Assessment of Connexin43 Hemichannel Functionality Based on Cytosolic Uptake of Yo-Pro1.

Connexin proteins are the building blocks of gap junctions and connexin hemichannels. Both provide a pathway for cellular communication. Gap junctions support intercellular communication mechanisms and regulate homeostasis. In contrast, open connexin hemichannels connect the intracellular compartment and the extracellular environment, and their activation fuels inflammation and cell death. The development of clinically applicable connexin hemichannel blockers for therapeutic purposes is therefore gaining momentum. This chapter describes a well-established protocol optimized for assessing connexin hemichannel activity by using the reporter dye Yo-Pro1.

Nanobody-based pannexin1 channel inhibitors reduce inflammation in acute liver injury.

BACKGROUND: The opening of pannexin1 channels is considered as a key event in inflammation. Pannexin1 channel-mediated release of adenosine triphosphate triggers inflammasome signaling and activation of immune cells. By doing so, pannexin1 channels play an important role in several inflammatory diseases. Although pannexin1 channel inhibition could represent a novel clinical strategy for treatment of inflammatory disorders, therapeutic pannexin1 channel targeting is impeded by the lack of specific, potent and/or in vivo-applicable inhibitors. The goal of this study is to generate nanobody-based inhibitors of pannexin1 channels. RESULTS: Pannexin1-targeting nanobodies were developed as potential new pannexin1 channel inhibitors. We identified 3 cross-reactive nanobodies that showed affinity for both murine and human pannexin1 proteins. Flow cytometry experiments revealed binding capacities in the nanomolar range. Moreover, the pannexin1-targeting nanobodies were found to block pannexin1 channel-mediated release of adenosine triphosphate. The pannexin1-targeting nanobodies were also demonstrated to display anti-inflammatory effects in vitro through reduction of interleukin 1 beta amounts. This anti-inflammatory outcome was reproduced in vivo using a human-relevant mouse model of acute liver disease relying on acetaminophen overdosing. More specifically, the pannexin1-targeting nanobodies lowered serum levels of inflammatory cytokines and diminished liver damage. These effects were linked with alteration of the expression of several NLRP3 inflammasome components. CONCLUSIONS: This study introduced for the first time specific, potent and in vivo-applicable nanobody-based inhibitors of pannexin1 channels. As demonstrated for the case of liver disease, the pannexin1-targeting nanobodies hold great promise as anti-inflammatory agents, yet this should be further tested for extrahepatic inflammatory disorders. Moreover, the pannexin1-targeting nanobodies represent novel tools for fundamental research regarding the role of pannexin1 channels in pathological and physiological processes.

Pannexin1 channels in the liver: an open enemy.

Pannexin1 proteins form communication channels at the cell plasma membrane surface, which allow the transfer of small molecules and ions between the intracellular compartment and extracellular environment. In this way, pannexin1 channels play an important role in various cellular processes and diseases. Indeed, a plethora of human pathologies is associated with the activation of pannexin1 channels. The present paper reviews and summarizes the structure, life cycle, regulation and (patho)physiological roles of pannexin1 channels, with a particular focus on the relevance of pannexin1 channels in liver diseases.

Effects of acute and chronic disease on cell junctions in mouse liver.

Cell junctions, including anchoring, occluding and communicating junctions, play an indispensable role in tissue architecture and homeostasis. Consequently, malfunctioning of cell junctions is linked with a wide range of disorders, including in liver. The present study was set up to investigate the effects of acute and chronic disease induced by chemical compounds on hepatic cell junctions in mice. Mice were either overdosed with paracetamol or repeatedly administered carbon tetrachloride followed by sampling at 24 hours or 8 weeks, respectively. mRNA and protein expression levels of adherens, gap and tight junction components were measured in liver using reverse transcription quantitative real-time polymerase chain reaction analysis and immunoblot techniques, respectively. It was found that protein levels of the adherens junction building blocks ß-catenin and γ-catenin, the gap junction components Cx26 and Cx32, and the tight junction constituent zonula occludens 2 were decreased, while mRNA levels of the adherens junction building block E-cadherin, and the tight junction constituent zonula occludens 2 and claudin 1 were upregulated following paracetamol overdosing. Repeated administration of carbon tetrachloride increased protein levels of E-cadherin, ß-catenin, Cx26, Cx32, Cx43 and claudin 1. The latter was reflected at the mRNA level. In conclusion, acute and chronic liver disease have different effects on cell junctions in liver.

Connexin-Based Channels in the Liver.

Connexin proteins oligomerize in hexameric structures called connexin hemichannels, which then dock to form gap junctions. Gap junctions direct cell-cell communication by allowing the exchange of small molecules and ions between neighboring cells. In this way, hepatic gap junctions support liver homeostasis. Besides serving as building blocks for gap junctions, connexin hemichannels provide a pathway between the intracellular and the extracellular environment. The activation of connexin hemichannels is associated with acute and chronic liver pathologies. This article discusses the role of gap junctions and connexin hemichannels in the liver. © 2022 American Physiological Society. Compr Physiol 12:1-17, 2022.

