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TMEM16A is a Ca2+-activated Cl−channel in the apical membrane of biliary epithelial cells, known as cholangiocytes, which contributes importantly to ductular bile formation. Whereas cholangiocyte TMEM16A activity is regulated by extracellular ATP-binding membrane purinergic receptors, channel expression is regulated by interleukin-4 (IL-4) through an unknown mechanism. Therefore, the aim of the present study was to identify the signaling pathways involved in TMEM16A expression and cholangiocyte secretion. Studies were performed in polarized normal rat cholangiocyte monolayers, human Mz-Cha-1 biliary cells, and cholangiocytes isolated from murine liver tissue. The results demonstrate that all the biliary models expressed the IL-4Rα/IL-13Rα1 receptor complex. Incubation of cholangiocytes with either IL-13 or IL-4 increased the expression of TMEM16A protein, which was associated with an increase in the magnitude of Ca2+-activated Cl−currents in response to ATP in single cells and the short-circuit current response in polarized monolayers. The IL-4- and IL-13-mediated increase in TMEM16A expression was also associated with an increase in STAT6 phosphorylation. Specific inhibition of JAK-3 inhibited the increase in TMEM16A expression and the IL-4-mediated increase in ATP-stimulated currents, whereas inhibition of STAT6 inhibited both IL-4- and IL-13-mediated increases in TMEM16A expression and ATP-stimulated secretion. These studies demonstrate that the cytokines IL-13 and IL-4 regulate the expression and function of biliary TMEM16A channels through a signaling pathway involving STAT6. Identification of this regulatory pathway provides new insight into biliary secretion and suggests new targets to enhance bile formation in the treatment of cholestatic liver disorders.NEW & NOTEWORTHY The Ca2+-activated Cl−channel transmembrane member 16A (TMEM16A) has emerged as an important regulator of biliary secretion and hence, ductular bile formation. The present studies represent the initial description of the regulation of TMEM16A expression in biliary epithelium. Identification of this regulatory pathway involving the IL-4 and IL-13 receptor complex and JAK-3 and STAT-6 signaling provides new insight into biliary secretion and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.

Myofibroblasts are the main cell types producing extracellular matrix proteins in a variety of fibrotic diseases. Therefore, they are useful targets for studies of intracellular communication and gene therapeutical approaches in scarring diseases. An artificial promoter containing the -702 bp regulatory sequence of the alpha-smooth muscle actin (SMA) gene linked to the first intron enhancer sequence of the beta-actin gene and the beta-globin intron-exon junction was constructed and tested for myofibroblast-dependent gene expression using the green fluorescent protein as a reporter. Reporter expression revealed myofibroblast-specific function in hepatic and renal myofibroblasts, in vitro. In addition, differentiation-dependent activation of the SMA-beta-actin promoter hybrid was shown after induction of myofibroblastic features in mesangial cells by stretching treatment. Furthermore, wound healing experiments with SMA-beta-actin promoter reporter mice demonstrated myofibroblast-specific action, in vivo. In conclusion, the -702 bp regulatory region of the SMA promoter linked to enhancing beta-actin and beta-globin sequences benefits from its small size and is suggested as a promising tool to target myofibroblasts as the crucial cell type in various scarring processes.

In liver wound healing, transforming growth factor-β (TGF-β) plays a critical role in stellate cell activation as well as signaling cascades in the fibrogenic response to injury. We postulate that the TGF-β-dependent downstream signaling pathway may vary according to the mechanism of stellate cell activation; this study was undertaken to ascertain whether the downstream signaling pathways mediated by TGF-β vary in different liver injury models. We measured Smad3 and MAP kinase activation after isolating stellate cells from rat livers injured by either bile duct ligation (BDL) or repeated carbon tetrachloride (CCl 4 ) administration. Phospho-Smad3 was dramatically up-regulated in stellate cells after CCl 4 injury, but not after BDL-induced injury. TGF-β signaling in stellate cells activated after BDL was mediated prominently through ERK activation, whereas activation induced by CCl 4 injury or culture led to a cross-signaling mechanism involving both Smad3 and p38. The divergent Smad signaling pathways observed appeared to be attributable to the differential regulation of the early growth response gene-1 (Egr-1), an apparent negative transcriptional factor for Smad3 in our system. In addition, inhibition of ERK activation in stellate cells from BDL-injured liver led to a decrease in expression of endothelin-converting enzyme-1, a critical regulator of endothelin-1. We speculate that TGF-β signaling proceeds through differential signaling pathways depending on the mechanism of liver injury that leads to stellate cell activation.

