Knockdown of tight junction protein claudin-2 prevents bile canalicular formation in WIF-B9 cells
The polarization of hepatocytes involves formation of functionally distinct sinusoidal (basolateral) and bile canalicular (apical) plasma membrane domains that are separated by tight junctions. Although various molecular mechanisms and signaling cascades including polarity complex proteins may contribute to bile canalicular formation in hepatocytes, the role of tight junction proteins in bile canalicular formation remains unclear. To investigate the role of the integral tight junction protein claudin-2 in bile canalicular formation, we depleted claudin-2 expression by siRNA in the polarized hepatic cell line WIF-B9 after treatment with or without phenobarbital. When WIF-B9 cells were treated with phenobarbital, claudin-2 expression and tight junction strands were markedly increased together with induction of canalicular formation with a biliary secretion function. Knockdown of claudin-2 prevented bile canalicular formation after treatment with or without phenobarbital. Furthermore, knockdown of claudin-2 caused a change from a hepatic polarized phenotype to a simple polarized phenotype, together with upregulation of pLKB1, pMAPK, pAkt and pp38 MAPK, but not pMLC, PTEN or cdc42, and an increase of intracellular vacuoles, which were present before bile canalicular formation. These results suggest that claudin-2 may affect not only the bile canalicular seal but also bile canalicular formation.
KeywordsHepatic cell polarity Bile canaliculi Tight junctions siRNA Phenobarbital Signal transduction
We thank Dr. Doris Cassio (Univ Paris-Sud) for the WIF-B9 cells and Ms. E. Suzuki (Sapporo Medical University) for technical support. This work was supported by Grants-in-Aid from the National Project”Knowledge Cluster Initiative” (2nd stage, “Sapporo Biocluster Bio-S”), the Ministry of Education, Culture, Sports Science, and Technology, and the Ministry of Health, Labour and Welfare of Japan, the Akiyama Foundation, Japan Science and Technology Agency and the Long-Range Research Initiative Project of the Japan Chemical Industry Association.
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