and characteristics of a cancer stem cell, which is required for a rational strategy for prevention and therapy for cancer, does not follow a linear path. Lastly, it will be speculated that there exists evidence of two distinct types of cancer stem cells, one that has its origin in an organ-specific adult stem cell that is ‘initiated’ in the stem cell stage, expressing the Oct4A gene and not expressing any connexin gene or having functional gap junctional intercellular communication (GJIC). The other cancer stem cell is derived from a stem cell that is initiated early after the Oct4A gene is suppressed and the connexin gene is expressed, which starts early differentiation, but it is blocked from terminal differentiation.

On the potential origin and characteristics of cancer stem cells.

The 'cancer stem cell' hypothesis has pointed to a specific target for new cancer therapies. The hypothesis is based on the observation that only the 'cancer stem cell' among the other heterogeneous cancer cells can sustain the growth of the cancer. The goal is to identify biomarkers of 'cancer stem cells' to distinguish them from the 'cancer non-stem cells' and normal adult tissue-specific stem cells. This analyst posits a hypothesis that, although all cancers originated from a single cell, there exist two types of 'cancer stem cells' either by the 'Stem Cell hypothesis' or from the 'De-differentiation hypothesis'. It is proposed that there exist two different 'cancer stem cells'. Some 'cancer stem cells' (a) lack the expression of connexins or gap junction genes and lack any form of gap junctional intercellular communication (GJIC) or (b) they have the expressed connexin-coded proteins for functional GJIC but are dysfunctional by some expressed oncogene. This is consistent with the Loewenstein hypothesis that a universal characteristic of cancer cells is they do not have growth control, nor terminally differentiate. This review speculates the normal organ-specific adult stem cell, that is 'initiated', is the origin of the 'cancer stem cells' with expressed Oct4A gene and no expressed connexin genes; whereas the other cancer stem cell has no expressed Oct4A genes but expressed connexin gene, whose coded protein is dysfunctional. Hence. both types of 'cancer stem cells' lack GJIC, for two different reasons, the selective therapies have to be different for these different cell types.

Transforming growth factor-\u03b21-induced N-cadherin drives cell\u2013cell communication through connexin43 in osteoblast lineage

Gap junction (GJ) has been indicated to have an intimate correlation with adhesion junction. However, the direct interaction between them partially remains elusive. In the current study, we aimed to elucidate the role of N-cadherin, one of the core components in adhesion junction, in mediating connexin 43, one of the functional constituents in gap junction, via transforming growth factor-β1(TGF-β1) induction in osteoblasts. We first elucidated the expressions of N-cadherin induced by TGF-β1 and also confirmed the upregulation of Cx43, and the enhancement of functional gap junctional intercellular communication (GJIC) triggered by TGF-β1 in both primary osteoblasts and MC3T3 cell line. Colocalization analysis and Co-IP experimentation showed that N-cadherin interacts with Cx43 at the site of cell–cell contact. Knockdown of N-cadherin by siRNA interference decreased the Cx43 expression and abolished the promoting effect of TGF-β1 on Cx43. Functional GJICs in living primary osteoblasts and MC3T3 cell line were also reduced. TGF-β1-induced increase in N-cadherin and Cx43 was via Smad3 activation, whereas knockdown of Smad3 signaling by using siRNA decreased the expressions of both N-cadherin and Cx43. Overall, these data indicate the direct interactions between N-cadherin and Cx43, and reveal the intervention of adhesion junction in functional gap junction in living osteoblasts.

CTGF facilitates cell‐cell communication in chondrocytes via PI3K/Akt signalling pathway

PurposesGap junction intercellular communication (GJIC) is essential for articular cartilage to respond appropriately to physical or biological stimuli and maintain homeostasis. Connective tissue growth factor (CTGF), identified as an endochondral ossification genetic factor, plays a vital role in cell proliferation, migration and adhesion. However, how CTGF regulates GJIC in chondrocytes is still unknown. This study aims to explore the effects of CTGF on GJIC in chondrocytes and its potential biomechanism.Materials and methodsqPCR was performed to determine the expression of gene profile in the CCN family in chondrocytes. After CTGF treatment, CCK‐8 assay and scratch assay were performed to explore cell proliferation and migration. A scrape loading/dye transfer assay was adopted to visualize GJIC in living chondrocytes. Western blot analysis was done to detect the expression of Cx43 and PI3K/Akt signalling. Immunofluorescence staining was used to show protein distribution. siRNA targeting CTGF was used to detect the influence on cell‐cell communication.ResultsThe CTGF (CCN2) was shown to be the highest expressed member of the CCN family in chondrocytes. CTGF facilitated functional gap junction intercellular communication in chondrocytes through up‐regulation of Cx43 expressions. CTGF activated PI3K/Akt signalling to promote Akt phosphorylation and translocation. Suppressing CTGF also reduced the expression of Cx43. The inhibition of PI3K/Akt signalling decreased the expressions of Cx43 and thus impaired gap junction intercellular communication enhanced by CTGF.ConclusionsFor the first time, we provide evidence to show CTGF facilitates cell communication in chondrocytes via PI3K/Akt signalling pathway.

