Endoplasmic Reticulum stress induces hepatic stellate cell apoptosis and contributes to fibrosis resolution.

BACKGROUND: Survival of hepatic stellate cells (HSCs) is a hallmark of liver fibrosis, while the induction of HSC apoptosis may induce recovery. Activated HSC are resistant to many pro-apoptotic stimuli. To this issue, the role of Endoplasmic Reticulum (ER) stress in promoting apoptosis of HSCs and consequently fibrosis resolution is still debated. AIM: To evaluate the potential ER stress-mediated apoptosis of HSCs and fibrosis resolution METHODS: HSCs were incubated with the ER stress agonists, tunicamycin or thapsigargin. In vivo, HSC were isolated from normal, bile duct-ligated (BDL) and bile duct-diverted (BDD) rats. RESULTS: In activated HSC, the specific inhibitor of ER stress-induced apoptosis, calpastatin, is significantly increased vs. quiescent HSCs. Calpain is conversely reduced in activated HSCs. This pattern of protein expression provides HSCs resistance to the ER stress signals of apoptosis (apoptosis-resistant phenotype). However, both tunicamycin and thapsigargin are able to induce apoptosis in HSCs in vitro, completely reversing the calpain/calpastatin pattern expression. Furthermore, in vivo, the fibrosis resolution observed in rat livers subjected to bile duct ligation (BDL) and subsequent bile duct diversion (BDD), leads to fibrosis resolution through a mechanism of HSCs apoptosis, potentially associated with ER stress: in fact, BDD rat liver shows an increased number of apoptotic HSCs associated with reduced calapstatin and increased calpain protein expression, leading to an apoptosis-sensible phenotype. CONCLUSIONS: ER stress sensitizes HSC to apoptosis both in vitro and in vivo. Thus, ER stress represents a key target to trigger cell death in activated HSC and promotes fibrosis resolution.

Prolactin stimulates the proliferation of normal female cholangiocytes by differential regulation of Ca2+-dependent PKC isoforms.

BACKGROUND: Prolactin promotes proliferation of several cells. Prolactin receptor exists as two isoforms: long and short, which activate different transduction pathways including the Ca2+-dependent PKC-signaling. No information exists on the role of prolactin in the regulation of the growth of female cholangiocytes. The rationale for using cholangiocytes from female rats is based on the fact that women are preferentially affected by specific cholangiopathies including primary biliary cirrhosis. We propose to evaluate the role and mechanisms of action by which prolactin regulates the growth of female cholangiocytes. RESULTS: Normal cholangiocytes express both isoforms (long and short) of prolactin receptors, whose expression increased following BDL. The administration of prolactin to normal female rats increased cholangiocyte proliferation. In purified normal female cholangiocytes, prolactin stimulated cholangiocyte proliferation, which was associated with increased [Ca2+]i levels and PKCbeta-I phosphorylation but decreased PKCalpha phosphorylation. Administration of an anti-prolactin antibody to BDL female rats decreased cholangiocyte proliferation. Normal female cholangiocytes express and secrete prolactin, which was increased in BDL rats. The data show that prolactin stimulates normal cholangiocyte growth by an autocrine mechanism involving phosphorylation of PKCbeta-I and dephosphorylation of PKCalpha. CONCLUSION: We suggest that in female rats: (i) prolactin has a trophic effect on the growth of normal cholangiocytes by phosphorylation of PKCbeta-I and dephosphorylation of PKCalpha; and (iii) cholangiocytes express and secrete prolactin, which by an autocrine mechanism participate in regulation of cholangiocyte proliferation. Prolactin may be an important therapeutic approach for the management of cholangiopathies affecting female patients.

Vascular endothelial growth factor stimulates rat cholangiocyte proliferation via an autocrine mechanism.

