5 mouse livers, which are comprised of several different liver cell lineages (Supporting Fig. 4A-C).19 These cells, named Hepo-2, Y27632 exhibited low HAI expression (Supporting Fig. 4A,C). Using these Hepo-2 cells we found that interleukin (IL)-β, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β)-1

stimulated HAI-1 expression, whereas TNF-α and HGF marginally induced HAI-2 expression (Fig. 3A). In addition, the expression of an inflammation-related enzyme activated in BA liver,28 COX-2, was significantly elevated in the cells treated with the above factors (Supporting Fig. 4D), and a COX-2 inhibitor, celecoxib, efficiently blocked these stimulatory effects on both HAIs (Fig. 3B). Furthermore, among various bile acids, deoxycholic and lithocholic acids, but not cholic or chenodeoxycholic acid, significantly stimulated HAI-1 expression but not HAI-2 (Fig. 3C). Deoxycholic acid induced the HAI-1 expression in a dose-dependent manner (Fig. 3D). Taken together, these data indicate that selective factors enriched in BA livers activate HAI expression, possibly through the increased expression of COX-2. BMS-777607 supplier To test whether increased HAI-1 or HAI-2 expression plays a role in the fibrosis process in BA livers, portal fibroblasts (PFs) (Fig. 4A) and stellate cells,20 two major cell types responsible for cholestasis-related fibrosis,29, 30 were treated with conditioned media from Hep3B cells stably

Teicoplanin overexpressing HAI-1 or HAI-2 (Fig. 4B) or control media (green fluorescent protein [GFP] or vector) to assay their effects on the fibrogenic activity. Conditioned media containing HAI-1 or HAI-2 significantly increased mRNA levels of collagen I and IV, two common types of collagen expressed in the ductular reaction in BA livers,31 in both cells compared with controls (Fig. 4C). Western blot analysis further confirmed that the conditioned media containing HAI-1 or HAI-2 enhanced the protein levels of collagen I in PFs (Supporting Fig. 5A,B). We also found that conditioned media containing either HAI-1 or HAI-2 significantly increased

PF cell migration, but only HAI-2 significantly increased stellate cell migration (Supporting Fig. 5C). Moreover, colorimetric cell viability (MTT) assays revealed that the HAI-1-rich, and probably HAI-2-rich (from one of two clones) conditioned media, significantly increased the survival of both fibroblasts (Supporting Fig. 5D). Furthermore, recombinant HAI-2 protein (Fig. 4D) significantly up-regulated the expression of Co11a1 and Col4a1 in PFs and the Co11a1 expression in stellate cells. Both HAIs were also expressed in BA livers in cell clusters or single cells with much CK19 and little or no AFP expression (Fig. 2C, arrows; Supporting Fig. 3D), which probably represent a subset of HSCs.15, 24 Thus, we hypothesized that HAI-1 and/or -2 may also have functions in HSCs. To prove this, we examined HAI expression in the developing livers of legally aborted fetuses.