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Gap junctions and non-neoplastic liver disease

Open AccessPublished:May 18, 2012DOI:https://doi.org/10.1016/j.jhep.2012.02.036

      Summary

      Because of their critical role as goalkeepers of hepatic homeostasis, gap junctions are frequent targets in liver disease. This concept has been demonstrated on many occasions in the light of hepatocarcinogenesis. Relatively little focus has been put on the fate of gap junctions in other liver pathologies, including hepatitis, liver fibrosis and cirrhosis, cholestasis and hepatic ischemia and reperfusion injury. The present paper provides an in-depth description of the multiple changes in expression, localization and function of connexins, the molecular constituents of gap junctions. The use of connexins as biomarkers and therapeutic targets in liver disease is also illustrated.

      Abbreviations:

      ALT (alanine aminotransferase), AST (aspartate aminotransferase), cdc42 (cell division cycle 42), Cx (connexin), GJIC (gap junctional intercellular communication), IFNγ (interferon gamma), IL-1(β)/2/6 (interleukin 1 (beta)/2/6), LPS (lipopolysaccharide), NF-κβ (nuclear factor kappa beta), (p38) MAPK ((p38) mitogen-activated protein kinase), mRNA (messenger ribonucleic acid), Panx (pannexin), TNFα (tumor necrosis factor alpha)