Hepatocyte lipoapoptosis induced by saturated free fatty acids (FFA) contributes to hepatic inflammation in lipotoxic liver injury, and the cellular mechanisms involved have not been defined. Recent studies have shown that apoptosis in nonhepatic cells stimulates ATP release via activation of pannexin1 (panx1), and extracellular ATP functions as a proinflammatory signal for recruitment and activation of the inflammatory cells. However, it is not known whether lipoapoptosis stimulates ATP release in liver cells. We found that lipoapoptosis induced by saturated FFA stimulated ATP release in liver cells that increased extracellular ATP concentration by more than fivefold above the values observed in healthy cells. This sustained pathophysiological ATP release was not dependent on caspase-3/7 activation. Inhibition of c-Jun NH2-terminal kinase (JNK), a key mediator of lipoapoptosis, with SP600125 blocked pathophysiological ATP release in a dose-dependent manner. RT-PCR analysis indicated that panx1 is expressed in hepatocytes and multiple liver cell lines. Notably, inhibition of panx1 expression with short hairpin (sh)RNA inhibited in part pathophysiological ATP release. Moreover, lipoapoptosis stimulated uptake of a membrane impermeable dye YoPro-1 (indicative of panx1 activation), which was inhibited by panx1 shRNA, probenecid, and mefloquine. These results suggest that panx1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated FFA. Thus panx1 may play an important role in hepatic inflammation by mediating an increase in extracellular ATP concentration in lipotoxic liver injury.

Liver wound healing is a coordinated response to injury caused by infections (hepatitis) or toxins (alcohol) or other processes where activation of hepatic stellate cells are a central component. During stellate cell activation, a major phenotypic transformation occurs which leads to increased production of increased extracellular matrix proteins and smooth muscle α-actin the results is organ dysfunction due to gross architectural disruption and impaired blood flow. Endothelin-1 (ET-1) is produced in increased amounts and the cellular source of ET-1 shifts from endothelial cells to stellate cells during liver injury thus setting a feedback loop which accentuates further activation, stellate cell proliferation, and production of extracellular matrix proteins. Therapy directed at intervening the ET-1 signaling pathway has significant therapeutic potential in patients with liver disease.

Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.

In the liver, adenosine triphosphate (ATP) is an extracellular signaling molecule that is released into bile and stimulates a biliary epithelial cell secretory response via engagement of apical P2 receptors. The molecular identities of the ion channels involved in ATP-mediated secretory responses have not been fully identified. Intermediate-conductance Ca2+-activated K+ channels (IK) have been identified in biliary epithelium, but functional data are lacking. The aim of these studies therefore was to determine the location, function, and regulation of IK channels in biliary epithelial cells and to determine their potential contribution to ATP-stimulated secretion. Expression of IK-1 mRNA was found in both human Mz-Cha-1 biliary cells and polarized normal rat cholangiocyte (NRC) monolayers, and immunostaining revealed membrane localization with a predominant basolateral signal. In single Mz-Cha-1 cells, exposure to ATP activated K+ currents, increasing current density from 1.6 ± 0.1 to 7.6 ± 0.8 pA/pF. Currents were dependent on intracellular Ca2+ and sensitive to clotrimazole and TRAM-34 (specific IK channel inhibitors). Single-channel recording demonstrated that clotrimazole-sensitive K+ currents had a unitary conductance of 46.2 ± 1.5 pS, consistent with IK channels. In separate studies, 1-EBIO (an IK activator) stimulated K+ currents in single cells that were inhibited by clotrimazole. In polarized NRC monolayers, ATP significantly increased transepithelial secretion which was inhibited by clotrimazole. Lastly, ATP-stimulated K+ currents were inhibited by the P2Y receptor antagonist suramin and by the inositol 1,4,5-triphosphate (IP3) receptor inhibitor 2-APB. Together these studies demonstrate that IK channels are present in biliary epithelial cells and contribute to ATP-stimulated secretion through a P2Y-IP3 receptor pathway.