7A), whereas hepsin was predominantly found in hepatocytes (Fig. 7B). In contrast, the HGFA expression pattern almost overlapped with that of HAI-2 (Fig. 7C,D). Similarly, the majority of N8 cells were found to also coexpress

HAI-2 Paclitaxel concentration and HGFA (Fig. 8A). Coimmunoprecipitation confirmed that HAI-2 interacts with HGFA in N8 cells (Fig. 8B). Furthermore, we evaluated the knockdown effect of HGFA and/or HAI-2 on N8 cell differentiation. Knockdown of HGFA alone decreased the expression of the majority of hepatocyte markers, but increased the expression of cholangiocyte marker genes Aqp1 and Notch 1 (Supporting Fig. 7A). Remarkably, HGFA knockdown significantly decreased the effect of HAI-2 knockdown on hepatocyte differentiation compared with HAI-2 knockdown alone (Fig. 8C). On the contrary, knockdown of HGFA enhanced the effect of HAI-2 knockdown on inducing cholangiocyte differentiation (Fig.

8C). To further dissect the possible pathway(s) that mediated the signals involved in HAI-2 knockdown-induced hepatic differentiation, we examined whether PD98059, a MEK1 inhibitor, and LY294002, a PI3K inhibitor, could alter the impact of HAI-2 knockdown on hepatic differentiation. PD98059 partly blocked the effects produced by HAI-2 knockdown, Avelestat (AZD9668) resulting in decreased expression of three out of four hepatocyte markers and three out of five cholangiocyte markers assayed (Supporting Fig. 7B), whereas LY294002 efficiently PD-0332991 clinical trial antagonized HAI-2 knockdown-induced expression of all but one of these genes (Supporting Fig. 7B). Taken together, our results suggest that HGFA is the most likely target protease for HAI-2 to modulate hepatic differentiation into hepatocytes, but not cholangiocytes; both PI3K and MEK1 pathways may mediate some effect of HAI-2 knockdown on bi-lineage differentiation of N8 cells. The hypothetic effects of

persistent overexpression of both HAIs in livers with cholangiopathies are summarized in Fig. 8D. Our present study has established that HAI-1 and HAI-2 expression is up-regulated in cholangiocyte precursors and probably HSCs in BA livers and that this up-regulation is correlated with disease progression. Furthermore, we propose that elevation of HAI-1 and -2 in livers with BA or other cholangiopathies may recapitulate some of their functions in early liver development, but their persistent overexpression may be unfavorable for hepatocyte differentiation and enhance fibrosis. We showed that both HAIs are involved in enhancing the fibrogenic activity of PFs and stellate cells.

Induction of hepatitis was associated with a time-dependent up-regulation of hepatic PBEF messenger RNA (mRNA) expression. As shown in Fig. 2A, PBEF expression

was induced 4.4-fold after 2 hours, 15.5-fold after 4 hours, and peaked after 6 hours showing a 46.3-fold expression. mRNA selleck chemical data were confirmed by way of western blot analysis (Fig. 2B). In accordance with the mRNA data, protein expression peaked after 6 hours. Cellular origins were determined by way of immmunofluorescent microscopy. Apart from hepatocytes, PBEF colocalized with F4/80-positive Kupffer cells (Fig. 2C) and CD31-positive liver sinusoidal endothelial cells (Fig. 2D). To study the role of mouse PBEF in mediating ConA-induced hepatitis, we first introduced the PBEF gene into mouse livers using hydrodynamic delivery. Hydrodynamic perfusion represents an effective method for in vivo gene transfer into mouse livers.27 The mouse PBEF gene was cloned into a pCI-neo

mammalian expression vector constitutively expressing murine PBEF gene under control of the cytomegalovirus immediate-early enhancer/promoter region (pCI-Pbef1). The same vector containing a nonsense sequence was used as a control (pCI-Ctrl). Quantitative reverse-transcription GS-1101 in vivo polymerase chain reaction (RT-PCR) analysis showed that PBEF mRNA was efficiently overexpressed in mouse livers 24 hours after injection (Supporting Fig. 1A). RT-PCR results were further confirmed by way of western blot analysis (Supporting Fig. 1B). Of note, circulating PBEF concentrations in pCI-Pbef1–injected animals were increased eight-fold compared with pCI-control injected mice (Supporting Fig. 1C). Experiments included four groups: (1) mice receiving the PBEF1-overexpressing plasmid (pCI-Pbef1) and 12.5 mg/kg ConA (Fig. 3A), (2) mice receiving the control plasmid (pCI-Ctrl) and mafosfamide 12.5 mg/kg ConA (Fig. 3B), (3) mice injected with the pCI-Pbef1 and saline (Fig. 3C), and (4) mice injected with the pCI-Ctrl

and saline (Fig. 3D). With respect to liver pathology, we found more severe lesions in group 1 (pCI-Pbef1 + ConA) compared with group 2 (pCI-Ctrl + ConA) (Fig. 3A,B). No necrotic areas were present in saline-injected control mice (group 3 and group 4) (Fig. 3C,D). Correspondingly, liver damage as measured by the release of liver enzymes (AST and ALT) was significantly higher in group 1 (pCI-Pbef1 + ConA) compared with group 2 (pCI-Ctrl + ConA) (Fig. 3E). AST and ALT tended to be higher in group 3 (pCI-Pbef1 + saline) versus group 4 (pCI-Ctrl + saline), with P values of 0.108 and 0.124, respectively (Fig. 3E). Potential differences in hepatic inflammatory cell infiltration were determined by way of immunohistochemistry. A vivid, comparable T cell infiltration was found in group 1 (pCI-Pbef1 + ConA) and group 2 (pCI-Ctrl + ConA), but not in ConA-naïve groups 3 and 4 (Supporting Fig. 1D).