Connexin 43 confers chemoresistance through activating PI3K.

Circumventing chemoresistance is crucial for effectively treating cancer including glioblastoma, a lethal brain cancer. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to chemotherapy; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we report that Cx43, but not other connexins, is highly expressed in a subpopulation of glioblastoma and Cx43 mRNA levels strongly correlate with poor prognosis and chemoresistance in this population, making Cx43 the prime therapeutic target among all connexins. Depleting Cx43 or treating cells with αCT1-a Cx43 peptide inhibitor that sensitizes glioblastoma to the chemotherapy temozolomide-inactivates phosphatidylinositol-3 kinase (PI3K), whereas overexpression of Cx43 activates this signaling. Moreover, αCT1-induced chemo-sensitization is counteracted by a PI3K active mutant. Further research reveals that αCT1 inactivates PI3K without blocking the release of PI3K-activating molecules from membrane channels and that Cx43 selectively binds to the PI3K catalytic subunit ß (PIK3CB, also called PI3Kß or p110ß), suggesting that Cx43 activates PIK3CB/p110ß independent of its channel functions. To explore the therapeutic potential of simultaneously targeting Cx43 and PIK3CB/p110ß, αCT1 is combined with TGX-221 or GSK2636771, two PIK3CB/p110ß-selective inhibitors. These two different treatments synergistically inactivate PI3K and sensitize glioblastoma cells to temozolomide in vitro and in vivo. Our study has revealed novel mechanistic insights into Cx43/PI3K-mediated temozolomide resistance in glioblastoma and demonstrated that targeting Cx43 and PIK3CB/p110ß together is an effective therapeutic approach for overcoming chemoresistance.

Novel Protocols for Scalable Production of High Quality Purified Small Extracellular Vesicles from Bovine Milk.

Extracellular Vesicles (EVs) are cell-secreted nanovesicles that have unique potential for encapsulating and targeting "difficult-to-drug" therapeutic cargos. Milk provides an enriched source of EVs, and of particular interest to the drug delivery field, small EVs. Small EVs are distinguished from large EVs by membrane components, biogenesis mechanism and downstream functionality - in particular, small EVs are primarily composed of exosomes, which show high stability in vivo and naturally function in the targeted delivery of biological materials to cells. Moreover, bovine milk is abundantly produced by the dairy industry, widely consumed, and generally well tolerated by humans. Importantly, there is evidence that milk exosomes and small EVs are efficiently taken up into the circulation from the gut, providing the opportunity for their use in administration of therapeutics such as microRNAs or peptides not typically available via an oral route. Unfortunately, present methods for isolation do not efficiently separate EVs from milk proteins, resulting in contamination that is not desirable in a clinical-grade therapeutic. Herein, we present novel EV purification methods focused on optimized timing and levels of temperature and divalent cation chelation. Incorporation of these solubilization steps into centrifugation- and tangential flow filtration-based methods provide large amounts of purified small EVs at ultra-dense concentrations, which are substantially free from contaminating milk proteins. Remarkably, these ultra-dense isolates equal 10 to 15% of the starting volume of milk indicating a prodigious rate of small EV production by mammary glands. Our approach enables gentle, scalable production of ultrastructurally and functionally intact small EVs from milk, providing a path to their industrial scale purification for oral delivery of therapeutic biologics and small drugs.

The adhesion function of the sodium channel beta subunit (ß1) contributes to cardiac action potential propagation.

Computational modeling indicates that cardiac conduction may involve ephaptic coupling - intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that ß1(SCN1B) -mediated adhesion scaffolds trans-activating NaV1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential ß1 localization at the perinexus, where it co-locates with NaV1.5. Smart patch clamp (SPC) indicated greater sodium current density (INa) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, ßadp1, potently and selectively inhibited ß1-mediated adhesion, in electric cell-substrate impedance sensing studies. ßadp1 significantly widened perinexi in guinea pig ventricles, and selectively reduced perinexal INa, but not whole cell INa, in myocyte monolayers. In optical mapping studies, ßadp1 precipitated arrhythmogenic conduction slowing. In summary, ß1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies.

Connexin 43 Inhibition Sensitizes Chemoresistant Glioblastoma Cells to Temozolomide.

Resistance of glioblastoma (GBM) to the front-line chemotherapeutic agent temozolomide (TMZ) continues to challenge GBM treatment efforts. The repair of TMZ-induced DNA damage by O-6-methylguanine-DNA methyltransferase (MGMT) confers one mechanism of TMZ resistance. Paradoxically, MGMT-deficient GBM patients survive longer despite still developing resistance to TMZ. Recent studies indicate that the gap junction protein connexin 43 (Cx43) renders GBM cells resistant to TMZ through its carboxyl terminus (CT). In this study, we report insights into how Cx43 promotes TMZ resistance. Cx43 levels were inversely correlated with TMZ sensitivity of GBM cells, including GBM stem cells. Moreover, Cx43 levels inversely correlated with patient survival, including as observed in MGMT-deficient GBM patients. Addition of the C-terminal peptide mimetic αCT1, a selective inhibitor of Cx43 channels, sensitized human MGMT-deficient and TMZ-resistant GBM cells to TMZ treatment. Moreover, combining αCT1 with TMZ-blocked AKT/mTOR signaling, induced autophagy and apoptosis in TMZ-resistant GBM cells. Our findings suggest that Cx43 may offer a biomarker to predict the survival of patients with MGMT-independent TMZ resistance and that combining a Cx43 inhibitor with TMZ could enhance therapeutic responses in GBM, and perhaps other TMZ-resistant cancers.

