In a variety of cells, bile acids have been shown to function as intracellular signals and to profoundly alter cellular functions such as proliferation, differentiation, secretion, and apoptosis[56-61]. These studies have shown that cellular uptake is required for bile acids to signal cellular processes. In cells not expressing a bile acid transporter, (which normally do not respond to the presence of bile acids in the media) experimental expression of a bile acid transporter activates de novo bile acid signaling. Once intracellular, bile acids at concentrations of less that 1 mmol/L, have been shown to alter intracellular Ca2+, PKC, MEK, ERK and PI3K pathways in hepatocytes or cholangiocytes[16,59,61,63-66]. Through these downstream signals, bile acids have been shown to alter cell proliferation, secretion, apoptosis and gene expression[8,16,61,63-67]. In addition, bile acids have been shown to induce activation (by phosphorylation) of the EGF receptor in hepatocytes and cholangiocytes. The signaling effects of bile acids should be distinguished from the toxic effects of bile acids, where bile acid in high concentration, through changes in membrane lipids induces, in a nonspecific manner, cellular damage.
Bile acid signaling of cholangiocyte secretion
It had previously been observed that ursodeoxycholic acid increases biliary bicarbonate excretion into bile. Shimokura et al found that ursodeoxycholic acid directly increases cholangiocyte secretion. They demonstrated ursodeoxycholic acid increases intracellular Ca2+ and increases chloride channel activity in a malignant cho-langiocyte cell line. The ursodeoxycholate effect on chloride channel activity was dependent on the increase in intracellular Ca2+. Our studies have shown that in freshly isolated cholangiocytes, taurocholic acid or taurolithocholic acid (1-20 mmol/L) increases secretin-stimulated cAMP levels and secretin-stimulated Cl-/HCO3 exchanger activity. These effects were dependent on taurocholate uptake by ASBT, since the stimulatory effect of taurocholate was not present in the absence of Na+. Dependence of bile acid effects on cholangiocyte secretion and ASBT transport activity, we found the Km for ASBT in cholangiocytes to be close to the concentration where in bile acids exert their maximum response in cholangiocytes. Similar to their effect in vitro, taurocholate or taurolithocholate feeding to normal rats for 7 d increased secretin-stimulated cAMP and Cl-/HCO3- exchanger activity in cholangiocytes and resulted in an increase of secretin-stimulated ductal bile flow in vitro. The studies show that both in vitro and in vivo, bile acids can augment secretin-stimulated ductal bile flow but the intracellular signals responsible for this effect have not been completely elucidated. Recently, cAMPindependent, bile activation of CFTR was shown to be present in ileal cells. Like cholangiocytes, ASBT-dependent bile acid uptake was required for CFTR activation but the molecular mechanism for CFTR activation was not disclosed in this study.
In contrast to the stimulatory effects of taurocholate and taurolithocholate, ursodeoxycholate inhibits secretin-stimulated cAMP synthesis and Cl-/HCO3-exchanger activity in isolated cholangiocytes and secretin-stimulated ductal bile flow in vivo. Inhibition of cholangiocyte secretion by ursodeoxycholic acid was found to be dependent on the ability of ursodeoxycholic acid to increase intracellular calcium and activate PKCalpha in cholangiocytes. We have proposed that the stimulatory and inhibitory effects of taurocholate and ursodeoxycholate, respectively on cholangiocyte secretion is due to their differing ability to activate intracellular Ca2+ and to activate different PKC isoforms.
Bile acid signaling of cholangiocyte proliferation
In vitro, taurocholate and taurolithocholate in 1-20 mmol/L concentrations increase H3-histone expression (a maker of cholangiocyte proliferation). Feeding taurocholate or taurolithocholate to rats increases 3H-thymindine uptake in cholangiocytes, increases bile duct mass 2 to 3 fold and consistent with a ductal hyperplasia there is accentuation of secretin-stimulated cAMP synthesis and ductal bile flow. The effects of taurocholate and taurolithocholate on bile duct proliferation in the bile acid feeding models occur in the absence hepatic inflammation. Recent studies show that taurocholate can induce phosphorylation of EGF receptor in cholangiocytes, similar to that reported in hepatocytes and the transactivation of the EGF receptor requires transforming growth factor alpha and matrix metalloproteinase.
