Methods. Cell culture experiments were performed in H69 cells. Determination of adenosine receptors was assessed by RT-PCR, and immunohistochemistry. Assessment of IL-6 expression was determined by qRT-PCR and ELISA. The role of the A2b receptor as a regulator of IL-6 release in H69 cells was tested by inhibition with the A2b-specific antagonist MRS-1754 and A2b-specific siRNA. The functional role of A2b in liver regeneration was tested by partial hepatectomy performed in A2b −/− and wt mice. Results. At the mRNA level, A2b was the primary adenosine receptor
expressed by H69 cells, and A2b was localized at the plasma membrane level. Since the A2b receptor is uniquely coupled to Gs and Gq proteins, we tested the effects of adenosine on cAMP and GDC-0199 Ca2+ generation.
We found that adenosine upregulated cAMP and generated Ca2+i signals. Adenosine upregulated IL-6 mRNA and IL-6 protein released by H69 cells, and this was blocked by MRS-1754 and A2b-specific siRNA. Interestingly, IL-6 upregulation was blocked by inhibition of Ca2+i but not cAMP. In response to partial hepatectomy, A2b knockout mice exhibited blunted and delayed regeneration without change in survival. Conclusions. This study provides evidence of a novel pathway in which extracellular adenosine induces intracellular cAMP and Ca2+ signals, of which the latter stimulates IL-6 upreguation. Adenosine-sensitive upregulation of IL-6 is important but not necessary in the injury response to partial hepatectomy. Disclosures: The following people have nothing to disclose: BAY 80-6946 cost Elise G. Lavoie, Jessica R. Goree, Michel Fausther, Jonathan A. Dranoff Background: Primary sclerosing cholangitis (PSC) is a chronic liver disease that is characterized by cholestasis and inflammation of cholangiocytes, resulting in bile duct strictures, which affects the entire biliary epithelium. MDR2−/− mice are used as a model of human PSC; however, the full characterization of
the biliary epithelium in this model is undefined. During cholestatic injury induced by bile duct ligation (BDL), 上海皓元医药股份有限公司 there is an upregulation of histamine (HA) secretion, histidine decar-boxylase (HDC) expression and vascular endothelial growth factor (VEGF) secretion and expression in cholangiocytes. We have shown that miR-125b expression is downregulated in BDL mice and BDL-induced liver injury is regulated by the miR-125b/HDC/HA/VEGF axis. Our study aims to (i) characterize biliary proliferation/damage in MDR2−/− mice at various ages; and (ii) determine if the miR-125b/HDC/HA/VEGF axis mediates biliary proliferation/damage in MDR2−/− mice. Methods: MDR2−/− and matching wild type (WT) were sacrificed from 1 to 36 weeks of age. We collected serum, cholangiocytes and liver blocks. By immunohistochemistry (IHC), we measured intrahepatic bile duct mass (IBDM) using CK-19 and proliferation by PCNA in liver sections.