Effects of Drugs Formerly Suggested for COVID-19 Repurposing on Pannexin1 Channels.

Although many efforts have been made to elucidate the pathogenesis of COVID-19, the underlying mechanisms are yet to be fully uncovered. However, it is known that a dysfunctional immune response and the accompanying uncontrollable inflammation lead to troublesome outcomes in COVID-19 patients. Pannexin1 channels are put forward as interesting drug targets for the treatment of COVID-19 due to their key role in inflammation and their link to other viral infections. In the present study, we selected a panel of drugs previously tested in clinical trials as potential candidates for the treatment of COVID-19 early on in the pandemic, including hydroxychloroquine, chloroquine, azithromycin, dexamethasone, ribavirin, remdesivir, favipiravir, lopinavir, and ritonavir. The effect of the drugs on pannexin1 channels was assessed at a functional level by means of measurement of extracellular ATP release. Immunoblot analysis and real-time quantitative reversetranscription polymerase chain reaction analysis were used to study the potential of the drugs to alter pannexin1 protein and mRNA expression levels, respectively. Favipiravir, hydroxychloroquine, lopinavir, and the combination of lopinavir with ritonavir were found to inhibit pannexin1 channel activity without affecting pannexin1 protein or mRNA levels. Thusthree new inhibitors of pannexin1 channels were identified that, though currently not being used anymore for the treatment of COVID-19 patients, could be potential drug candidates for other pannexin1-related diseases.

Effects of Drugs Formerly Proposed for COVID-19 Treatment on Connexin43 Hemichannels.

Connexin43 (Cx43) hemichannels form a pathway for cellular communication between the cell and its extracellular environment. Under pathological conditions, Cx43 hemichannels release adenosine triphosphate (ATP), which triggers inflammation. Over the past two years, azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, and ritonavir have been proposed as drugs for the treatment of the coronavirus disease 2019 (COVID-19), which is associated with prominent systemic inflammation. The current study aimed to investigate if Cx43 hemichannels, being key players in inflammation, could be affected by these drugs which were formerly designated as COVID-19 drugs. For this purpose, Cx43-transduced cells were exposed to these drugs. The effects on Cx43 hemichannel activity were assessed by measuring extracellular ATP release, while the effects at the transcriptional and translational levels were monitored by means of real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblot analysis, respectively. Exposure to lopinavir and ritonavir combined (4:1 ratio), as well as to remdesivir, reduced Cx43 mRNA levels. None of the tested drugs affected Cx43 protein expression.

Expression and Functionality of Connexin-Based Channels in Human Liver Cancer Cell Lines.

Liver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis. Among such mechanisms is cellular communication mediated by connexin-based gap junctions. The present study investigated changes in connexin expression and gap junction functionality in liver cancer in vitro. For this purpose, seven human liver cancer cell lines, as well as primary human hepatocytes, were subjected to connexin and gap junction analysis at the transcriptional, translational and activity level. Real-time quantitative reverse transcription polymerase chain reaction analysis showed enhanced expression of connexin43 in the majority of liver cancer cell lines at the expense of connexin32 and connexin26. Some of these changes were paralleled at the protein level, as evidenced by immunoblot analysis and in situ immunocytochemistry. Gap junctional intercellular communication, assessed by the scrape loading/dye transfer assay, was generally low in all liver cancer cell lines. Collectively, these results provide a full scenario of modifications in hepatocyte connexin production and gap junction activity in cultured liver cancer cell lines. The findings may be valuable for the selection of neoplastic hepatocytes for future mechanistic investigation and testing of anti-cancer drugs that target connexins and their channels.

Connexin-Based Channel Activity Is Not Specifically Altered by Hepatocarcinogenic Chemicals.

Connexin-based channels play key roles in cellular communication and can be affected by deleterious chemicals. In this study, the effects of various genotoxic carcinogenic compounds, non-genotoxic carcinogenic compounds and non-carcinogenic compounds on the expression and functionality of connexin-based channels, both gap junctions and connexin hemichannels, were investigated in human hepatoma HepaRG cell cultures. Expression of connexin26, connexin32, and connexin43 was evaluated by means of real-time reverse transcription quantitative polymerase chain reaction analysis, immunoblot analysis and in situ immunostaining. Gap junction functionality was assessed via a scrape loading/dye transfer assay. Opening of connexin hemichannels was monitored by measuring extracellular release of adenosine triphosphate. It was found that both genotoxic and non-genotoxic carcinogenic compounds negatively affect connexin32 expression. However, no specific effects related to chemical type were observed at gap junction or connexin hemichannel functionality level.