Abstract 505: Adenovirus Targets Connexin43 Gap Junction Expression and Function During Infection

Introduction: Myocarditis leads to pathologic remodeling of the myocardium and arrhythmia. A leading etiological agent of viral myocarditis is human Adenovirus type 5 (Ad5). Individuals affected are at a higher risk of arrhythmogenesis which can lead to sudden cardiac death. Gap junctions, comprised of connexin proteins, provide direct intercellular communication between cardiomyocytes to facilitate electrical coupling. Connexin43 (Cx43) is the predominant connexin expressed in the working myocardium and is tightly regulated by post-translational modifications. Gap junctions are also known to propagate the innate antiviral immune response but the impact of Ad5 infection on Cx43 expression and function is essentially unexplored. Hypothesis: During Ad5 infection Cx43 gap junctions are directly targeted to limit intercellular communication thus precipitating an arrhythmogenic substrate. Methods: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), keratinocytes (HaCaT), and lentivirus-transduced HaCaTs stably overexpressing Cx43 were infected with Ad5. Biochemical fractionation and pulse chase assays were implemented to determine gap junction and Cx43 stability. Cx43 protein levels and phosphorylation status were determined by western blotting and immunoprecipitation experiments. Confocal immunofluorescence microscopy was employed determine localization and functional of Cx43 during Ad5 infection. Results: Cx43 expression is targeted early in infection with remaining protein localized at the cell-cell boarder during infection. Phosphorylation of Cx43-Ser368 was detected during early Ad5 infection implicating reduced gap junction conductance. In contrast phosphorylation of Cx43-Ser262 is reduced during Ad5 infection with a concomitant increase in protein half-life despite losses in functional gap junction intercellular communication. Conclusion: Cx43 gap junction structures are stabilized during Ad5 infection but directly targeted by specific phosphorylation events to effect channel closure. Insight provided into mechanisms by which Ad5 manipulates Cx43 regulation will provide novel therapeutic avenues to target gap junction function in a broad spectrum of heart disease states.

Gadolinium Chloride Restores the Function of the Gap Junctional Intercellular Communication between Hepatocytes in a Liver Injury.

Background: Gadolinium chloride (GdCl3) has been reported to attenuate liver injury caused by a variety of toxicants. Gap junctional intercellular communication (GJIC) is thought to be essential in controlling liver homeostasis and pathology. Here we evaluate the effects of GdCl3 on functional GJIC and connexin expression in mouse models and primary hepatocytes. Methods: Mice were administered GdCl3 intraperitoneally the day before a carbon tetrachloride (CCl4) injection or bile duct ligation (BDL) operation. Primary hepatocytes were treated with CCl4 or lipopolysaccharides (LPS), with or without GdCl3. A scrape loading/dye transfer assay was performed to assess the GJIC function. The expression of connexins was examined by real-time reverse transcription polymerase chain reaction (RT-PCR), western blot and immunofluorescent staining. Results: CCl4 treatment or the BDL operation led to the dysfunction of GJIC and a down-regulation of Cx32 and Cx26 in injured liver. GdCl3 administration restored GJIC function between hepatocytes by facilitating the transfer of fluorescent dye from one cell into adjacent cells via GJIC, and markedly prevented the decrease of Cx32 and Cx26 in injured liver. In primary hepatocytes, CCl4 or LPS treatment induced an obvious decline of Cx32 and Cx26, whereas GdCl3 pretreatment prevented the down-regulation of connexins. In vivo GdCl3 protected hepatocytes and attenuated the liver inflammation and fibrosis in liver injury mouse models. Conclusion: GdCl3 administration protects functional GJIC between hepatocytes, and prevents the decrease of connexin proteins at mRNA and protein levels during liver injury, leading to the alleviation of chronic liver injury.