BACKGROUND & AIMS: Vascular endothelial growth factor (VEGF) is secreted by several epithelia and modulates cellular functions by autocrine and paracrine mechanisms. The role of VEGF in cholangiocyte pathophysiology is unknown. We evaluated the role of VEGF in the regulation of cholangiocyte proliferation in rats that underwent bile duct ligation. METHODS: The expression of VEGF-A and VEGF-C and their receptors in cholangiocytes from normal and BDL rats was evaluated. Normal or BDL rats were treated with recombinant-VEGF-A or recombinant-VEGF-C or anti-VEGF antibodies, and proliferation of cholangiocytes was evaluated in situ by morphometry and in vitro by proliferating cell nuclear antigen immunoblots and MTS assay. In vitro, normal rat cholangiocyte cultures were stimulated with r-VEGF-A or r-VEGF-C and proliferation and signal transduction were evaluated. RESULTS: We found that (1) cholangiocytes express messenger RNA and protein for VEGF-A, VEGF-C, VEGF receptor 2 (VEGFR-2), and VEGF receptor 3 (VEGFR-3) and secrete VEGF; (2) secretion of VEGF and expression of VEGFR-2 and VEGFR-3 increases in BDL cholangiocytes; (3) blocking VEGF in vivo by anti-VEGF-A or anti-VEGF-C antibodies decreases cholangiocyte proliferation; (4) the in vivo administration of r-VEGF-A or r-VEGF-C induces cholangiocyte proliferation in normal rats; and (5) in vitro, VEGF-A increases normal rat cholangiocyte culture proliferation by activation of inositol 1,4,5-triphosphate/Ca2+/protein kinase C alpha and phosphorylation of Src/ERK1/2. CONCLUSIONS: Cholangiocytes secrete VEGF and express VEGFR-2 and VEGFR-3, all of which are amplified in BDL cholangiocytes. VEGF induces cholangiocyte proliferation by activation of inositol 1,4,5-triphosphate/[Ca2+]i/protein kinase C alpha and phosphorylation of Src/ERK1/2. VEGF mediates the adaptive proliferative response of cholangiocytes to cholestasis.

gamma-Aminobutyric acid inhibits cholangiocarcinoma growth by cyclic AMP-dependent regulation of the protein kinase A/extracellular signal-regulated kinase 1/2 pathway.

We studied the effect of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), in the regulation of cholangiocarcinoma growth. We determined the in vitro effect of GABA on the proliferation of the cholangiocarcinoma cell lines (Mz-ChA-1, HuH-28, and TFK-1) and evaluated the intracellular pathways involved. The effect of GABA on migration of Mz-ChA-1 cells was also evaluated. In vivo, Mz-ChA-1 cells were s.c. injected in athymic mice, and the effects of GABA on tumor size, tumor cell proliferation, apoptosis, collagen quantity, and the expression of vascular endothelial growth factor-A (VEGF-A) and VEGF-C (cancer growth regulators) were measured after 82 days. GABA decreased in vitro cholangiocarcinoma growth in a time-dependent and dose-dependent manner, by both cyclic AMP/protein kinase A- and D-myo-inositol-1,4,5-thriphosphate/Ca(2+)-dependent pathways, leading to down-regulation of extracellular signal-regulated kinase 1/2 phosphorylation. Blocking of GABA(A), GABA(B), and GABA(C) receptors prevented GABA inhibition of cholangiocarcinoma proliferation. GABA inhibited Mz-ChA-1 cell migration and, in vivo, significantly decreased tumor volume, tumor cell proliferation, and VEGF-A/C expression whereas increasing apoptosis compared with controls. An increase in collagen was evident in GABA-treated tumors. GABA decreases biliary cancer proliferation and reduces the metastatic potential of cholangiocarcinoma. GABA may represent a therapeutic agent for patients affected by malignancies of the biliary tract.

Tannic acid inhibits cholangiocyte proliferation after bile duct ligation via a cyclic adenosine 5',3'-monophosphate-dependent pathway.

Chronic cholestatic diseases are characterized by morphological changes involving cholangiocyte proliferation and functional alterations of secretory capacity. The plant polyphenol tannic acid inhibits the growth of malignant human cholangiocytes. However, the mechanisms by which tannic acid limits excessive cholangiocyte proliferation are unknown. In this study we assessed the effect of tannic acid on cholangiocyte proliferation after bile duct ligation in rats. Tannic acid feeding decreased cholangiocyte proliferation and ductal mass in vivo after bile duct ligation. These changes were associated with functional changes in bile secretion and with decreases of intracellular cyclic adenosine 5',3'-monophosphate. The anti-proliferative effect of tannic acid was associated with a reduction of ERK1,2 phosphorylation. Additionally, tannic acid feeding decreased protein kinase A phosphorylation and activity. Similar changes were observed in isolated cholangiocytes during in vitro incubation with tannic acid. Furthermore, forskolin abolished the anti-proliferative effect of tannic acid on cholangiocyte proliferation after bile duct ligation. In conclusion, the anti-proliferative effects of tannic acid in cholangiocytes involve modulation of ERK1,2 by a cyclic adenosine 5',3'-monophosphate-protein kinase A-dependent pathway. These data suggest that tannic acid may be useful in limiting excessive cholangiocyte proliferation and modulating secretion during cholestasis.