      Keywords

      Introduction

      Gap junctions gather at the cell plasma membrane surface in areas called plaques. They arise from the interaction of two hemichannels of neighboring cells, that on their turn are hexamers of connexin (Cx) proteins. Connexins all share a similar structure, consisting of four transmembrane domains, two extracellular loops, one cytoplasmic loop, one cytoplasmic carboxytail and one cytoplasmic aminotail (Fig. 1) [
      • Vinken M.
      • Decrock E.
      • De Vuyst E.
      • Ponsaerts R.
      • D’Hondt C.
      • Bultynck G.
      • et al.
      Connexins: sensors and regulators of cell cycling.
      ,
      • Vinken M.
      • Doktorova T.
      • Decrock E.
      • Leybaert L.
      • Vanhaecke T.
      • Rogiers V.
      Gap junctional intercellular communication as a target for liver toxicity and carcinogenicity.
      ,
      • Vinken M.
      • Henkens T.
      • De Rop E.
      • Fraczek J.
      • Vanhaecke T.
      • Rogiers V.
      Biology and pathobiology of gap junctional channels in hepatocytes.
      ]. The predominant connexin species in the liver is Cx32, which is abundantly expressed by hepatocytes and to a lesser extent by sinusoidal endothelial cells. The latter cells, as well as the stellate cells, produce also small quantities of Cx26 [
      • Fischer R.
      • Reinehr R.
      • Lu T.P.
      • Schonicke A.
      • Warskulat U.
      • Dienes H.P.
      • et al.
      Intercellular communication via gap junctions in activated rat hepatic stellate cells.
      ], while Cx43 is detectable in Kupffer cells, stellate cells, sinusoidal endothelial cells, and cholangiocytes [
      • Fischer R.
      • Reinehr R.
      • Lu T.P.
      • Schonicke A.
      • Warskulat U.
      • Dienes H.P.
      • et al.
      Intercellular communication via gap junctions in activated rat hepatic stellate cells.
      ,
      • Berthoud V.M.
      • Iwanij V.
      • Garcia A.M.
      • Saez J.C.
      Connexins and glucagon receptors during development of rat hepatic acinus.
      ,
      • Bode H.P.
      • Wang L.
      • Cassio D.
      • Leite M.F.
      • St-Pierre M.V.
      • Hirata K.
      • et al.
      Expression and regulation of gap junctions in rat cholangiocytes.
      ]. However, the presence of functional gap junctions has only been demonstrated in hepatocytes and stellate cells [
      • Fischer R.
      • Reinehr R.
      • Lu T.P.
      • Schonicke A.
      • Warskulat U.
      • Dienes H.P.
      • et al.
      Intercellular communication via gap junctions in activated rat hepatic stellate cells.
      ]. Gap junctional intercellular communication (GJIC) includes the passive exchange between adjacent cells of small and hydrophilic substances, such as second messengers [
      • Vinken M.
      • Decrock E.
      • De Vuyst E.
      • Ponsaerts R.
      • D’Hondt C.
      • Bultynck G.
      • et al.
      Connexins: sensors and regulators of cell cycling.
      ,
      • Vinken M.
      • Doktorova T.
      • Decrock E.
      • Leybaert L.
      • Vanhaecke T.
      • Rogiers V.
      Gap junctional intercellular communication as a target for liver toxicity and carcinogenicity.
      ,
      • Vinken M.
      • Henkens T.
      • De Rop E.
      • Fraczek J.
      • Vanhaecke T.
      • Rogiers V.
      Biology and pathobiology of gap junctional channels in hepatocytes.
      ], and is regulated by a plethora of mechanisms, including connexin phosphorylation [
      • Solan J.L.
      • Lampe P.D.
      Connexin43 phosphorylation: structural changes and biological effects.
      ]. As such, hepatic GJIC, in particular between hepatocytes, has been shown to drive a number of essential processes, namely albumin secretion [
      • Yang J.
      • Ichikawa A.
      • Tsuchiya T.
      A novel function of connexin 32: marked enhancement of liver function in a hepatoma cell line.
      ], glycogenolysis [
      • Nelles E.
      • Butzler C.
      • Jung D.
      • Temme A.
      • Gabriel H.D.
      • Dahl U.
      • et al.
      Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.
      ,
      • Stumpel F.
      • Ott T.
      • Willecke K.
      • Jungermann K.
      Connexin 32 gap junctions enhance stimulation of glucose output by glucagon and noradrenaline in mouse liver.
      ], ammonia detoxification [
      • Yang J.
      • Ichikawa A.
      • Tsuchiya T.
      A novel function of connexin 32: marked enhancement of liver function in a hepatoma cell line.
      ], bile secretion [
      • Nathanson M.H.
      • Rios-Velez L.
      • Burgstahler A.D.
      • Mennone A.
      Communication via gap junctions modulates bile secretion in the isolated perfused rat liver.
      ,
      • Temme A.