Cancer Prevention and Therapy of Two Types of Gap Junctional Intercellular Communication⁻Deficient "Cancer Stem Cell".

Early observations showed a lack of growth control and terminal differentiation with a lack of gap junctional intercellular communication (GJIC). Subsequent observations showed that epigenetic tumor promoters and activated oncogenes, which block gap junction function, provide insights into the multi-stage, multi-mechanism carcinogenic process. With the isolation of embryonic induced pluri-potent stem cells and organ-specific adult stem cells, gap junctions were linked to early development. While tumors and tumor cell lines are a heterogeneous mixture of “cancer stem cells” and “cancer non-stem cells”, the cancer stem cells seem to be of two types, namely, they express (a) no connexin genes or (b) connexin genes, but do not have functional GJIC. These observations suggest that these “cancer stem cells” originate from normal adult stem cells or from the de-differentiation or re-programming of somatic differentiated cells. This “Concept Paper” provides a hypothesis that “cancer stem cells” either originate from (a) organ-specific adult stem cells before the expression of the connexin genes or (b) organ-specific adult stem cells that just express gap junction genes but that the connexin proteins are rendered dysfunctional by activated oncogenes. Therefore, cancer prevention and therapeutic strategies must account for these two different types of “cancer stem cell”.

TGF-β1 promotes gap junctions formation in chondrocytes via Smad3/Smad4 signalling.

ObjectivesConnexin‐mediated functional gap junction intercellular communication (GJIC) has a vital role in development, homeostasis and pathology. Transforming growth factor‐β1 (TGF‐β1), as one of the most vital factors in chondrocytes, promotes cartilage precursor cell differentiation and chondrocyte proliferation, migration and metabolism. However, how TGF‐β1 mediates GJIC in chondrocytes remains unclear. This study aims to determine the influence of TGF‐β1 on GJIC in mouse chondrocytes and its underlying mechanism.MethodsqPCR and mRNA microarray were used to verify the expression of genes in the TGF‐β and connexin families in cartilage and chondrocytes. A scrape loading/dye transfer assay was performed to explore GJIC. Western blot analysis was used to detect connexin43 (Cx43) and Smad signalling components. Immunofluorescence staining was performed to characterize protein distribution.ResultsThe TGF‐β1 mRNA was the highest expressed member of the TGFβ super family in cartilage. TGF‐β1 promoted functional GJIC through increased expression of Cx43. TGF‐β1‐mediated GJIC required the participation of TGF‐β type I receptor. TGF‐β1 activated Smad3 and Smad4 signalling to facilitate their nuclear translocation. The Smad3 and Smad4 signalling proteins bound to the promoter of Gja1 and thus initiated Cx43 gene expression.ConclusionsFor the first time, these results revealed a vital role of TGF‐β1 in cell‐cell communication in chondrocytes via gap junction formation. We describe the regulatory mechanism, the involvement of TGF‐β type I receptor and the nuclear translocation of Smad3/4.

Gap Junction Intercellular Communication Positively Regulates Cisplatin Toxicity by Inducing DNA Damage through Bystander Signaling.

The radiation-induced bystander effect (RIBE) can increase cellular toxicity in a gap junction dependent manner in unirradiated bystander cells. Recent reports have suggested that cisplatin toxicity can also be mediated by functional gap junction intercellular communication (GJIC). In this study using lung and ovarian cancer cell lines, we showed that cisplatin cytotoxicity is mediated by cellular density. This effect is ablated when GJA1 or Connexin 43 (Cx43) is targeted, a gap junction gene and protein, respectively, leading to cisplatin resistance but only at high or gap junction forming density. We also observed that the cisplatin-mediated bystander effect was elicited as DNA Double Strand Breaks (DSBs) with positive H2AX Ser139 phosphorylation (γH2AX) formation, an indicator of DNA DSBs. These DSBs are not observed when gap junction formation is prevented. We next showed that cisplatin is not the “death” signal traversing the gap junctions by utilizing the cisplatin-GG intrastrand adduct specific antibody. Finally, we also showed that cells deficient in the structure-specific DNA endonuclease ERCC1-ERCC4 (ERCC1-XPF), an important mediator of cisplatin resistance, further sensitized when treated with cisplatin in the presence of gap junction forming density. Taken together, these results demonstrate the positive effect of GJIC on increasing cisplatin cytotoxicity.