IL-6 activates serum and glucocorticoid kinase via p38alpha mitogen-activated protein kinase pathway.

Interleukin-6 (IL-6) has been implicated as an autocrine factor involved in growth of several human cancers, such as tumors arising from the biliary tract or cholangiocarcinoma. In malignant biliary tract epithelia, IL-6 activates the p38 MAPK pathway, which mediates a dominant survival signaling pathway. Serum and glucocorticoid-stimulated kinase (SGK) has been implicated as a survival kinase, but its role in survival signaling by IL-6 is unknown. After IL-6 stimulation, p38 MAPK activation preceded phosphorylation of SGK at Ser78. Pretreatment with the pharmacological inhibitors of p38 MAPK SB-203580 or SB-202190 blocked IL-6-induced SGK phosphorylation at Ser78 and SGK activation. Overexpression of p38alpha increased constitutive SGK phosphorylation at Ser78, whereas dominant negative p38alpha MAPK blocked IL-6-induced SGK phosphorylation and nuclear translocation. Interestingly, in addition to stimulating SGK phosphorylation, both IL-6 stimulation and p38alpha MAPK overexpression increased SGK mRNA and protein expression. An increase in p38 MAPK and SGK occurred following enforced expression of IL-6 in vivo. Furthermore, inhibition of SGK expression by siRNA increased toxicity due to chemotherapeutic drugs. Taken together, these data identify SGK as both a downstream kinase substrate as well as a transcriptionally regulated gene target of p38 MAPK in response to IL-6 and support a role of SGK during survival signaling by IL-6 in human cancers, such as cholangiocarcinoma.

Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.

BACKGROUND & AIMS: The biliary tree is the target of cholangiopathies that are chronic cholestatic liver diseases characterized by loss of proliferative response and enhanced apoptosis of cholangiocytes, the epithelial cells lining the biliary tree. The endogenous factors that regulate cholangiocyte proliferation are poorly understood. Therefore, we studied the role of the neuroendocrine hormone serotonin as a modulator of cholangiocyte proliferation. METHODS: The presence of the serotonin 1A and 1B receptors on cholangiocytes was evaluated. We then tested whether the activation of such receptors by the administration of the selective agonists modifies cholangiocyte proliferation and functional activity both in vivo and in vitro. In addition, the intracellular signal mediating the serotonin receptor action in cholangiocytes was characterized. We studied the expression and secretion of serotonin by cholangiocytes and the effects of the neutralization of the secreted hormone on the growth of the biliary tree. RESULTS: Cholangiocytes express the serotonin 1A and 1B receptors. Their activation markedly inhibits the growth and choleretic activity of the biliary tree in the bile duct-ligated rat, a model of chronic cholestasis. Such changes are mediated by enhanced d -myo-inositol 1,4,5-triphosphate/Ca 2+ /protein kinase C signaling and the consequent inhibition of the adenosine 3',5'-cyclic monophosphate/protein kinase A/Src/extracellular signal-regulated kinase 1/2 cascade. Cholangiocytes secrete serotonin, the blockage of which enhances cholangiocyte proliferation in the course of cholestasis. CONCLUSIONS: We observed the existence of an autocrine loop based on serotonin that limits the growth of the biliary tree in the course of chronic cholestasis. Our novel findings might open new approaches for the management of cholangiopathies.

Alpha-1 adrenergic receptor agonists modulate ductal secretion of BDL rats via Ca(2+)- and PKC-dependent stimulation of cAMP.

