1C). Immunoblot analysis confirmed that hepatocyte populations from regenerating livers were enriched with cells that expressed keratin (K)7, a marker of immature hepatocytes (Fig. 4B). Hepatocytic cells from regenerating livers also expressed Ihh and Shh ligands (Fig. 4B), and immunostaining of 48-hour cytospins from regenerating (but not

sham-operated) livers co-localized expression buy Hydroxychloroquine of Shh and albumin (Supporting Fig. 1D). Thus, the aggregate data provide conclusive evidence that hepatocytic cells expressing progenitor markers and Hh ligands or target genes increase as the liver regenerates after PH. Hh ligands are known to promote the proliferation of various progenitors. Therefore, it was important to determine whether the proliferative activity of hepatocytic or ductular cells increased as these populations became enriched with Hh-responsive cells. Mice received a single injection of BrdU 2 hours before sacrifice to label cells that were engaged in DNA synthesis. The numbers of hepatocytes and ductular cells with BrdU nuclear staining increased significantly after PH (Fig. MLN2238 4C, D). As with Gli2-staining (Fig. 4A), BrdU nuclear staining peaked first in hepatocytes, and then in ductular cells. PH also increased nuclear accumulation

of Ki67, another S-phase marker, in both cell populations (Supporting Fig. 2A, B). Thus, increased proliferative activity in hepatocyte and ductular cell populations closely paralleled their enrichment with Hh-responsive cells. To determine how Hh-pathway activation impacts regenerative responses, post-PH, mice were treated with cyclopamine, a specific Smo antagonist that abrogates Hh signal transduction32

or vehicle (olive oil) before PH and at regular intervals (every 24 hours) after PH. As expected, cyclopamine did not prevent induction of Hh ligands (data not shown). However, it attenuated induction of Gli1 and Gli2 mRNAs (Fig. 5A) and proteins (Fig. 5B), and inhibited mRNA/protein expression of sFRP1 (Fig. 5A) and Ptc (Fig. 5B), two other Gli-regulated genes. Inhibiting Hh MCE公司 signaling also reduced mRNA or protein expression of various progenitor markers, such as AFP, Fn14, and keratin (K)19 after PH (Fig. 5C), and prevented cells that expressed AE1/AE3 or muscle pyruvate kinase (Mpk) (other progenitor markers) from accumulating in the liver (Fig. 5D). In addition, it attenuated fibrogenic repair, as evidenced by decreased expression of α-SMA and collagen mRNAs, α-SMA protein, and picrosirius red staining (Fig. 5E). Cyclopamine inhibition of Hh-regulated responses was associated with significantly reduced survival after PH.