Abstract 4023: Gap junctions contribute to anchorage-independent clustering of breast cancer cells

Abstract Purpose: Cancer cell cluster formation is a key process involved during primary tumor growth, metastatic cells dissemination and secondary tumor evolution. We previously reported that cell-cell adhesion proteins such as E-Cadherin and desmosomal proteins are involved in cell aggregation independently of cell migration or matrix adhesion (Saias et al. 2015). Here we investigate the involvement of gap junction intercellular communication (GJIC) during anchorage-independent clustering of MCF7 breast adenocarcinoma cells. Experimental Design: Live cell imaging and image processing was used to evaluate MCF7 clustering in the presence of pharmacological inhibitors and to perform a LOPAC® small bioactive compound library screening. Flow cytometry was performed to monitor calcein transfer assay and to evaluate GJIC. Results: We first demonstrate that functional GJIC are established in the early phase of aggregation. We next show that pharmacological inhibition of GJIC using Tonabersat and Meclofenamate both reduces calcein transfer and delays clustering of MCF7 cells. We found that treatment with Latrunculin A, an actin cytoskeleton-disrupting agent, inhibits MCF7 clustering and impairs calcein transfer. In a screen for inhibitors of cell aggregation using a small-compound chemical library we identified Brefeldin A, an inhibitor of vesicular trafficking, and showed that it also impairs calcein transfer. Conclusion: Our results demonstrate that GJIC are involved at the earliest stages of the anchorage-independent clustering process. They shed new light on the regulatory mechanisms that could modulate the formation of clusters of circulating tumor cells.

Gap junctions contribute to anchorage-independent clustering of breast cancer cells

BackgroundCancer cell aggregation is a key process involved in the formation of clusters of circulating tumor cells. We previously reported that cell-cell adhesion proteins, such as E-cadherin, and desmosomal proteins are involved in cell aggregation to form clusters independently of cell migration or matrix adhesion. Here, we investigated the involvement of gap junction intercellular communication (GJIC) during anchorage-independent clustering of MCF7 breast adenocarcinoma cells.MethodsWe used live cell image acquisition and analysis to monitor the kinetics of MCF7 cell clustering in the presence/absence of GJIC pharmacological inhibitors and to screen a LOPAC® bioactive compound library. We also used a calcein transfer assay and flow cytometry to evaluate GJIC involvement in cancer cell clustering.ResultsWe first demonstrated that functional GJIC are established in the early phase of cancer cell aggregation. We then showed that pharmacological inhibition of GJIC using tonabersat and meclofenamate delayed MCF7 cell clustering and reduced calcein transfer. We also found that brefeldin A, an inhibitor of vesicular trafficking, which we identified by screening a small compound library, and latrunculin A, an actin cytoskeleton-disrupting agent, both impaired MCF7 cell clustering and calcein transfer.ConclusionsOur results demonstrate that GJIC are involved from the earliest stages of anchorage-independent cancer cell aggregation. They also give insights into the regulatory mechanisms that could modulate the formation of clusters of circulating tumor cells.

Cancer-Associated Fibroblasts Accelerate Malignant Progression of Non-Small Cell Lung Cancer via Connexin 43-Formed Unidirectional Gap Junctional Intercellular Communication

