Emma A. Kruglov

Portal fibroblasts regulate the proliferation of bile duct epithelia via expression of NTPDase2

Bile duct epithelia are the target of a number of “cholangiopathies” characterized by disordered bile ductular proliferation. Although mechanisms for bile ductular proliferation are unknown, recent evidence suggests that extracellular nucleotides regulate cell proliferation via activation of P2Y receptors. Portal fibroblasts may regulate bile duct epithelial P2Y receptors via expression of the ecto-nucleotidase NTPDase2. Thus, we tested the hypothesis that portal fibroblasts regulate bile duct epithelial proliferation via expression of NTPDase2. We generated a novel co-culture model of Mz-ChA-1 human cholangiocarcinoma cells and primary portal fibroblasts. Cell proliferation was measured by bromodeoxyuridine uptake. NTPDase2 expression was assessed by immunofluorescence and quantitative real-time reverse transcription PCR. NTPDase2 expression in portal fibroblasts was blocked using short interfering RNA. NTPDase2 overexpression in portal myofibroblasts isolated from bile duct-ligated rats was achieved by cDNA transfection. Co-culture of Mz-ChA-1 cells with portal fibroblasts decreased their proliferation to 26% of control. Similar decreases in Mz-ChA-1 proliferation were induced by the soluble ecto-nucleotidase apyrase and the P2 receptor inhibitor suramin. The proliferation of Mz-ChA-1 cells returned to baseline when NTPDase2 expression in portal fibroblasts was inhibited using NTPDase2-specific short interfering RNA. Untransfected portal myofibroblasts lacking NTPDase2 had no effect on Mz-ChA-1 proliferation, yet portal myofibroblasts transfected with NTPDase2 cDNA inhibited Mz-ChA-1 proliferation. We conclude that portal fibroblasts inhibit bile ductular proliferation via expression of NTPDase2 and blockade of P2Y activation. Loss of NTPDase2 may mediate the bile ductular proliferation typical of obstructive cholestasis. This novel cross-talk signaling pathway may mediate pathologic alterations in bile ductular proliferation in other cholangiopathic conditions.

Autocrine release of TGF-β by portal fibroblasts regulates cell growth

Portal fibroblasts (PF) are a newly isolated population of fibrogenic cells in the liver postulated to play a significant role in early biliary fibrosis. Because transforming growth factor‐β (TGF)‐β is a key growth factor in fibrosis, we characterized the response of PF to TGF‐β. We demonstrate that PF produce significant amounts of TGF‐β2 and, unlike activated hepatic stellate cells (HSC), express all three TGF‐β receptors and are growth inhibited by TGF‐β1 and TGF‐β2. Fibroblast growth factor (FGF)‐2, but not platelet derived growth factor (PDGF), causes PF proliferation. These data suggest a mechanism whereby HSC eclipse PF as the dominant myofibroblast population in biliary fibrosis.

Isolation of Primary Rat Liver Fibroblasts

Introduction One of the major advances in liver research in the past decade was the ability to isolate distinct liver cell populations. Although there are established methods of isolating hepatocytes, cholangiocytes, and stellate cells, before this study no technique for liver fibroblast isolation had been devised. Consequently, we developed a technique to isolate primary rat liver fibroblasts. Methods Fibroblasts were isolated from a freshly perfused rat liver with a modification of the procedure for isolation of rat cholangiocytes. Cell markers were assessed with the use of confocal immunofluorescence. Cell morphology was assessed with transmission electron microscopy. Expression of procollagen-1 was assessed by reverse transcription polymerase chain reaction. Results The appearance of cells with fibroblast morphology was first noted at 48 hours, and almost all cells in culture had fibroblast morphology at 96 hours. Putative fibroblasts stained for vimentin, but not for smooth muscle actin, von Willebrand factor, or cytokeratins. Cell morphology was consistent with that of fibroblasts and showed no features of epithelial, endothelial, or smooth muscle cells. Liver fibroblasts expressed procollagen-1 mRNA. Conclusion Primary isolated rat fibroblasts can be produced from a freshly perfused rat liver with a modification of standard cell culture methods. The role of fibroblasts in liver physiology can now be studied directly.