      • Stumpel F.
      • Sohl G.
      • Rieber E.P.
      • Jungermann K.
      • Willecke K.
      • et al.
      Dilated bile canaliculi and attenuated decrease of nerve-dependent bile secretion in connexin32-deficient mouse liver.
      ], and biotransformation [
      • Neveu M.J.
      • Babcock K.L.
      • Hertzberg E.L.
      • Paul D.L.
      • Nicholson B.J.
      • Pitot H.C.
      Colocalized alterations in connexin32 and cytochrome P450IIB1/2 by phenobarbital and related liver tumor promoters.
      ,
      • Shoda T.
      • Mitsumori K.
      • Onodera H.
      • Toyoda K.
      • Uneyama C.
      • Takada K.
      • et al.
      Liver tumor-promoting effect of beta-naphthoflavone, a strong CYP 1A1/2 inducer, and the relationship between CYP 1A1/2 induction and Cx32 decrease in its hepatocarcinogenesis in the rat.
      ]. Moreover, gap junctions are also key players in liver development [
      • Vinken M.
      • Henkens T.
      • De Rop E.
      • Fraczek J.
      • Vanhaecke T.
      • Rogiers V.
      Biology and pathobiology of gap junctional channels in hepatocytes.
      ], liver cell growth [
      • Vinken M.
      • Decrock E.
      • De Vuyst E.
      • Ponsaerts R.
      • D’Hondt C.
      • Bultynck G.
      • et al.
      Connexins: sensors and regulators of cell cycling.
      ], and liver cell death [
      • Decrock E.
      • Vinken M.
      • De Vuyst E.
      • Krysko D.V.
      • D‘Herde K.
      • Vanhaeck T.
      • et al.
      Connexin-related signaling in cell death: to live or let die?.
      ]. Their ubiquitous involvement in the maintenance of hepatic homeostasis may explain why gap junctions are frequently targeted in stress situations. Thus far, this has been well documented during liver toxicity [
      • Vinken M.
      • Doktorova T.
      • Decrock E.
      • Leybaert L.
      • Vanhaecke T.
      • Rogiers V.
      Gap junctional intercellular communication as a target for liver toxicity and carcinogenicity.
      ,
      • Chipman J.K.
      • Mally A.
      • Edwards G.O.
      Disruption of gap junctions in toxicity and carcinogenicity.
      ] and hepatocarcinogenesis [
      • Vinken M.
      • Doktorova T.
      • Decrock E.
      • Leybaert L.
      • Vanhaecke T.
      • Rogiers V.
      Gap junctional intercellular communication as a target for liver toxicity and carcinogenicity.
      ,
      • Chipman J.K.
      • Mally A.
      • Edwards G.O.
      Disruption of gap junctions in toxicity and carcinogenicity.
      ,
      • Schwarz M.
      • Wanke I.
      • Wulbrand U.
      • Moennikes O.
      • Buchmann A.
      Role of connexin32 and beta-catenin in tumor promotion in mouse liver.
      ]. Indeed, several excellent review papers have described the fate of connexins and their channels in liver cancer [
      • King T.J.
      • Bertram J.S.
      Connexins as targets for cancer chemoprevention and chemotherapy.
      ,
      • Mesnil M.
      • Crespin S.
      • Avanzo J.L.
      • Zaidan-Dagli M.L.
      Defective gap junctional intercellular communication in the carcinogenic process.
      ,
      • Ruch R.J.
      • Trosko J.E.
      The role of oval cells and gap junctional intercellular communication in hepatocarcinogenesis.
      ,
      • Trosko J.E.
      • Chang C.C.
      Mechanism of up-regulated gap junctional intercellular communication during chemoprevention and chemotherapy of cancer.
      ,
      • Yamasaki H.
      • Krutovskikh V.
      • Mesnil M.
      • Columbano A.
      • Tsuda H.
      • Ito N.
      Gap junctional intercellular communication and cell proliferation during rat liver carcinogenesis.
      ,
      • Yamasaki H.
      • Krutovskikh V.
      • Mesnil M.
      • Tanaka T.
      • Zaidan-Dagli M.L.
      • Omori Y.
      Role of connexin (gap junction) genes in cell growth control and carcinogenesis.
      ]. Less attention has been paid to the role of liver gap junctions in a number of other relevant non-cancerous clinical situations, including cholestasis, liver fibrosis and cirrhosis, hepatitis and systemic inflammation, and hepatic liver ischemia and reperfusion injury. A state-of-the-art overview of these features is provided in the current paper, with emphasis on gap junctions composed of Cx26, Cx32 and Cx43.
      Figure thumbnail gr1
      Fig. 1Molecular architecture of gap junctions. Gap junctions are grouped in plaques at the cell plasma membrane surface of two apposed cells, and are composed of 12 connexin proteins, organized as two hexameric hemichannels. The connexin protein is organized as four transmembrane domains (TM), two extracellular loops (EL), one cytoplasmic loop (CL), one cytoplasmic aminotail (NT) and one cytoplasmic carboxytail (CT).