Background/Aims: Gap junctions, which are assembled by connexins, can directly connect the cytoplasm of adjacent cells and enable gap junctional intercellular communication (GJIC) as well as metabolic coupling between neighboring cells. Here, we investigated the role of connexin 43 (Cx43) and its derived GJIC in the interplay between non-small cell lung cancer (NSCLC) cells and cancer-associated fibroblasts (CAFs). Methods: CAFs and NSCLC cells were co-cultured with direct contact and separated using flow cytometry. Glucose uptake, lactate production, and the expression and activity of PKM-2 and LDH-A in sorted CAFs were measured by a colorimetric assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Meanwhile, E-cadherin and N-cadherin expression and the migration and invasion of sorted NSCLC cells were detected by western blotting, wound width, and Transwell assays. Pyruvate, acetyl-CoA, and citric acid levels, ATP levels, and LDH-B and α-KG activity in sorted NSCLC cells were determined by a colorimetric or fluorometric assay and ELISA, respectively. Functional GJIC between cells and the subcellular location of connexins were detected by a “Parachute” assay and immunofluorescence. Levels of α-SMA, Cx43, and LDH-B in tissue from patients with NSCLC were determined by immunohistochemistry. Results: Cx43 accumulated in the plasma membrane, which favored the assembly of asymmetric unidirectional GJIC from CAFs to NSCLC cells. CAFs underwent increased aerobic glycolysis and promoted the epithelial-mesenchymal transition, migration, and invasion of NSCLC cells. In contrast, NSCLC cells experienced enhanced oxidative phosphorylation upon CAF stimulation, with an increase in ATP generation and thereby activation of the PI3K/Akt and MAPK/ERK pathways. Metabolic coupling between CAFs and NSCLC cells was under the strict control of Cx43-formed unidirectional GJIC. Patients with high tri-expression of α-SMA, Cx43, and LDH-B had the shortest overall survival and relapse-free survival compared with those with individual overexpression or high bi-expression. Conclusion: Cx43-formed unidirectional GJIC plays a critical role in mediating close metabolic cooperation between CAFs and NSCLC cells to support the malignant progression of NSCLC.

Study of Gap Junctions in Human Embryonic Stem Cells.

Gap junctional intercellular communication (GJIC) has been described in different cell types including stem cells and has been involved in different biological events. GJIC is required for mouse embryonic stem cell maintenance and proliferation and various studies suggest that functional GJIC is a common characteristic of human embryonic stem cells (hESC) maintained in different culture conditions. This chapter introduces methods to study gap junctions in hESC, from expression of gap junction proteins to functional study of GJIC in hESC proliferation, apoptosis, colony growth, and pluripotency.

Human cytomegalovirus immediate early proteins promote degradation of connexin 43 and disrupt gap junction communication: implications for a role in gliomagenesis

A lack of gap junctional intercellular communication (GJIC) is common in cancer. Many oncogenic viruses have been shown to downregulate the junctional protein connexin 43 (Cx43) and reduce GJIC. Human cytomegalovirus (HCMV) is a ubiquitous, species-specific betaherpesvirus that establishes life-long latency after primary infection. It encodes two viral gene products, immediate early (IE) proteins IE1 and IE2, which are crucial in viral replication and pathogenesis of many diseases. Emerging evidence demonstrates that HCMV DNA and proteins are highly prevalent in glioblastoma multiforme (GBM) and in other tumors, but HCMV's role in tumorigenesis remains obscure. In the present study, we examined the effects of HCMV infection on Cx43 expression and GJIC as well as the viral mechanism mediating the effects in human GBM cells and tissue samples. We found that HCMV downregulated Cx43 protein, resulting in disruption of functional GJIC as assayed by fluorescent dye transfer assay. We show that both HCMV-IE72 and IE86 mediate downregulation of Cx43 by silencing RNA targeting either IE72 or IE86 coupled with ganciclovir. This finding was further validated by transfection with expression vectors encoding IE72 or IE86, and we show that viral-mediated Cx43 depletion involved proteasomal degradation. Importantly, we also observed that the Cx43 protein levels and IE staining correlated inversely in 10 human GBM tissue specimens. Thus, HCMV regulates Cx43 expression and GJIC, which may contribute to gliomagenesis.

Abstract 519: Attenuation of mammary tumor growth by gap junction enhancer.

Abstract Normal cells have functional gap junctional intercellular communication (GJIC), while cancer cells exhibit many defects in cellular communication that contribute to the loss of tissue homeostasis. It is believed that restoring GJIC is linked to drug sensitivity and reduction of tumorigenicity. Previous studies show that GJIC and neoplastic cellular growth are inversely related and small molecule targeting gap junction can alter tumor growth. A dose of 25 mg/kg gap junction enhancer (PQ) can cause a 77% decrease of xenograft T47D breast tumors. In addition these small molecules can induce a decrease of tumor growth by 110%, 129%, and 73% in the pre, early, and late stages of tumor development of the transgenic mouse strain, FVB/N-Tg(MMTV-PyVT)634Mul/J. Western blot analysis shows that the gap junction protein, Cx43, was highly expressed in the PQ-treated tumors compared to the controls. Data suggests that enhancement of GJIC attenuates tumor growth; specifically Cx43 plays a significant role in tumorigenesis. Establishment of a continuous line of mammary tumor cells from the malignant neoplasm of a female transgenic mouse demonstrates that PQs can cause an increase of Cx43 expression and subsequently trigger a decrease of cell viability. Overall, targeting gap junction by small molecule PQ has shed light to the dynamic of connexins and gap junctions in mammary tumors. Citation Format: Stephanie N. Shishido, Thu A. Nguyen. Attenuation of mammary tumor growth by gap junction enhancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 519. doi:10.1158/1538-7445.AM2013-519

