Met provides essential signals for liver regeneration

Abstract

Genetic analysis in mice has demonstrated a crucial role of the Met tyrosine kinase receptor and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), in development of the liver, muscle, and placenta. Here, we use conditional mutagenesis in mice to analyze the function of Met during liver regeneration, using the Mx-cre transgene to introduce the mutation in the adult. After partial hepatectomy in mice carrying the Mx-cre-induced Met mutation, regeneration of the liver is impaired. Comparison of signal transduction pathways in control and mutant livers indicates that Met and other signaling receptors cooperate to fully activate particular signaling molecules, for instance, the protein kinase Akt. However, activation of the Erk1/2 kinase during liver regeneration depends exclusively on Met. Signaling crosstalk is thus an important aspect of the regulation of liver regeneration. Analysis of cell cycle progression of hepatocytes in conditional Met mutant mice indicates a defective exit from quiescence and diminished entry into S phase. Impaired liver regeneration is accompanied by compensatory physiological responses that include prolonged up-regulation of HGF/SF and IL-6 in peripheral blood. Our data demonstrate that the HGF/SF/Met signaling system is essential not only during liver development but also for the regeneration of the organ in the adult.

Fig. 1.

Elimination of Met in the liver of mice. (A) Schematic representation of the targeting strategy used to generate the Met^flox allele. Exon 15 of Met (indicated in red) encodes the ATP-binding site of the Met kinase. Yellow boxes indicate exons 14 and 16; loxP sites are shown by arrowheads. NEO, neomycin resistance gene; BHI, BamHI. (B) Southern blot analysis of genomic DNA from the liver of Mx-Cre; Met^flox/+ (lanes 1 and 3) or Mx-Cre; Met^flox /– (lanes 2 and 4) animals before (lanes 1 and 2) and after (lanes 3 and 4) pIpC injections. (C) Autophosphorylation of Met precipitated from liver extract of Mx-Cre; Met^flox /+ (lane 1) or Mx-Cre; Met^flox /– (lanes 2 and 3) 2 days after pIpC injections.

Fig. 2.

Histology of the liver of mice that carry an Mx-cre-induced mutation of Met. Liver sections of control (A and C) and conditional Met mutant (B and D) mice stained with hematoxylin and eosin 3 weeks (A and B) and 6 months (C and D) after the induction of the mutation. Note the large numbers of vesicles in the liver of aged (D) but not of young (B) conditional Met mutants. Oil red O staining of liver sections from control (E) and aged (F) conditional Met mutant mice.

Fig. 3.

Liver size, hepatocyte proliferation, and apoptosis after partial hepatectomy in conditional Met mutant mice. (A) Ratio of liver/body weight in individual control and conditional Met mutant mice before and after partial hepatectomy. (B) BrdUrd-positive nuclei per field in the liver of control (white bars) and conditional Met mutant mice (black bars) at the indicated times after partial hepatectomy. (C) Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive nuclei per 30 fields in the liver of control (white bars) and conditional Met mutant mice (black bars) at the indicated times after partial hepatectomy.

Fig. 4.

Cell cycle progression in the regenerating liver of conditional Met mutant mice. (A) Western blot analysis of phospho-c-jun and c-fos in control and conditional Met mutant liver at the indicated time points after partial hepatectomy. (B) Western blot analysis of cyclin D1, E, and A, p21Cip1/Waf1, phospho-pRb, PCNA, phospho-cdk1, cdk4, and p53 in control and conditional Met mutant liver at the indicated time points after partial hepatectomy. β-Actin was analyzed as a loading control. Note that the signal for cyclin D1 corresponds to the lower band (36 kDa).

Fig. 5.

Circulating blood levels of HGF/SF and IL-6 after partial hepatectomy in conditional Met mutant mice. HGF/SF (A) and IL-6 (B) concentrations were analyzed by ELISA in the blood of control (white bars) and conditional Met mutant mice (black bars) at the indicated times after partial hepatectomy.

Fig. 6.

Activation of signaling cascades in the regenerating liver of conditional Met mutant mice. Western blot analysis of phosho-Erk1/2, total Erk1/2 protein, phospho-Akt and total Akt protein, phospho-GSK3-β, total GSK3-β protein, phospho-STAT3, and total STAT3 protein in control and conditional Met mutant liver is shown at the indicated time points after partial hepatectomy.