      Gap junctions in cholestasis

      Cholestasis denotes any situation of impaired bile secretion with concomitant accumulation of potentially noxious cholephiles in the liver or in the systemic circulation [
      • Wagner M.
      • Zollner G.
      • Trauner M.
      New molecular insights into the mechanisms of cholestasis.
      ]. Depending on the site of the obstruction, a distinction is made between intrahepatic and extrahepatic cholestasis. In humans, cholestasis can be induced by a variety of factors, including several drug treatments and hereditary mutations in drug transporter genes, and may become manifested in a number of conditions, such as primary sclerosing cholangitis and primary biliary cirrhosis [
      • Rodriguez-Garay E.A.
      Cholestasis: human disease and experimental animal models.
      ,
      • Zollner G.
      • Trauner M.
      Mechanisms of cholestasis.
      ]. A popular experimental model of extrahepatic biliary obstruction is common bile duct ligation in rat [
      • Rodriguez-Garay E.A.
      Cholestasis: human disease and experimental animal models.
      ]. A handful of early studies described a reversible decrease of the hepatic gap junction number upon bile duct ligation [
      • De Vos R.
      • Desmet V.J.
      Morphologic changes of the junctional complex of the hepatocytes in rat liver after bile duct ligation.
      ,
      • Metz J.
      • Aoki A.
      • Merlo M.
      • Forssmann W.G.
      Morphological alterations and functional changes of interhepatocellular junctions induced by bile duct ligation.
      ,
      • Metz J.
      • Bressler D.
      Reformation of gap and tight junctions in regenerating liver after cholestasis.
      ]. Later on, this was repeatedly demonstrated to be accompanied by a rapid drop in amounts of Cx32 mRNA [
      • Fallon M.B.
      • Nathanson M.H.
      • Mennone A.
      • Saez J.C.
      • Burgstahler A.D.
      • Anderson J.M.
      Altered expression and function of hepatocyte gap junctions after common bile duct ligation in the rat.
      ] and protein levels [
      • Fallon M.B.
      • Nathanson M.H.
      • Mennone A.
      • Saez J.C.
      • Burgstahler A.D.
      • Anderson J.M.
      Altered expression and function of hepatocyte gap junctions after common bile duct ligation in the rat.
      ,
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      ,
      • Kojima T.
      • Yamamoto T.
      • Murata M.
      • Lan M.
      • Takano K.
      • Go M.
      • et al.
      Role of the p38 MAP-kinase signaling pathway for Cx32 and claudin-1 in the rat liver.
      ,
      • Traub O.
      • Druge P.M.
      • Willecke K.
      Degradation and resynthesis of gap junction protein in plasma membranes of regenerating liver after partial hepatectomy or cholestasis.
      ], a process mediated by the p38 mitogen-activated protein kinase (p38 MAPK) [
      • Fallon M.B.
      • Nathanson M.H.
      • Mennone A.
      • Saez J.C.
      • Burgstahler A.D.
      • Anderson J.M.
      Altered expression and function of hepatocyte gap junctions after common bile duct ligation in the rat.
      ]. Hepatic Cx26 immunoreactivity also decreases following bile duct ligation [
      • Fallon M.B.
      • Nathanson M.H.
      • Mennone A.
      • Saez J.C.
      • Burgstahler A.D.
      • Anderson J.M.
      Altered expression and function of hepatocyte gap junctions after common bile duct ligation in the rat.
      ,
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      ], though its mRNA content rather increases [
      • Fallon M.B.
      • Nathanson M.H.
      • Mennone A.
      • Saez J.C.
      • Burgstahler A.D.
      • Anderson J.M.
      Altered expression and function of hepatocyte gap junctions after common bile duct ligation in the rat.
      ]. In contrast, Cx43 protein is positively affected in this model [
      • Fallon M.B.
      • Nathanson M.H.
      • Mennone A.
      • Saez J.C.
      • Burgstahler A.D.
      • Anderson J.M.
      Altered expression and function of hepatocyte gap junctions after common bile duct ligation in the rat.
      ] and it has been reported that bile duct ligated heterozygous Cx43+/− knock-out mice display less hepatic vein angiogenesis, while other parameters, such as biliary duct hyperplasia, remain unchanged [
      • Teixeira T.F.
      • da Silva T.C.
      • Fukumasu H.
      • de Lima C.E.
      • Lucia Zaidan Dagli J.L.
      • Guerra J.L.
      Histological alterations in the livers of Cx43-deficient mice submitted to a cholestasis model.
      ]. Unlike Cx26 and Cx32, no changes in Cx43 steady-state protein levels are observed in the choledochocaval fistula rat model of complete biliary retention [
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      ]. Effects on Cx43 are also absent in estrogen-treated rats, which typically develop intrahepatic cholestasis, and Cx32 production even increases in this experimental model [
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      ]. However, induction of intrahepatic cholestasis in rats by phalloidin administration adversely influences gap junctions and Cx32-positive spots, first in the pericentral region and subsequently throughout the entire liver acinus [
      • Ohta M.
      • Okanoue T.
      • Takami S.
      • Nagao Y.
      • Mori T.
      • Hori N.
      • et al.
      Morphological alterations of gap junctions in phalloidin-treated rat livers.
      ]. Such contradicting results illustrate that the outcome of the induced cholestasis response on hepatic connexins depends on the nature of the trigger used. Furthermore, the mechanisms that underlie the effects of cholestasis on gap junctions remain obscure. Boucherie and colleagues reported that cholestatic bile acids, such as taurolithocholic acid, taurolithocholicsulfate acid, and taurochenodeoxycholic acid, dose-dependently and reversibly inhibited GJIC between cultured primary rat hepatocyte couplets. Choleretic bile acids, including taurocholic acid and tauroursodeoxycholic acid, do not affect gap junction activity in these cells. Taurolithocholicsulfate acid also suppresses GJIC between normal rat cholangiocytes, which only express Cx43, but not in cervical cancer cells stably transfected with Cx32 or Cx26. This shows not only that the presence of bile transporters is crucial for inhibiting gap junctions, but also that this effect is not connexin-specific. How this ultimately leads to suppression of GJIC is unclear, though increases in calcium or hydrogen concentration or protein kinase C-mediated phosphorylation have been definitely excluded [
      • Boucherie S.
      • Koukoui O.
      • Nicolas V.
      • Combettes L.
      Cholestatic bile acids inhibit gap junction permeability in rat hepatocyte couplets and normal rat cholangiocytes.
      ].