Specificity in the participation of connexin proteins in flow-induced endothelial gap junction communication

Endothelial cell (EC) dysfunction and atherosclerotic plaque formation coincide with human circulatory regions where blood flow is altered (disturbed). In areas of undisturbed uniform blood flow, including the majority of the vasculature, the vessel wall is relatively atherosclerotic lesion-resistant with normal endothelium. The molecular mechanisms of blood flow regulation of EC function and atherogenesis are unclear. We hypothesize that EC dysfunction potentiating atherosclerosis is related to disturbed flow (DF)-induced EC gap junctional intercellular communication (GJIC) changes via the gap junction connexin (Cx) 37, 40, and 43 proteins, which are involved in EC proliferation and vasoactivity that are known to be altered in atherosclerosis. We investigated human EC GJIC using an in vitro model of the hemodynamic features found in atherosclerotic-prone DF regions in vivo. Using dye transfer assays, Cx-specific mimetic peptide inhibitors, proliferation assays, and immunocytochemistry, we correlated functional GJIC via gap junction channels formed by hemichannels composed of the two most abundant endothelial Cx-Cx40 and Cx43-to EC proliferation and expression of vasoactive endothelial-type nitric oxide synthase (eNOS). We found that, in uniform flow conditions, substantial GJIC was conducted through gap junctions containing Cx40 hemichannels and correlated to a nonproliferative EC phenotype and membrane localization of eNOS, similar to physiological conditions. In DF, GJIC was largely attained through Cx43 hemichannel-containing gap junctions, EC phenotype was proliferative (attributed to loss of contact inhibition), and intracellular eNOS was more abundant than membrane eNOS, typical of atherosclerotic sites in vivo. This is the first in vitro study to demonstrate local hemodynamically defined Cx protein specificity in human EC GJIC with a potential role in endothelial dysfunction characteristic of early atherosclerosis.

Intrinsic properties of tumour cells have a key impact on the bystander effect mediated by genetically engineered mesenchymal stromal cells

Engineered mesenchymal stromal cells (MSC) have been used in many preclinical studies of gene directed enzyme/prodrug therapy. We aimed to compare the efficacy of two most frequently used systems, as well as evaluate the extent of a bystander effect mediated by therapeutic MSC towards cell lines derived from different tumours. Two approaches were compared: (i) herpes simplex virus thymidine kinase (TK)/ganciclovir (GCV) and (ii) yeast cytosine deaminase fused with uracil phosphoribosyltransferase (CD::UPRT)/5-fluorocytosine (5-FC). The cytotoxic effect mediated by therapeutic MSC was evaluated in direct co-culture by a fluorimetric assay. The expression profile of tumour cells was analyzed by a quantitative polymerase chain reaction, and the ability of gap-junctional intercellular communication (GJIC) was evaluated by a dye transfer assay. Both systems were effective only on glioblastoma cells (8-MG-BA). The CD::UPRT-MSC/5-FC system showed efficiency on melanoma A375 cells. We decreased the sensitivity of 8-MG-BA cells and A375 cells to the CD::UPRT-MSC/5-FC system by pharmacological inhibition of thymidylate synthase, and we achieved a similar result in A375 cells by inhibition of thymidine phosphorylase. Although we demonstrated functional GJIC in A375 cells, TK-MSC were ineffective in mediating the bystander effect similarly to HeLa cells, which were also relatively resistant to CD::UPRT-MSC/5-FC treatment. TK-MSC/GCV treatment had a strong cytotoxic effect on MDA-MB-231 cells (breast carcinoma), whereas CD::UPRT-MSC/5-FC treatment failed as a result of overexpression of the gene for ABCC11. Transfection of the MDA-MB-231 cell line with small interference RNA specific to ABCC11 led to a significantly increased sensitivity to the CD::UPRT-MSC/5-FC approach. GJIC, expression of enzymes involved in drug metabolism and ABC transporters correlate with the response of tumour cells to treatment by MSC-expressing prodrug-converting genes.