Acetylcholine potentiates secretin-stimulated ductal secretion by Ca(2+)-calcineurin-mediated modulation of adenylyl cyclase. D2 dopaminergic receptor agonists inhibit secretin-stimulated ductal secretion via activation of protein kinase C (PKC)-gamma. No information exists regarding the effect of adrenergic receptor agonists on ductal secretion in a model of cholestasis induced by bile duct ligation (BDL). We evaluated the expression of alpha-1A/1C, -1beta and beta-1 adrenergic receptors in liver sections and cholangiocytes from normal and BDL rats. We evaluated the effects of the alpha-1 and beta-1 adrenergic receptor agonists (phenylephrine and dobutamine, respectively) on bile and bicarbonate secretion and cholangiocyte IP(3) and Ca(2+) levels in normal and BDL rats. We measured the effect of phenylephrine on lumen expansion in intrahepatic bile duct units (IBDUs) and cyclic adenosine monophosphate (cAMP) levels in cholangiocytes from BDL rats in the absence or presence of BAPTA/AM and Gö6976 (a PKC-alpha inhibitor). We evaluated if the effects of phenylephrine on ductal secretion were associated with translocation of PKC isoforms leading to increased protein kinase A activity. Alpha-1 and beta-1 adrenergic receptors were present mostly in the basolateral domain of cholangiocytes and, following BDL, their expression increased. Phenylephrine, but not dobutamine, increased secretin-stimulated choleresis in BDL rats. Phenylephrine did not alter basal but increased secretin-stimulated IBDU lumen expansion and cAMP levels, which were blocked by BAPTA/AM and Go6976. Phenylephrine increased IP(3) and Ca(2+) levels and activated PKC-alpha and PKC-beta-II. In conclusion, coordinated regulation of ductal secretion by secretin (through cAMP) and adrenergic receptor agonist activation (through Ca(2+)/PKC) induces maximal ductal bicarbonate secretion in liver diseases. (Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

cAMP stimulates the secretory and proliferative capacity of the rat intrahepatic biliary epithelium through changes in the PKA/Src/MEK/ERK1/2 pathway.

BACKGROUND/AIMS: To evaluate if increased cholangiocyte cAMP levels alone are sufficient to enhance cholangiocyte proliferation and secretion. METHODS: Normal rats were treated in vivo with forskolin for two weeks. Cholangiocyte apoptosis, proliferation and secretion were evaluated. Purified cholangiocytes from normal rats were treated in vitro with forskolin in the absence or presence of Rp-cAMPs (a PKA inhibitor), PP2 (an Src inhibitor) or PD98059 (a MEK inhibitor). Subsequently, we evaluated cholangiocyte proliferation by determination of proliferating cellular nuclear antigen (PCNA) protein expression by immunoblots. We evaluated if the effects of forskolin on cholangiocyte functions are associated with changes in the cAMP/PKA/Src/MEK/ERK1/2 pathway. RESULTS: Chronic administration of forskolin to normal rats increased the number of ducts, cAMP levels, and secretin-induced choleresis compared to controls. Forskolin-induced increases in cholangiocyte proliferation and secretion were devoid of cholangiocyte necrosis, inflammation and apoptosis. In vitro, in pure isolated cholangiocytes, forskolin increased cholangiocyte proliferation, which was ablated by Rp-cAMPs, PP2 and PD98059. The effects of forskolin on cholangiocyte proliferation were associated with increased activity of PKA, Src Tyrosine 139 (Tyr 139) and ERK1/2. CONCLUSIONS: Modulation of the PKA/Src/MEK/ERK1/2 pathway may be important in the regulation of cholangiocyte growth and secretion observed in cholestatic liver diseases.

Tauroursodeoxycholate inhibits human cholangiocarcinoma growth via Ca2+-, PKC-, and MAPK-dependent pathways.

Tauroursodeoxychate (TUDCA) is used for the treatment of cholangiopathies including primary sclerosing cholangitis, which is considered the primary risk factor for cholangiocarcinoma. The effect of TUDCA on cholangiocarcinoma growth is unknown. We evaluated the role of TUDCA in the regulation of growth of the cholangiocarcinoma cell line Mz-ChA-1. TUDCA inhibited the growth of Mz-ChA-1 cells in concentration- and time-dependent manners. TUDCA inhibition of cholangiocarcinoma growth was blocked by BAPTA-AM, an intracellular Ca(2+) concentration ([Ca(2+)](i)) chelator, and H7, a PKC-alpha inhibitor. TUDCA increased [Ca(2+)](i) and membrane translocation of the Ca(2+)-dependent PKC-alpha in Mz-ChA-1 cells. TUDCA inhibited the activity of MAPK, and this inhibitory effect of TUDCA was abrogated by BAPTA-AM and H7. TUDCA did not alter the activity of Raf-1 and B-Raf and the phosphorylation of MAPK p38 and JNK/stress-activated protein kinase. TUDCA inhibits Mz-ChA-1 growth through a signal-transduction pathway involving MAPK p42/44 and PKC-alpha but independent from Raf proteins and MAPK p38 and JNK/stress-activated protein kinases. TUDCA may be important for the treatment of cholangiocarcinoma.