In Vitro Liver Toxicity Testing of Chemicals: A Pragmatic Approach.

The liver is among the most frequently targeted organs by noxious chemicals of diverse nature. Liver toxicity testing using laboratory animals not only raises serious ethical questions, but is also rather poorly predictive of human safety towards chemicals. Increasing attention is, therefore, being paid to the development of non-animal and human-based testing schemes, which rely to a great extent on in vitro methodology. The present paper proposes a rationalized tiered in vitro testing strategy to detect liver toxicity triggered by chemicals, in which the first tier is focused on assessing general cytotoxicity, while the second tier is aimed at identifying liver-specific toxicity as such. A state-of-the-art overview is provided of the most commonly used in vitro assays that can be used in both tiers. Advantages and disadvantages of each assay as well as overall practical considerations are discussed.

Mechanisms Underlying Connexin Hemichannel Activation in Disease.

Gap junctions and connexin hemichannels mediate intercellular and extracellular communication, respectively. While gap junctions are seen as the "good guys" by controlling homeostasis, connexin hemichannels are considered as the "bad guys", as their activation is associated with the onset and dissemination of disease. Open connexin hemichannels indeed mediate the transport of messengers between the cytosol and extracellular environment and, by doing so, fuel inflammation and cell death in a plethora of diseases. The present mini-review discusses the mechanisms involved in the activation of connexin hemichannels during pathology.

Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor.

Cell plasma membrane proteins are considered as gatekeepers of the cell and play a major role in regulating various processes. Transport proteins constitute a subclass of cell plasma membrane proteins enabling the exchange of molecules and ions between the extracellular environment and the cytosol. A plethora of human pathologies are associated with the altered expression or dysfunction of cell plasma membrane transport proteins, making them interesting therapeutic drug targets. However, the search for therapeutics is challenging, since many drug candidates targeting cell plasma membrane proteins fail in (pre)clinical testing due to inadequate selectivity, specificity, potency or stability. These latter characteristics are met by nanobodies, which potentially renders them eligible therapeutics targeting cell plasma membrane proteins. Therefore, a therapeutic nanobody-based strategy seems a valid approach to target and modulate the activity of cell plasma membrane transport proteins. This review paper focuses on methodologies to generate cell plasma membrane transport protein-targeting nanobodies, and the advantages and pitfalls while generating these small antibody-derivatives, and discusses several therapeutic nanobodies directed towards transmembrane proteins, including channels and pores, adenosine triphosphate-powered pumps and porters.

Cholestasis Differentially Affects Liver Connexins.

Connexins are goal keepers of tissue homeostasis, including in the liver. As a result, they are frequently involved in disease. The current study was set up to investigate the effects of cholestatic disease on the production of connexin26, connexin32 and connexin43 in the liver. For this purpose, bile duct ligation, a well-known trigger of cholestatic liver injury, was applied to mice. In parallel, human hepatoma HepaRG cell cultures were exposed to cholestatic drugs and bile acids. Samples from both the in vivo and in vitro settings were subsequently subjected to assessment of mRNA and protein quantities as well as to in situ immunostaining. While the outcome of cholestasis on connexin26 and connexin43 varied among experimental settings, a more generalized repressing effect was seen for connexin32. This has also been observed in many other liver pathologies and could suggest a role for connexin32 as a robust biomarker of liver disease and toxicity.

Canonical and Non-Canonical Roles of Connexin43 in Cardioprotection.

Since the mid-20th century, ischemic heart disease has been the world's leading cause of death. Developing effective clinical cardioprotection strategies would make a significant impact in improving both quality of life and longevity in the worldwide population. Both ex vivo and in vivo animal models of cardiac ischemia/reperfusion (I/R) injury are robustly used in research. Connexin43 (Cx43), the predominant gap junction channel-forming protein in cardiomyocytes, has emerged as a cardioprotective target. Cx43 posttranslational modifications as well as cellular distribution are altered during cardiac reperfusion injury, inducing phosphorylation states and localization detrimental to maintaining intercellular communication and cardiac conduction. Pre- (before ischemia) and post- (after ischemia but before reperfusion) conditioning can abrogate this injury process, preserving Cx43 and reducing cell death. Pre-/post-conditioning has been shown to largely rely on the presence of Cx43, including mitochondrial Cx43, which is implicated to play a major role in pre-conditioning. Posttranslational modifications of Cx43 after injury alter the protein interactome, inducing negative protein cascades and altering protein trafficking, which then causes further damage post-I/R injury. Recently, several peptides based on the Cx43 sequence have been found to successfully diminish cardiac injury in pre-clinical studies.

Targeting gap junctional intercellular communication by hepatocarcinogenic compounds.