The Spatial Distribution of Inositol 1,4,5-Trisphosphate Receptor Isoforms Shapes Ca2+ Waves

Cytosolic Ca2+ is a versatile second messenger that can regulate multiple cellular processes simultaneously. This is accomplished in part through Ca2+ waves and other spatial patterns of Ca2+ signals. To investigate the mechanism responsible for the formation of Ca2+ waves, we examined the role of inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in Ca2+ wave formation. Ca2+ signals were examined in hepatocytes, which express the type I and II InsP3R in a polarized fashion, and in AR4-2J cells, a nonpolarized cell line that expresses type I and II InsP3R in a ratio similar to what is found in hepatocytes but homogeneously throughout the cell. Expression of type I or II InsP3R was selectively suppressed by isoform-specific DNA antisense in an adenoviral delivery system, which was delivered to AR4-2J cells in culture and to hepatocytes in vivo. Loss of either isoform inhibited Ca2+ signals to a similar extent in AR4-2J cells. In contrast, loss of the basolateral type I InsP3R decreased the sensitivity of hepatocytes to vasopressin but had little effect on the initiation or spread of Ca2+ waves across hepatocytes. Loss of the apical type II isoform caused an even greater decrease in the sensitivity of hepatocytes to vasopressin and resulted in Ca2+ waves that were much slower and delayed in onset. These findings provide evidence that the apical concentration of type II InsP3Rs is essential for the formation of Ca2+ waves in hepatocytes. The subcellular distribution of InsP3R isoforms may critically determine the repertoire of spatial patterns of Ca2+ signals.

Regulation of Multidrug Resistance-Associated Protein 2 by Calcium Signaling in Mouse Liver

Multidrug resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca2+) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5‐triphosphate receptor (InsP3R2) regulates Ca2+ release in the canalicular region of hepatocytes. However, the role of InsP3R2 and of Ca2+ signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP3R2‐mediated Ca2+ signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild‐type (WT) and InsP3R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time‐lapse imaging of Ca2+ signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)‐Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP3R2 was concentrated in the canalicular region of WT mice but absent in InsP3R2 KO livers, whereas expression and localization of InsP3R1 was preserved, and InsP3R3 was absent from both WT and KO livers. Ca2+ signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP3R2. Canalicular secretion of the organic anion 5‐chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP3R2 KO mice. Finally, ATP increased GFP‐Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA. Conclusion: InsP3R2‐mediated Ca2+ signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane. HEPATOLOGY 2010

Type 2 inositol 1,4,5-trisphosphate receptor modulates bile salt export pump activity in rat hepatocytes.

Bile salt secretion is mediated primarily by the bile salt export pump (Bsep), a transporter on the canalicular membrane of the hepatocyte. However, little is known about the short‐term regulation of Bsep activity. Ca2+ regulates targeting and insertion of transporters in many cell systems, and Ca2+ release near the canalicular membrane is mediated by the type II inositol 1,4,5‐trisphosphate receptor (InsP3R2), so we investigated the possible role of InsP3R2 in modulating Bsep activity. The kinetics of Bsep activity were monitored by following secretion of the fluorescent Bsep substrate cholylglycylamido‐fluorescein (CGamF) in rat hepatocytes in collagen sandwich culture, an isolated cell system in which structural and functional polarity is preserved. CGamF secretion was nearly eliminated in cells treated with Bsep small interfering RNA (siRNA), demonstrating specificity of this substrate for Bsep. Secretion was also reduced after chelating intracellular calcium, inducing redistribution of InsP3R2 by depleting the cell membrane of cholesterol, or reducing InsP3R function by either knocking down InsP3R2 expression using siRNA or pharmacologic inhibition using xestospongin C. Confocal immunofluorescence showed that InsP3R2 and Bsep are in close proximity in the canalicular region, both in rat liver and in hepatocytes in sandwich culture. However, after knocking down InsP3R2 or inducing its dysfunction with cholesterol depletion, Bsep redistributed intracellularly. Finally, InsP3R2 was lost from the pericanalicular region in animal models of estrogen‐ and endotoxin‐induced cholestasis. Conclusion: These data provide evidence that pericanalicular calcium signaling mediated by InsP3R2 plays an important role in maintaining bile salt secretion through posttranslational regulation of Bsep, and suggest that loss or redistribution of InsP3R2 may contribute to the pathophysiology of intrahepatic cholestasis. (HEPATOLOGY 2011;)