      Gap junctions in liver fibrosis and cirrhosis

      Figure thumbnail fx4

      Gap junctions in hepatitis and systemic inflammation

      Several pathological conditions in the liver, including fibrosis and cirrhosis, and cholestasis, are associated with inflammation. As such, low Cx32 protein levels have been detected in liver tissue from hepatitis patients [
      • Nakashima Y.
      • Ono T.
      • Yamanoi A.
      • El-Assal O.N.
      • Kohno H.
      • Nagasue N.
      Expression of gap junction protein connexin32 in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.
      ,
      • Yamaoka K.
      • Nouchi T.
      • Kohashi T.
      • Marumo F.
      • Sato C.
      Expression of gap junction protein connexin 32 in chronic liver diseases.
      ]. An acute status of inflammation can be experimentally induced in animals by injection of lipopolysaccharide (LPS), a component isolated from the outer membrane of Gram-negative bacteria that acts as an endotoxin. Similar to hepatitis patients, decreased liver Cx32 amounts have been measured in mice [
      • Temme A.
      • Ott T.
      • Haberberger T.
      • Traub O.
      • Willecke K.
      Acute-phase response and circadian expression of connexin26 are not altered in connexin32-deficient mouse liver.
      ] and rats [
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      ,
      • Correa P.R.
      • Guerra M.T.
      • Leite M.F.
      • Spray D.C.
      • Nathanson M.H.
      Endotoxin unmasks the role of gap junctions in the liver.
      ,
      • Gingalewski C.
      • Wang K.
      • Clemens M.G.
      • De Maio A.
      Posttranscriptional regulation of connexin 32 expression in liver during acute inflammation.
      ] as well as in isolated perfused livers [
      • De Maio A.
      • Gingalewski C.
      • Theodorakis N.G.
      • Clemens M.G.
      Interruption of hepatic gap junctional communication in the rat during inflammation induced by bacterial lipopolysaccharide.
      ] treated with LPS. This is paralleled by identical changes at the mRNA level [
      • Temme A.
      • Ott T.
      • Haberberger T.
      • Traub O.
      • Willecke K.
      Acute-phase response and circadian expression of connexin26 are not altered in connexin32-deficient mouse liver.
      ,
      • Gingalewski C.
      • Wang K.
      • Clemens M.G.
      • De Maio A.
      Posttranscriptional regulation of connexin 32 expression in liver during acute inflammation.
      ,
      • De Maio A.
      • Gingalewski C.
      • Theodorakis N.G.
      • Clemens M.G.
      Interruption of hepatic gap junctional communication in the rat during inflammation induced by bacterial lipopolysaccharide.
      ,
      • Theodorakis N.G.
      • De Maio A.
      Cx32 mRNA in rat liver: effects of inflammation on poly(A) tail distribution and mRNA degradation.
      ], which in turn results from increased Cx32 mRNA degradation [
      • Gingalewski C.
      • Wang K.
      • Clemens M.G.
      • De Maio A.
      Posttranscriptional regulation of connexin 32 expression in liver during acute inflammation.
      ,
      • Theodorakis N.G.
      • De Maio A.
      Cx32 mRNA in rat liver: effects of inflammation on poly(A) tail distribution and mRNA degradation.
      ] by shortening of its poly(A) tail [
      • Theodorakis N.G.
      • De Maio A.
      Cx32 mRNA in rat liver: effects of inflammation on poly(A) tail distribution and mRNA degradation.
      ]. Less consistent results have been obtained for Cx26, which can be either downregulated [
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      ,
      • De Maio A.
      • Gingalewski C.
      • Theodorakis N.G.
      • Clemens M.G.
      Interruption of hepatic gap junctional communication in the rat during inflammation induced by bacterial lipopolysaccharide.
      ] or upregulated [
      • Temme A.
      • Ott T.
      • Haberberger T.
      • Traub O.
      • Willecke K.
      Acute-phase response and circadian expression of connexin26 are not altered in connexin32-deficient mouse liver.
      ], whether or not accompanied by modifications in its mRNA production [
      • Temme A.
      • Ott T.
      • Haberberger T.
      • Traub O.
      • Willecke K.
      Acute-phase response and circadian expression of connexin26 are not altered in connexin32-deficient mouse liver.
      ,
      • De Maio A.
      • Gingalewski C.
      • Theodorakis N.G.
      • Clemens M.G.
      Interruption of hepatic gap junctional communication in the rat during inflammation induced by bacterial lipopolysaccharide.
      ]. This becomes even more pronounced when using other inflammatory triggers, such as the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6), both in vitro [
      • Temme A.
      • Traub O.
      • Willecke K.
      Downregulation of connexin32 protein and gap-junctional intercellular communication by cytokine-mediated acute-phase response in immortalized mouse hepatocytes.
      ] and in vivo [
      • Temme A.
      • Ott T.
      • Haberberger T.
      • Traub O.
      • Willecke K.
      Acute-phase response and circadian expression of connexin26 are not altered in connexin32-deficient mouse liver.
      ]. In cultures of primary rat hepatocytes [
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      ,
      • Yamamoto T.
      • Kojima T.
      • Murata M.
      • Takano K.
      • Go M.
      • Chiba H.
      • et al.
      IL-1beta regulates expression of Cx32, occludin, and claudin-2 of rat hepatocytes via distinct signal transduction pathways.
      ] and immortalized mouse hepatocytes [
      • Temme A.
      • Traub O.
      • Willecke K.
      Downregulation of connexin32 protein and gap-junctional intercellular communication by cytokine-mediated acute-phase response in immortalized mouse hepatocytes.
      ], these pro-inflammatory cytokines suppress GJIC and it has been demonstrated that the concomitant reduction of Cx32 steady-state levels is mediated by a MAPK/nuclear factor kappa beta (NF-κβ) signaling cascade [
      • Yamamoto T.
      • Kojima T.
      • Murata M.
      • Takano K.
      • Go M.
      • Chiba H.
      • et al.
      IL-1beta regulates expression of Cx32, occludin, and claudin-2 of rat hepatocytes via distinct signal transduction pathways.
      ]. In contrast, in primary rat stellate cultures and primary rat Kupffer cell cultures exposed to IL-1β [
      • Fischer R.
      • Reinehr R.
      • Lu T.P.
      • Schonicke A.
      • Warskulat U.
      • Dienes H.P.
      • et al.
      Intercellular communication via gap junctions in activated rat hepatic stellate cells.
      ] and LPS/interferon gamma (IFNγ) [
      • Eugenin E.A.
      • Gonzalez H.E.
      • Sanchez H.A.
      • Branes M.C.
      • Saez J.C.
      Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro.
      ], respectively, GJIC becomes more intensified. This is associated with increased Cx43 production, both at the translational and at the transcriptional level [
      • Fischer R.
      • Reinehr R.
      • Lu T.P.
      • Schonicke A.
      • Warskulat U.
      • Dienes H.P.
      • et al.
      Intercellular communication via gap junctions in activated rat hepatic stellate cells.
      ,
      • Eugenin E.A.
      • Gonzalez H.E.
      • Sanchez H.A.
      • Branes M.C.
      • Saez J.C.
      Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro.
      ]. In fact, upon inflammatory challenge, Cx43 protein moves from the cytoplasm to the cell plasma membrane surface in Kupffer cells, where it starts to form functional gap junctions [
      • Eugenin E.A.
      • Gonzalez H.E.
      • Sanchez H.A.
      • Branes M.C.
      • Saez J.C.
      Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro.
      ]. Increases in Cx43 protein amounts have been also observed during liver inflammation in vivo [
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      ,
      • Eugenin E.A.
      • Gonzalez H.E.
      • Sanchez H.A.
      • Branes M.C.
      • Saez J.C.
      Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro.
      ]. It is thought that this is part of the activation of the macrophage activity of Kupffer cells during inflammation, taking care of debris clearance and apoptosis of macrophages and damaged hepatocytes [
      • Eugenin E.A.
      • Gonzalez H.E.
      • Sanchez H.A.
      • Branes M.C.
      • Saez J.C.
      Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro.
      ].

