Portal Fibroblasts Regulate the Proliferation of Bile Duct Epithelia via Expression of NTPDase2

Abstract

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 MzChA-1 human cholangiocarcinoma cells and primary portal fibroblasts. Cell proliferation was measured by bromodeoxyuridine uptake. NTPDase2 expression was assessed by immunofluorescence and quantitative realtime 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.

Cellular function and molecular structure of ecto-nucleotidases

“…Knockdown of NTPDase2 abrogated the inhibitory effect of portal fibroblasts on P2Y receptor-mediated bile ductular proliferation in liver [182]. These and other studies unanimously demonstrate that NTPDases function to constitutively attenuate the impact of nucleotides on their receptors, an effect abolished by application of inhibitors or by deletion of the enzyme.…”

Expression of mediators of purinergic signaling in human liver cell lines

“…For instance, purinergic signals regulate proliferation [12][13][14] and glucose release [15] in hepatocytes; secretion [16,17], proliferation [18], and mechanosensation [19] in cholangiocytes; and fibrogenic activity [20,21] and properties in HSC [22,23]. Of note, the purinergic signaling system is quite diverse, including multiple subtypes of G proteincoupled receptors for adenosine P1 receptors [24] and nucleotide P2Y receptors [25], nucleotide P2X ligand-gated ion channels [26], and a variety of plasma membrane-bound ecto-enzymes that regulate extracellular nucleotide/ nucleoside levels, namely ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases) [27,28], CD73/ecto-5′-nucleotidase [29,30], and tissue-non-specific alkaline phosphatase (TNAP) [31].…”

IL-6 downregulates transcription of NTPDase2 via specific promoter elements

“…NTPDase2 is expressed by PF that surround intrahepatic bile ducts. PF expressing NTPDase2 markedly block the proliferation of bile duct epithelia, whereas those lacking NTPDase2 expression have no effect on the proliferation of bile duct epithelia (21). Bile ductular proliferation is upregulated by biliary cirrhosis (3,46), and NTPDase2 transcription and expression are down-regulated by biliary cirrhosis (10).…”

“…In the liver, P2Y receptors are of particular importance in such diverse processes as bile secretion (12), cell volume autoregulation (53), and cell growth (21,49). Since P2Y receptor activation has important downstream consequences, the regulation of P2Y receptors is of great interest.…”

“…NTPDase2 is specifically downregulated in biliary cirrhosis in both rats and humans (10). Recently, we demonstrated that NTPDase2 critically regulates bile duct epithelial growth via catalysis of extracellular nucleotides and that this regulation is lost in biliary cirrhosis (21). However, the molecular mechanism by which NTPDase2 is downregulated in biliary cirrhosis is unknown.…”