fig1

Figure 1. Historical data on the disrupted TJ structure in intestinal kindlin-2-deleted mice, histology of human PSC, and PC transport in the polarized human biliary tumor cell line Mz-ChA-1. (A) On the left, an electron micrograph displays altered TJ architecture in intestinal kindlin-2-deleted mice^[17]. On the right, a liver section (HE staining) of human PSC shows the characteristic onion-skin type of periductal fibrosis with cuboidal-shaped cholangiocytes indicating TJ disruption^[22]; (B) Substrate transport was assessed using the polarized biliary epithelial tumor cell line Mz-ChA-1 in a transwell tissue culture system^[38]. Shown is the equilibrium distribution of PC versus inulin when applied equally at 100 mM to the apical and basal compartments. Over 1-hour incubation, vectorial transport of PC was directed apically, depleting PC at the basal side. In contrast, a significantly higher accumulation of inulin was registered at the basal side; (C) Substrate specificity was examined by assessing apical transport for 1-hour after basal application of different substrates at a 10 mM concentration; (D) The mechanism of apical PC translocation after basal application (10 mM) involved ionic driving forces generated by the apical application of salts and substrates. Anionic and cationic partners permeate the membrane at different velocities, leaving a positive or negative-charged environment at the apical cell surface. The involvement of the responsible anion exporters CFTR and AE2 was proven by their downregulation using respective siRNAs in comparison to scrambled siRNA. PC (10 mM) was applied to the basal compartment; (E) Evidence of TJ involvement in apical transport of PC after their chemical disruption by acetaldehyde vapor or the indicated PPAR-γ inhibitors T0070907 and GW9962. The involvement of the responsible TJ proteins was evaluated by indicated siRNA preincubation in comparison to scrambled siRNA. PC (10 mM) was applied to the basal compartment in polarized Mz-ChA-1 cells. Means ± SD, n = 6. Significances were calculated against PBS (control). ***P < 0.001. PSC: Primary sclerosing cholangitis; TJ: tight junction; PC: phosphatidylcholine; PBS: phosphate buffered saline; CFTR: cystic fibrosis transmembrane conductance regulator; AE2: anion exchange protein 2; PPAR-γ: peroxisome proliferator-activated receptor-γ.