      Gap junctions in liver ischemia and reperfusion injury

      Hepatic ischemia and reperfusion injury occurs in a variety of clinical situations, including trauma, liver resection surgery, and liver transplantation. The mechanism of liver damage after ischemia and reperfusion is complex and relies on an interplay of multiple pathways [
      • Dogan S.
      • Aslan M.
      Hepatic ischemia-reperfusion injury and therapeutic strategies to alleviate cellular damage.
      ,
      • Klune J.R.
      • Tsung A.
      Molecular biology of liver ischemia/reperfusion injury: established mechanisms and recent advancements.
      ,
      • Zhai Y.
      • Busuttil R.W.
      • Kupiec-Weglinski J.W.
      Liver ischemia and reperfusion injury: new insights into mechanisms of innate-adaptive immune-mediated tissue inflammation.
      ]. Changes in hepatocellular gap junction ultrastructure are known to take place during ischemia in rat in vivo [
      • Schellens J.P.
      • Blange T.
      • de Groot K.
      Gap junction ultrastructure in rat liver parenchymal cells after in vivo ischemia.
      ]. In a rat model of partial liver ischemia and reperfusion, Cx26 and Cx32 expression levels decreased during ischemia, but were induced during the early reperfusion phase, which holds true especially for Cx26. In later phases, deterioration of connexin production was noticed. Identical changes were seen in concentration of calcium, which is known to move intercellularly through gap junctions [
      • Nakashima Y.
      • Kohno H.
      • ON E.L.-A.
      • Dhar D.K.
      • Ono T.
      • Yamanoi A.
      • et al.
      Sequential changes of connexin32 and connexin26 in ischemia-reperfusion of the liver in rats.
      ]. In a similar experimental model, it was found that the reductions in Cx32 mRNA and protein amounts occur at different time points and are driven by different posttranscriptional and posttranslational mechanisms in ischemic and non-ischemic liver areas during reperfusion [
      • Gingalewski C.
      • De Maio A.
      Differential decrease in connexin 32 expression in ischemic and nonischemic regions of rat liver during ischemia/reperfusion.
      ]. In addition to a pronounced inflammatory response, a major factor that contributes to ischemia and reperfusion injury includes the generation of oxygen-derived free radicals [
      • Dogan S.
      • Aslan M.
      Hepatic ischemia-reperfusion injury and therapeutic strategies to alleviate cellular damage.
      ,
      • Klune J.R.
      • Tsung A.
      Molecular biology of liver ischemia/reperfusion injury: established mechanisms and recent advancements.
      ,
      • Zhai Y.
      • Busuttil R.W.
      • Kupiec-Weglinski J.W.
      Liver ischemia and reperfusion injury: new insights into mechanisms of innate-adaptive immune-mediated tissue inflammation.
      ]. In this regard, hydrogen peroxide caused a suppression of Cx32 protein levels and GJIC in cultures of primary rat hepatocytes [
      • Fukuda T.
      • Ikejima K.
      • Hirose M.
      • Takei Y.
      • Watanabe S.
      • Sato N.
      Taurine preserves gap junctional intercellular communication in rat hepatocytes under oxidative stress.
      ]. Hydrogen peroxide and paraquat also induced oxidative stress and blocked GJIC in primary mouse hepatocyte cultures [
      • Ruch R.J.
      • Klaunig J.E.
      Inhibition of mouse hepatocyte intercellular communication by paraquat-generated oxygen free radicals.
      ]. Administration of ochratoxin A to rats resulted in hepatotoxicity associated with mild oxidative stress and the downregulation of Cx26, Cx32, and Cx43 expression [
      • Gagliano N.
      • Donne I.D.
      • Torri C.
      • Migliori M.
      • Grizzi F.
      • Milzani A.
      • et al.
      Early cytotoxic effects of ochratoxin A in rat liver: a morphological, biochemical and molecular study.
      ]. Cx32 production was equally compromised by hepatic oxidative stress induced by administration of furan to rats [
      • Hickling K.C.
      • Hitchcock J.M.
      • Oreffo V.
      • Mally A.
      • Hammond T.G.
      • Evans J.G.
      • et al.
      Evidence of oxidative stress and associated DNA damage, increased proliferative drive, and altered gene expression in rat liver produced by the cholangiocarcinogenic agent furan.
      ]. Oxidative stress in primary hepatocyte cultures was found to be associated with different cis/trans regulation of the Cx32 gene [
      • Morsi A.S.
      • Godfrey R.E.
      • Chipman J.K.
      • Minchin S.D.
      Characterisation of the connexin32 promoter and changes in response element complexes in rat liver and hepatocytes during culture associated with oxidative stress.
      ].