Cx43 associates with Na(v)1.5 in the cardiomyocyte perinexus.

Gap junctions (GJs) are aggregates of channels that provide for direct cytoplasmic connection between cells. Importantly, this connection is thought responsible for cell-to-cell transfer of the cardiac action potential. The GJ channels of ventricular myocytes are composed of connexin43 (Cx43). Interaction of Cx43 with zonula occludens-1 (ZO-1) is localized not only at the GJ plaque, but also to the region surrounding the GJ, the perinexus. Cx43 in the perinexus is not detectable by immunofluorescence, yet localization of Cx43/ZO-1 interaction to this region indicated the presence of Cx43. Therefore, we hypothesized that Cx43 occurs in the perinexus at a lower concentration per unit membrane than in the GJ itself, making it difficult to visualize. To overcome this, the Duolink protein-protein interaction assay was used to detect Cx43. Duolink labeling of cardiomyocytes localized Cx43 to the perinexus. Quantification demonstrated that signal in the perinexus was lower than in the GJ but significantly higher than in nonjunctional regions. Additionally, Duolink of Triton X-100-extracted cultures suggested that perinexal Cx43 is nonjunctional. Importantly, the voltage gated sodium channel Na(v)1.5, which is responsible for initiation of the action potential, was found to interact with perinexal Cx43 but not with ZO-1. This work provides a detailed characterization of the structure of the perinexus at the GJ edge and indicates that one of its potential functions in the heart may be in facilitating conduction of action potential.

Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1.

Connexin 43 (Cx43) is a gap junction (GJ) protein widely expressed in mammalian tissues that mediates cell-to-cell coupling. Intercellular channels comprising GJ aggregates form from docking of paired connexons, with one each contributed by apposing cells. Zonula occludens-1 (ZO-1) binds the carboxy terminus of Cx43, and we have previously shown that inhibition of the Cx43/ZO-1 interaction increases GJ size by 48 h. Here we demonstrated that increases in GJ aggregation occur within 2 h (∼Cx43 half-life) following disruption of Cx43/ZO-1. Immunoprecipitation and Duolink protein-protein interaction assays indicated that inhibition targets ZO-1 binding with Cx43 in GJs as well as connexons in an adjacent domain that we term the "perinexus." Consistent with GJ size increases being matched by decreases in connexons, inhibition of Cx43/ZO-1 reduced the extent of perinexal interaction, increased the proportion of connexons docked in GJs relative to undocked connexons in the plasma membrane, and increased GJ intercellular communication while concomitantly decreasing hemichannel-mediated membrane permeance in contacting, but not noncontacting, cells. ZO-1 small interfering RNA and overexpression experiments verified that loss and gain of ZO-1 function govern the transition of connexons into GJs. It is concluded that ZO-1 regulates the rate of undocked connexon aggregation into GJs, enabling dynamic partitioning of Cx43 channel function between junctional and proximal nonjunctional domains of plasma membrane.

ZO-1 determines adherens and gap junction localization at intercalated disks.

The disruption of the spatial order of electromechanical junctions at myocyte-intercalated disks (ICDs) is a poorly understood characteristic of many cardiac disease states. Here, in vitro and in vivo evidence is provided that zonula occludens-1 (ZO-1) regulates the organization of gap junctions (GJs) and adherens junctions (AJs) at ICDs. We investigated the contribution of ZO-1 to cell-cell junction localization by expressing a dominant-negative ZO-1 construct (DN-ZO-1) in rat ventricular myocytes (VMs). The expression of DN-ZO-1 in cultured neonatal VMs for 72 h reduced the interaction of ZO-1 and N-cadherin, as assayed by colocalization and coimmunoprecipitation, prompting cytoplasmic internalization of AJ and GJ proteins. DN-ZO-1 expression in adult VMs in vivo also reduced N-cadherin colocalization with ZO-1, a phenomenon not observed when the connexin-43 (Cx43)-ZO-1 interaction was disrupted using a mimetic of the ZO-1-binding ligand from Cx43. DN-ZO-1-infected VMs demonstrated large GJs at the ICD periphery and showed a loss of focal ZO-1 concentrations along plaque edges facing the disk interior. Additionally, there was breakdown of the characteristic ICD pattern of small interior and large peripheral GJs. Continuous DN-ZO-1 expression in VMs over postnatal development reduced ICD-associated Cx43 GJs and increased lateralized and cytoplasmic Cx43. We conclude that ZO-1 regulation of GJ localization is via an association with the N-cadherin multiprotein complex and that this is a key determinant of stable localization of both AJs and GJs at the ICD.

