Abstract
The E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC) is the key autoantigen in primary biliary cholangitis (PBC) and STAT3 is an inflammatory modulator that participates in the pathogenesis of many liver diseases. This study investigated whether PDC-E2 interacts with STAT3 in human cholangiocytes (NHC) and hepatocytes (Hep-G2) under cholestatic conditions induced by glyco-chenodeoxycholic acid (GCDC). GCDC induced PDC-E2 expression in the cytoplasmic and nuclear fraction of NHC, whereas in Hep-G2 cells PDC-E2 expression was induced only in the cytoplasmic fraction. GCDC-treatment stimulated phosphorylation of STAT3 in the cytoplasmic fraction of NHC. siRNA-mediated gene silencing of PDC-E2 reduced the expression of pY-STAT3 in NHC but not in HepG2 cells. Immunoprecipitation and a proximity ligation assay clearly demonstrated that GCDC enhanced pY-STAT3 binding to PDC-E2 in the nuclear and cytoplasmic fraction of NHC cells. Staining with Mitotracker revealed mitochondrial co-localization of PDC-E2/pS-STAT3 complexes in NHC and Hep-G2 cells. In cirrhotic PBC livers the higher expression of both PDC-E2 and pY-STAT3 was observed. The immunoblot analysis demonstrated the occurrence of double bands of PDC-E2 protein in control livers, which was associated with a lower expression of pY-STAT3. Our data indicate the interaction between PDC-E2 and phosphorylated STAT3 under cholestatic conditions, which may play a role in the development of PBC.
Highlights
The E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC) is the key autoantigen in primary biliary cholangitis (PBC) and STAT3 is an inflammatory modulator that participates in the pathogenesis of many liver diseases
We found that in normal human cholangiocytes (NHC) cells, glyco-chenodeoxycholic acid (GCDC) increased the level of PDC-E2 and p-STAT3 (Y705) in the cytoplasmic fraction by 4.3-fold and 3.6-fold vs. the control and the level of PDC-E2 in the nuclear fraction by 1.5fold vs. the control (p = 0.032) (Fig. 1A)
IP performed with antibodies against PDC-E2 followed by western blotting developed with anti-pY-STAT3 or anti-STAT3, showed an association of PDC-E2 with pY-STAT3 in both the cytoplasmic and nuclear fraction of NHC cells stimulated, or not, with GCDC, whereas an association of PDC-E2 with STAT3 presented mostly in the cytoplasmic fraction of cells treated with GCDC (Fig. 1B)
Summary
The E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC) is the key autoantigen in primary biliary cholangitis (PBC) and STAT3 is an inflammatory modulator that participates in the pathogenesis of many liver diseases. Abbreviations DTT Dithiothreitol GCDC Glyco-chenodeoxycholic acid PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffer saline PLA Proximity ligation assay PBC Primary biliary cholangitis PDC Pyruvate dehydrogenase complex SDS Sodium dodecyl sulfate STAT3 Signal transducer and activator of transcription factor 3 DAPI 4′,6-Diamidino-2-phenylindole dihydrochloride PIPES Piperazine-N,N′-bis (2-ethanesulfonic acid) HEPES N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) TBS Tris buffer saline TBS-T Tris buffer saline containing Tween 20 ALD Alcoholic liver disease TNF Tumor necrosis factor. The E2 component of PDC (PDC-E2) is the key autoantigen in primary biliary cholangitis (PBC), a chronic cholestatic liver disease with autoimmune phenome targeting intrahepatic bile duct lining cells, i.e., c holangiocytes[4]. In combination with the autoimmune reaction against cholangiocytes, leads to inflammation, cholangiocyte death, fibrosis, cirrhosis, and to liver failure[8]
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