Ectonucleotidase NTPDase2 Is Selectively Down-Regulated in Biliary Cirrhosis

Background Portal fibroblasts are newly identified, potentially fibrogenic liver cells that are distinct from hepatic stellate cells. The ectonucleotidase* nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is restricted to portal fibroblasts in the normal liver. However, the fate of NTPDase2 after bile duct ligation (BDL) is unknown. * Extracellular enzymes that mediate the degradation of adenosine triphosphate (ATP) and other nucleotides. Aims The aim of this study was to assess the effect of experimental rat and disease-mediated human biliary cirrhosis on NTP-Dase2 expression in the liver. Methods Cirrhosis was induced in experimental rats via BDL and carbon tetrachloride (CCl4) administration. Archived human liver biopsy specimens from normal liver, primary biliary cirrhosis, or hepatitis C cirrhosis were examined. Changes in expression of NTPDase2 were determined using confocal immunofluorescence, immunoblot, and real-time polymerase chain reaction. Results Confocal immunofluorescence demonstrated a decrease in NTPDase2 expression after BDL. Immunoblot and real-time polymerase chain reaction demonstrated a decrease in NTPDase2 expression by portal fibroblasts after BDL. No decrease in NTP-Dase2 protein was noted after CCl4 administration, and NTPDase2 messenger ribonucleic acid was markedly up-regulated after CCl4 administration. Confocal immunofluorescence demonstrated a shift of NTPDase2 expression from portal areas to central areas that colocalized with α-smooth muscle actin after CCl4 administration. In human biopsy specimens, NTPDase2 expression was lost in cirrhosis owing to primary biliary cirrhosis, whereas NTPDase2 expression was shifted to bridging fibrous bands in cirrhosis owing to hepatitis C. Conclusions Loss of NTPDase2 is a common pathway in both rat and human manifestations of biliary cirrhosis. Conversely, in nonbiliary cirrhosis, NTPDase2 is shifted from the portal area to bridging fibrous bands. Elucidations of the mechanisms regulating NTP-Dase2 expression may lead to new therapeutic approaches to fibrotic liver disease.

Inositol 1,4,5-trisphosphate receptor type II (InsP3R-II) is reduced in obese mice, but metabolic homeostasis is preserved in mice lacking InsP3R-II

Inositol 1,4,5-trisphosphate receptor type II (InsP3R-II) is the most prevalent isoform of the InsP3R in hepatocytes and is concentrated under the canalicular membrane, where it plays an important role in bile secretion. We hypothesized that altered calcium (Ca(2+)) signaling may be involved in metabolic dysfunction, as InsP3R-mediated Ca(2+) signals have been implicated in the regulation of hepatic glucose homeostasis. Here, we find that InsP3R-II, but not InsP3R-I, is reduced in the livers of obese mice. In our investigation of the functional consequences of InsP3R-II deficiency, we found that organic anion secretion at the canalicular membrane and Ca(2+) signals were impaired. However, mice lacking InsP3R-II showed no deficits in energy balance, glucose production, glucose tolerance, or susceptibility to hepatic steatosis. Thus, our results suggest that reduced InsP3R-II expression is not sufficient to account for any disruptions in metabolic homeostasis that are observed in mouse models of obesity. We conclude that metabolic homeostasis is maintained independently of InsP3R-II. Loss of InsP3R-II does impair secretion of bile components; therefore, we suggest that conditions of obesity would lead to a decrease in this Ca(2+)-sensitive process.