Cardiac expression patterns of endothelin-converting enzyme (ECE): implications for conduction system development.

The spatiotemporal distribution of the endothelin-converting enzyme (ECE) protein in the embryonic chick heart and the association of this polypeptide with the developing cardiac conduction system is described here for the first time. Further, we show how cardiac hemodynamic load directly affects ECE level and distribution. Endothelin (ET) is a cytokine involved in the inductive recruitment of Purkinje fibers. ET is produced by proteolytic cleavage of Big-ET by ECE. We generated an antibody against chick ECE recognizing a single band at approximately 70 kD to correlate the cardiac expression of this protein with that reported previously for its mRNA. ECE protein expression was more widespread compared to its mRNA, being present in endothelial cells, mesenchymal cells, and myocytes, and particularly enriched in the trabeculae and nascent ventricular conduction system. The myocardial expression was significantly modified under experimentally altered hemodynamic loading. In vivo, ET receptor blockade with bosentan delayed activation sequence maturation. These data support a role for ECE in avian cardiac conduction system differentiation and maturation.

The unstoppable connexin43 carboxyl-terminus: new roles in gap junction organization and wound healing.

Intercellular connectivity mediated by gap junctions (GJs) composed of connexin43 (Cx43) is critical to the function of excitable tissues such as the heart and brain. Disruptions to Cx43 GJ organization are thought to be a factor in cardiac arrhythmias and are also implicated in epilepsy. This article is based on a presentation to the 4th Larry and Horti Fairberg Workshop on Interactive and Integrative Cardiology and summarizes the work of Gourdie and his lab on Cx43 GJs in the heart. Background and perspective of recently published studies on the function of Cx43-interacting protein zonula occludens-(ZO)-1 in determining the organization of GJ plaques are provided. In addition how a peptide containing a PDZ-binding sequence of Cx43, developed as part of the work on cardiac GJ organization is also described, which has led to evidence for novel and unexpected roles for Cx43 in modulating healing following tissue injury.

Quantitative analysis of ZO-1 colocalization with Cx43 gap junction plaques in cultures of rat neonatal cardiomyocytes.

The gap junction (GJ) is an aggregate of intercellular channels that facilitates cytoplasmic interchange of ions, second messengers, and other molecules of less than 1000 Da between cells. In excitable organs such as heart and brain, GJs configure extended intercellular pathways for stable and long-term propagation of action potential. In a previous study in adult rat heart, we have shown that the Drosophila disks-large related protein ZO-1 shows low to moderate colocalization at myocyte borders with the GJ protein Cx43. In the present study, we detail a protocol for characterizing the pattern and level of colocalization of ZO-1 with Cx43 in cultures of neonatal myocytes at the level of individual GJ plaques. The data indicate that ZO-1 shows on average a partial 26.6% overlap (SD = 11.3%) with Cx43 GJ plaques. There is a strong positive correlation between GJ plaque size and area of ZO-1 colocalization, indicating that the level of associated ZO-1 scales with the area of the GJ plaque. Qualitatively, the most prominent colocalization occurs at the plaque perimeter. These studies may provide insight into the presently unknown biological function of ZO-1 interaction with Cx43.

Fusion of GFP to the carboxyl terminus of connexin43 increases gap junction size in HeLa cells.

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.

Wnt11 and Wnt7a are up-regulated in association with differentiation of cardiac conduction cells in vitro and in vivo.

The heart beat is coordinated by a precisely timed sequence of action potentials propagated through cells of the conduction system. Previously, we have shown that conduction cells in the chick embryo are derived from multipotent, cardiomyogenic progenitors present in the looped, tubular heart. Moreover, analyses of heterogeneity within myocyte clones and cell birth dating have indicated that elaboration of the conduction system occurs by ongoing, localized recruitment from within this multipotent pool. In this study, we have focused on a potential role for Wnt signaling in development of the cardiac conduction system. Treatment of embryonic myocytes from chick with endothelin-1 (ET-1) has been shown to promote expression of markers of Purkinje fiber cells. By using this in vitro model, we find that Wnt11 are Wnt7a are up-regulated in association with ET-1 treatment. Moreover, in situ hybridization reveals expression, although not temporal coincidence of, Wnt11 and Wnt7a in specialized tissues in the developing heart in vivo. Specifically, whereas Wnt11 shows transient and prominent expression in central elements of the developing conduction system (e.g., the His bundle), relative increases in Wnt7a expression emerge at sites consistent with the location of peripheral conduction cells (e.g., subendocardial Purkinje fibers). The patterns of Wnt11 and Wnt7a expression observed in vitro and in the embryonic chick heart appear to be consistent with roles for these two Wnts in differentiation of cardiac conduction tissues.