      Conclusions and perspectives

      Inherent to their key role in liver homeostasis, gap junction functionality is typically altered during aberration of this critical equilibrium as exemplified in the current review for a number of pathological situations (Table 1, Table 2, Table 3). Although exceptions do exist, it can be generalized from this literature survey that Cx32 expression progressively deteriorates upon progression of liver disease, while Cx43 production is gradually promoted. This becomes even more pronounced in liver cancer, which is frequently the end stage of chronic liver pathology [
      • Nakashima Y.
      • Ono T.
      • Yamanoi A.
      • El-Assal O.N.
      • Kohno H.
      • Nagasue N.
      Expression of gap junction protein connexin32 in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.
      ,
      • Oyamada M.
      • Krutovskikh V.A.
      • Mesnil M.
      • Partensky C.
      • Berger F.
      • Yamasaki H.
      Aberrant expression of gap junction gene in primary human hepatocellular carcinomas: increased expression of cardiac-type gap junction gene connexin 43.
      ]. Therefore, Cx43 could be considered as a hepatic stress connexin and could serve a diagnostic role as a biomarker in clinical settings. With respect to this, it has been recently demonstrated that alterations in connexin expression, particularly increased Cx43 production, is an early indicator of chronic kidney disease. Cx43 was hereby suggested to amplify calcium signaling and to spread inflammatory messengers [
      • Toubas J.
      • Beck S.
      • Pageaud A.L.
      • Huby A.C.
      • Mael-Ainin M.
      • Dussaule J.C.
      • et al.
      Alteration of connexin expression is an early signal for chronic kidney disease.
      ]. A similar scenario may take place in the liver. The exact role of gap junctions in hepatic stress situations and its functional implications are, however, unclear. Thus, no differences in the expression of positive (e.g. β-fibrinogen) and negative (e.g. albumin) acute phase transcripts were observed following administration of cytokines and LPS to Cx32-deficient mice compared with wild-type mice, suggesting that Cx32 does not play a role in experimental liver inflammation [
      • Temme A.
      • Ott T.
      • Haberberger T.
      • Traub O.
      • Willecke K.
      Acute-phase response and circadian expression of connexin26 are not altered in connexin32-deficient mouse liver.
      ]. Correa and colleagues showed that Cx32-based gap junctions are indispensable in model animals during the recovery from hypoglycemia and cholestasis upon LPS-triggered systemic inflammation [
      • Correa P.R.
      • Guerra M.T.
      • Leite M.F.
      • Spray D.C.
      • Nathanson M.H.
      Endotoxin unmasks the role of gap junctions in the liver.
      ]. On the other hand, increases in serum ALT and AST levels and the occurrence of cell death were less manifested in Cx32 dominant-negative mutant transgenic rats that received carbon tetrachloride in comparison with their wild-type counterparts, pointing to a role for GJIC in the dissemination of cell injury and cell death signals [
      • Asamoto M.
      • Hokaiwado N.
      • Murasaki T.
      • Shirai T.
      Connexin 32 dominant-negative mutant transgenic rats are resistant to hepatic damage by chemicals.
      ]. Further research in this direction is necessary and needs to take into account the possible involvement of new players in the gap junction arena, including hemichannels and pannexin channels. Indeed, hemichannels not only are gap junction building blocks, but also provide a route for communication between the cytoplasm and the extracellular environment [
      • Vinken M.
      • Vanhaecke T.
      • Rogiers V.
      Emerging roles of connexin hemichannels in gastrointestinal and liver pathophysiology.
      ]. A similar type of channels is formed by pannexins (Panx), that are connexin-like proteins of which three family members have been characterized in humans [
      • D’Hondt C.
      • Ponsaerts R.
      • De Smedt H.
      • Vinken M.
      • De Vuyst E.
      • De Bock M.
      • et al.
      Pannexin channels in ATP release and beyond: an unexpected rendezvous at the endoplasmic reticulum.
      ], with Panx1 and Panx2 being expressed in the liver [
      • Bruzzone R.
      • Hormuzdi S.G.
      • Barbe M.T.
      • Herb A.
      • Monyer H.
      Pannexins, a family of gap junction proteins expressed in brain.
      ]. Both hemichannels and pannexin channels are known to act as pathological pores, though specific information in relation to the liver is scarce [
      • Vinken M.
      • Decrock E.
      • De Vuyst E.
      • De Bock M.
      • Vandenbroucke R.E.
      • De Geest B.G.
      • et al.
      Connexin32 hemichannels contribute to the apoptotic-to-necrotic transition during Fas-mediated hepatocyte cell death.