Type 2 inositol trisphosphate receptor gene expression in hepatocytes is regulated by cyclic AMP

The type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) is the principal intracellular Ca2+ release channel in hepatocytes, and so is important for bile secretion and other functions. IP3R2 activity is regulated in part by post-translational modifications but little is known about transcriptional regulation of its expression. We found that both IP3R2 mRNA and protein levels in liver were increased during fasting. Treatment of hepatocytes with forskolin or 8-CPT-cAMP also increased IP3R2, and this was reduced by actinomycin D. Analysis of the IP3R2 promoter revealed five CREs, and CREB potently increased promoter activity. Mutation of CRE4 or CRE5 decreased induction by CREB, and ChIP assay showed recruitment of CREB to these sites. Adenylyl cyclase (AC) 6 and 9 were the principal AC isoforms detected in rat hepatocytes, and silencing either one decreased organic anion secretion, which depends on IP3R2. Secretion furthermore was increased by overnight but not acute treatment with forskolin or 8-CPT-cAMP. These findings provide evidence that IP3R2 expression is transcriptionally regulated by cAMP via CREB binding to CRE elements in its promoter. The findings furthermore suggest that this mechanism is relevant for hormonal regulation of bile secretion.

539 Inositol 1,4,5-Trisphosphate Receptor Type 2 Modulates Bsep Activity in Hepatocytes

Hepatic bile salt secretion is mediated primarily by the bile salt export pump (BSEP), an ABC transporter on the canalicular membrane of the hepatocyte. Defects in bile salt secretion lead to the profound consequences of intrahepatic cholestasis, sometimes culminating in liver failure. Studies on the short term regulation of BSEP have implicated cAMP and other signaling pathways, but have yielded contradictory results regarding calcium. In order to clarify the role of calcium in BSEP regulation, we examined pericanalicular calcium signaling mediated by the inositol 1,4,5-trisphosphate receptor type 2 (InsP3R2), which is the principal calcium release channel in hepatocytes and is concentrated in the peri-canalicular domain. BSEP and InsP3R2 expression and function were examined in rat liver and in rat hepatocytes in collagen sandwich culture. Using confocal immunofluorescence microscopy, InsP3R2 and BSEP were both found in the region of the canalicular membrane. BSEP activity was examined bymonitoring the kinetics of secretion of CGamF, a fluorescent BSEP substrate, into the sealed canalicular vacuoles of hepatocytes in sandwich culture. CGamF secretion was significantly inhibited by either (1) treating hepatocytes with the calcium chelator BAPTA for 30 minutes; (2) inducing redistribution of InsP3R2 away from the canalicular region by depleting the plasma membrane of cholesterol with 5 mM methyl-beta-cyclodextrin for 30 minutes; (3) knocking down InsP3R2 expression using a specific siRNA; or (4) knocking down BSEP expression using BSEP-specific siRNA as a positive control for the secretion assay. Moreover, knockdown of InsP3R2 significantly decreased bile salt secretion without altering BSEP expression or localization, as confirmed by immunofluorescence microscopy. In separate studies, basal bile salt secretion was reduced in livers from InsP3R2 KO mice, despite maintenance of BSEP localization and expression, as assessed by immunofluorescence microscopy and western blot, respectively. These data provide evidence that pericanalicular calcium signaling mediated by InsP3R2 plays an important role in maintaining bile salt secretion via post-translational regulation of BSEP.We postulate this regulationmay involvemodulating insertion of BSEP into the canalicular membrane .