Increased susceptibility of cholangiocytes to tumor necrosis factor-alpha cytotoxicity after bile duct ligation.

Tumor necrosis factor (TNF)-alpha plays a critical role in epithelial cell injury. However, the role of TNF-alpha in mediating cholangiocyte injury under physiological or pathophysiological conditions is unknown. Thus we assessed the effects of TNF-alpha alone or following sensitization by actinomycin D on cell apoptosis, proliferation, and basal and secretin-stimulated ductal secretion in cholangiocytes from normal or bile duct-ligated (BDL) rats. Cholangiocytes from normal or BDL rats were highly resistant to TNF-alpha alone. However, presensitization by actinomycin D increased apoptosis in cholangiocytes following BDL and was associated with an inhibition of proliferation and secretin-stimulated ductal secretion. Thus TNF-alpha mediates cholangiocyte injury and altered ductal secretion following bile duct ligation. These observations suggest that cholestasis may enhance susceptibility to cytokine-mediated cholangiocyte injury.

Selective Na+/H+ exchange inhibition by cariporide reduces liver fibrosis in the rat.

The aim of this study was to evaluate the effect of cariporide, a selective Na(+)/H(+) exchange inhibitor, on isolated and cultured hepatic stellate cells (HSCs) and in 2 in vivo models of rat liver fibrosis. Platelet-derived growth factor (PDGF)-induced HSC proliferation, evaluated by measuring the percentage of bromodeoxyuridine-positive cells, was significantly inhibited by cariporide, with a maximal effect at 10 micromol/L. Incubation with cariporide did not inhibit PDGF-induced extracellular-regulated kinase 1/2 (ERK1/2), Akt (a downstream component of the phosphatidylinositol [PI]-3 kinase pathway), and protein kinase C (PKC) activation but reduced PDGF-induced activation of the Na(+)/H(+) exchanger, with a maximal effect at 10 micromol/L. Rats treated with dimethylnitrosamine (DMN; 10 mg/kg) for 1 and 5 weeks received a diet with or without 6 ppm cariporide. Treatment with cariporide reduced the degree of liver injury, as determined by alanine aminotransferase (ALT) values, also when administered after the induction of hepatic damage. This was associated with reduced HSC activation and proliferation and reduced collagen deposition, as determined by morphometric evaluation of alpha-smooth muscle actin (SMA)/proliferating cell nuclear antigen-positive cells and percentage of Sirius red-positive parenchyma, respectively. Moreover, cariporide was also able to reduce alpha(1)I procollagen messenger RNA (mRNA) expression. Similar effects were observed in bile duct-ligated (BDL) rats. In conclusion, selective inhibition of the Na(+)/H(+) exchanger by cariporide may represent an effective therapeutic strategy in the treatment of hepatic fibrosis.

Effect of pirfenidone on rat hepatic stellate cell proliferation and collagen production.

BACKGROUND/AIMS: Pirfenidone has been recently shown to reduce dimethynitrosamine-induced liver fibrosis in the rat, but no information are available on the effect of this drug on cultured hepatic stellate cells (HSC). METHODS: HSC proliferation was evaluated by measuring bromodeoxyuridine incorporation; PDGF-receptor autophosphorylation, extracellular signal-regulated kinase (ERK1/2) and pp70(S6K) activation were evaluated by western blot; protein kinase C activation was evaluated by western blot and by ELISA; type I collagen accumulation and alpha1(I) procollagen mRNA expression were evaluated by ELISA and northern blot, respectively. RESULTS: Pirfenidone significantly inhibited PDGF-induced HSC proliferation, starting at a concentration of 1 microM, with a maximal effect at 1000 microM, without affecting HSC viability and without inducing apoptosis. The inhibition of PDGF-induced HSC proliferation was associated neither with variations in PDGF-receptor autophosphorylation, or with ERK1/2 and pp70(S6K) activation. On the other hand, pirfenidone was able to inhibit PDGF-induced activation of the Na(+)/H(+) exchanger, which is involved in PDGF-induced HSC proliferation in HSC, with a maximal effect at 1000 microM and inhibited PDGF-induced protein kinase C activation. Pirfenidone 100 and 1000 microM inhibited type I collagen accumulation in the culture medium induced by transforming growth factor(beta1) by 54% and 92%, respectively, as well as TGF(beta1)-induced alpha1(I) procollagen mRNA expression. RESULTS: Pirfenidone could be a new candidate for antifibrotic therapy in chronic liver diseases.