In contrast to taurocholate and taurolithocholate, ursodeoxycholate inhibits cholangiocyte proliferation both in vitro in isolated cholangiocytes and in vivo in bile duct ligated rats. The inhibition of cholangiocyte proliferation was found to be dependent on activation of PKCalpha and calcium-dependent pathways. The inhibitory effect of ursodeoxycholate on cholangiocyte proliferation may be one mechanism for the histological and biochemical improvement of diseases targeting the biliary tree (e.g. primary biliary cirrhosis) with ursodeoxycholate treatment. Inhibition of cholangiocyte proliferation may reduce the number of proliferating cholangiocytes that release proinflammatory cytokines or profibrotic signaling molecules such as platelet-derived growth factor. The lack of therapeutic effect for ursodeoxycholic acid in the late stage primary biliary cirrhosis may be at least partially related to lack of proliferating ducts (e.g. ductopenia) as the disease progresses. In a cholangiocarcinoma cell line, we demonstrated that ursodeoxycholic acid inhibits growth by inhibition of Raf through PKC-dependent mechanism. This study supports the need for clinical trials examining the effect of ursodeoxycholic acid in the promotion and progression of cholangiocarcinoma in patients with primary sclerosing cholangitis.
Bile acid signaling of cholangiocyte death and survival pathways
Previous studies in hepatocytes have shown bile acids may be either cytotoxic[75-78] or cytoprotective. Bile acid cytotoxicity may be induced by abrupt permeability of the inner mitochondrial membrane to ions leading to mitochondrial membrane permeability transition (MMPT), depolarization of the mitochondrial membrane potential and uncoupling of oxidative phosphorylation. The uncoupling of oxidative phosphorylation, if extensive, results in ATP depletion and cellular death by necrosis. Furthermore, the associated mitochondrial swelling has also been linked to redistribution of cytochrome c from the intermembrane space to the cytosol. In the cytosol, cytochrome c interacts with apoptotic protease-activating factor 1 to activate caspase 9 and subsequently to cause apoptosis. Bile salt-induced hepatocyte apoptosis also entails activation of the Fas death-receptor and subsequent activation of caspase 8 followed by activation of Bid which leads to mitochondrial dysfunction.
Alternatively, bile acids may provide cytoprotective effects. Heuman et al proposed that the protective effect of ursodeoxycholic acid in opposing the hepatotoxicity of bile acids was due to its direct interaction with plasma membranes of hepatocytes. Ursodeoxycholic acid may provide membrane stability via a physicochemical effect by reducing the toxic bile salt disruption of cholesterol-rich model membranes. More recent studies, revealed that ursodeoxycholic acid does not directly stabilize membranes but rather prevents hydrophobic bile acid-induced membrane disruption by alteration of the structure and composition of mixed micelles.
In cholangiocytes, Benedetti et al showed that in vitro, unconjugated but not conjugated bile acids induce ultrastructural evidence of cytotoxicity. These findings were not observed in vivo. Even taurine depleted livers did not show evidence of cytotoxicity despite having high concentration of unconjugated bile acids in bile. The authors concluded that bile acid toxicity, although potentially present in biliary epithelium, is prevented in the intact liver by the presence of active bile acid transport in cholangiocytes.
Our studies have shown that taurocholate feeding is protective against cholangiocyte apoptosis induced by either CCl4 or vagotomy. In CCl4 treated animals, cholangiocyte apoptosis, demonstrated by the presence of nuclear fragmentation, positive annexin staining and loss of cholangiocyte function (secretin-stimulated cAMP synthesis), was not observed in CCl4-treated rats that were fed taurocholate. Similarly, CCl4-induced apoptosis in vitro was ablated by pretreating cholangiocytes with 20 mmol/L taurocholate. The taurocholate inhibition of CCl4-induced apoptosis required activation of PKCalpha. Taurocholate feeding also prevents vagotomy induced cholangiocyte apoptosis. In the vagotomy-induced model, apoptosis is associated with loss of PI3K activity and activation of caspase activities. Taurocholate feeding prevented cholangiocyte apoptosis, loss of PI3K activity and activation of caspase activity. Thus, taurocholate is protective against CCl4- and vagotomy-induced cholangiocyte apoptosis by activation of the PKC and PI3K-dependent pathways, respectively.
Feeding ursodeoxycholate inhibits the ductal hyperplasia in BDL rats, however these studies showed that the effect of ursodeoxycholate was on inhibition of proliferation without increasing cholangiocyte apoptosis. In contrast, Que et al showed that ursodeoxycholate inhibits beauvericin-induced apoptosis in a cholangiocarcinoma cell line and that the inhibition was dependent on preventing cytochrome C release from mitochondria and subsequent activation of caspases.