6C). The liver is a major organ for HGF synthesis, but the decrease in the mature form of HGF in the hepsin−/− mice was not caused by decreased synthesis of pro-HGF, because western blotting analysis of liver lysates revealed that there was no significant difference in the level of pro-HGF in WT and hepsin−/− mice (Fig. 6D). Hepsin−/− mouse livers may therefore be defective in converting pro-HGF produced in the liver into mature HGF that is released into the serum after processing; such a decreased level of mature HGF would be expected to cause diminished

HGF signal transduction in the livers of hepsin−/− mice. Correspondingly, see more we observed that the level of c-Met phosphorylation (HGF activation site, residues Y1234 and Y1235, in the tyrosine kinase domain) was significantly decreased in hepsin−/− livers, as compared to WT livers, whereas the total c-Met level appeared unchanged (Fig. 6E). Furthermore, when both WT

and hepsin−/− mice were treated with an antibody against hepsin, only WT mice exhibited a decrease in HGF and phosphorylated c-Met (Supporting Fig. 17). All of these results indicate that the c-Met-signaling pathway was down-regulated in the hepsin−/− mouse liver because of the defect in pro-HGF Selleckchem R428 activation in the liver. It has been shown that HGF down-regulates the level of connexin expression in vitro.23 In addition, we observed increased connexin expression and decreased HGF/c-Met signaling in hepsin−/− mouse livers. Therefore, we hypothesized that

the decreased HGF level in hepsin−/− mice caused an increase in both the expression of connexins and hepatocyte size in the liver. To test this, we first analyzed the level of connexin expression in WT and hepsin−/− mouse livers treated with HGF or an antagonist of the HGF receptor, NK4. HGF treatment decreased the expression of connexins in hepsin−/− mice (Fig. 7A), whereas NK4 increased the expression of connexins in WT mice (Fig. 7B). Consistently, hepsin−/− mice had significantly enlarged liver sinusoids after HGF treatment (Fig. 8A), and WT mice had significantly narrowed liver sinusoids after NK4 treatment Erastin (Fig. 8B). A dose-dependent increase in the level of phosphorylated c-Met was also detected after HGF treatment (Supporting Fig. 18). Overall, these results suggest that hepsin regulates the liver architecture through the HGF/c-Met/connexin-signaling axis. The identification of novel phenotypes in our hepsin−/− mice establishes a strong connection in vivo between hepsin and the maintenance of liver architecture. We propose that hepsin deficiency reduces HGF maturation and downstream c-Met phosphorylation that is required for expressing proper levels of connexins, which are, in turn, critical for the maintenance of normal hepatocyte size and, ultimately, normal sinusoidal diameter (Supporting Fig. 19).