      ]. Furthermore, hepatic connexins are known to interact with tight junction proteins, including occludin and zonula occludens proteins, which contribute to the pathogenesis of liver disease, in particular cholestasis, upon dysregulation [
      • Kojima T.
      • Yamamoto T.
      • Murata M.
      • Chiba H.
      • Kokai Y.
      • Sawada N.
      Regulation of the blood-biliary barrier: interaction between gap and tight junctions in hepatocytes.
      ]. Besides serving diagnostic purposes, connexins and their channels are also interesting therapeutic targets. Dexamethasone, for instance, counteracts decreased Cx32 protein levels and GJIC induced by pro-inflammatory cytokines in cultured hepatocytes [
      • Temme A.
      • Traub O.
      • Willecke K.
      Downregulation of connexin32 protein and gap-junctional intercellular communication by cytokine-mediated acute-phase response in immortalized mouse hepatocytes.
      ]. Carbenoxolone, a prototypical GJIC inhibitor, suppressed DNA synthesis and collagen production in cultures of activated stellate cells [
      • Uyama N.
      • Shimahara Y.
      • Okuyama H.
      • Kawada N.
      • Kamo S.
      • Ikeda K.
      • et al.
      Carbenoxolone inhibits DNA synthesis and collagen gene expression in rat hepatic stellate cells in culture.
      ]. As dysregulated GJIC underlies a multitude of diseases, a large-scale search for specific GJIC modifying tools, especially those that are applicable in vivo, is currently ongoing [
      • Bodendiek S.B.
      • Raman G.
      Connexin modulators and their potential targets under the magnifying glass.
      ]. It can be expected that this will result in a number of agents that can be potentially used for the clinical treatment of liver diseases.
      Table 1Effects of cholestasis on liver gap junctions.
      • Robenek H.
      • Herwig J.
      • Themann H.
      The morphologic characteristics of intercellular junctions between normal human liver cells and cells from patients with extrahepatic cholestasis.
      ,
      • Robenek H.
      • Rassat J.
      • Themann H.
      A quantitative freeze-fracture analysis of gap and tight junctions in the normal and cholestatic human liver.
      ,
      • Snigerevskaia E.S.
      • Veselov V.S.
      Changes in the structure of the plasmalemma of hepatocytes and its specialized portions: intercellular junctions in man in complicated forms of cholelithiasis.
      ,
      • De Vos R.
      • Desmet V.
      Morphology of liver cell tight junctions in ethinyl estradiol induced cholestasis.
      ,
      • Grosser V.
      • Robenek H.
      • Rassat J.
      • Themann H.
      Ultrastructural study of cholestasis induced by longterm treatment with estradiol valerate. II. Gap junctional analysis.
      ,
      • van Hengel J.
      • D’Hooge P.
      • Hooghe B.
      • Wu X.
      • Libbrecht L.
      • De Vos R.
      • et al.
      Continuous cell injury promotes hepatic tumorigenesis in cdc42-deficient mouse liver.
      ,
      • Youson J.H.
      • Ellis L.C.
      • Ogilvie D.
      • Shivers R.R.
      Gap junctions and zonulae occludentes of hepatocytes during biliary atresia in the lamprey.
      Cdc42, cell division cycle 42; Cx, connexin; GJIC, gap junctional intercellular communication; p38 MAPK, p38 mitogen-activated protein kinase.
      ↑, upregulation; ↓, downregulation; ∼, no modification.
      Table 2Effects of hepatic fibrosis and cirrhosis on liver gap junctions.
      • James J.L.
      • Friend D.S.
      • MacDonald J.R.
      • Smuckler E.A.
      Alterations in hepatocyte plasma membrane in carbon tetrachloride poisoning: freeze-fracture analysis of gap junction and electron spin resonance analysis of lipid fluidity.
      ,
      • Ruch R.J.
      • Klaunig J.E.
      Effects of tumor promoters, genotoxic carcinogens and hepatocytotoxins on mouse hepatocyte intercellular communication.
      Cx, connexin; GJIC, gap junctional intercellular communication.
      ↑, upregulation; ↓, downregulation; ∼, no modification.
      Table 3Effects of hepatitis and systemic inflammation on liver gap junctions.
      Cx, connexin; GJIC, gap junctional intercellular communication; IFNγ, interferon gamma; IL-1(β)/2/6, interleukin 1 (beta)/2/6; LPS, lipopolysaccharide; NF-κβ, nuclear factor kappa beta; MAPK, mitogen-activated protein kinase; TNFα, tumor necrosis factor alpha.
      ↑, upregulation; ↓, downregulation; ∼, no modification.

      Conflict of interest

      The author declares that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Acknowledgements

      This work was financially supported by the grants of the Research Council of the Vrije Universiteit Brussel (OZR-VUB) and the Fund for Scientific Research Flanders (FWO-Vlaanderen).

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