Gap junctions in liver, as in other organs, play a critical role in tissue homeostasis. Inherently, these cellular constituents are major targets for systemic toxicity and diseases, including cancer. This review provides an overview of chemicals that compromise liver gap junctions, in particular biological toxins, organic solvents, pesticides, pharmaceuticals, peroxides, metals and phthalates. The focus in this review is placed upon the mechanistic scenarios that underlie these adverse effects. Further, the potential use of gap junctional activity as an in vitro biomarker to identify non-genotoxic hepatocarcinogenic chemicals is discussed.

Non-canonical roles of connexins.

Gap junctions mediate cellular communication and homeostasis by controlling the intercellular exchange of small and hydrophilic molecules and ions. Gap junction channels are formed by the docking of 2 hemichannels of adjacent cells, which in turn are composed of 6 connexin subunits. Connexin proteins as such can also control the cellular life cycle independent of their channel activities. This has been most demonstrated in the context of cell growth and cell death. Different mechanisms are involved mainly related to direct interaction with cell growth or cell death regulators, but also implying effects on the expression of cell growth and cell death regulators. The present paper focuses on these atypical roles of connexin proteins.

Increased Expression of Adherens Junction Components in Mouse Liver following Bile Duct Ligation.

Adherens junctions, consisting of cadherins and catenins, are a group of cell-to-cell junctions that mediate mechanistic linkage between neighboring cells. By doing so, adherens junctions ensure direct intercellular contact and play an indispensable role in maintaining tissue architecture. Considering these critical functions, it is not surprising that adherens junctions are frequently involved in disease. In the present study, the effects of bile duct ligation-a surgical procedure to experimentally induce cholestatic and fibrotic liver pathology-on hepatic adherens junctions were investigated in mice. In essence, it was found that liver mRNA and protein levels of E-cadherin, ß-catenin and γ-catenin drastically increase following bile duct ligation. These results could suggest a cytoprotective role for hepatic adherens junctions following bile duct ligation.

Primary hepatocytes and their cultures for the testing of drug-induced liver injury.

Drug-induced liver injury is a major reason for discontinuation of drug development and withdrawal of drugs from the market. Intensive efforts in the last decades have focused on the establishment and finetuning of liver-based in vitro models for reliable prediction of hepatotoxicity triggered by drug candidates. Of those, primary hepatocytes and their cultures still are considered the gold standard, as they provide an acceptable reflection of the hepatic in vivo situation. Nevertheless, these in vitro systems cope with gradual deterioration of the differentiated morphological and functional phenotype. The present paper gives an overview of traditional and more recently introduced strategies to counteract this dedifferentiation process in an attempt to set up culture models that can be used for long-term testing purposes. The relevance and applicability of such optimized cultures of primary hepatocytes for the testing of drug-induced cholestatic liver injury is demonstrated.

Industrial, Biocide, and Cosmetic Chemical Inducers of Cholestasis.

A frequent side effect of many drugs includes the occurrence of cholestatic liver toxicity. Over the past couple of decades, drug-induced cholestasis has gained considerable attention, resulting in a plethora of data regarding its prevalence and mechanistic basis. Likewise, several food additives and dietary supplements have been reported to cause cholestatic liver insults in the past few years. The induction of cholestatic hepatotoxicity by other types of chemicals, in particular synthetic compounds, such as industrial chemicals, biocides, and cosmetic ingredients, has been much less documented. Such information can be found in occasional clinical case reports of accidental intake or suicide attempts as well as in basic and translational study reports on mechanisms or testing of new therapeutics in cholestatic animal models. This paper focuses on such nonpharmaceutical and nondietary synthetic chemical inducers of cholestatic liver injury, in particular alpha-naphthylisocyanate, 3,5-diethoxycarbonyl-1,4-dihydrocollidine, methylenedianiline, paraquat, tartrazine, triclosan, 2-octynoic acid, and 2-nonynoic acid. Most of these cholestatic compounds act by similar mechanisms. This could open perspectives for the prediction of cholestatic potential of chemicals.

Connexin and Pannexin (Hemi)Channels: Emerging Targets in the Treatment of Liver Disease.

Connexin proteins are the building blocks of hemichannels, which dock further between adjacent cells to form gap junctions. Gap junctions control the intercellular exchange of critical homeostasis regulators. By doing so, gap junctions control virtually all aspects of the hepatic life cycle. In the last decade, it has become clear that connexin hemichannels also provide a pathway for cellular communication on their own independent of their role as structural precursors of gap junctions, namely between the cytosol of an individual cell and its extracellular environment. In contrast to gap junctions, connexin hemichannels become particularly active in liver disease by facilitating inflammation and cell death. This equally holds true for cellular channels composed of pannexins, being connexin-like proteins recently identified in the liver that gather in structures reminiscent of hemichannels. This paper gives an overview of the involvement of connexin-based and pannexin-based channels in noncancerous liver disease.