Changes in WNT/beta-catenin pathway during regulated growth in rat liver regeneration

Abstract

The wnt/beta-catenin pathway is important during embryogenesis and carcinogenesis. beta-Catenin interaction with E-cadherin has been shown to be crucial in cell-cell adhesion. We report novel findings in the wnt pathway during rat liver regeneration after 70% partial hepatectomy using Western blot analyses, immunoprecipitation studies, and immunofluorescence. We found wnt-1 and beta-catenin proteins to be predominantly localized in hepatocytes. Immediately following partial hepatectomy, we observed an initial increase in beta-catenin protein during the first 5 minutes with its translocation to the nucleus. We show this increase to be the result of decreased degradation of beta-catenin (decrease in serine phosphorylated beta-catenin) as seen by immunoprecipitation studies. We observed activation of beta-catenin degradation complex comprising of adenomatous polyposis coli gene product (APC) and serine-phosphorylated axin protein, beginning at 5 minutes after hepatectomy, leading to its decreased levels after this time. Quantitative changes observed in E-cadherin protein during liver regeneration are, in general, reverse to those seen in beta-catenin. In addition, using immunoprecipitation, we observe elevated levels of tyrosine-phosphorylated beta-catenin at 6 hours onward. Thus, changes in the wnt pathway during regulated growth seem to tightly regulate cytosolic beta-catenin levels and may be contributing to induce cell proliferation and target gene expression. Furthermore, these changes might also be intended to negatively regulate cell-cell adhesion for structural reorganization during the process of liver regeneration.

Fig. 1

Wnt-1, β-catenin and E-cadherin proteins localize predominantly in the hepatocytes of an adult rat liver. (A) Western blot demonstrates wnt-1 in hepatocytes of a resting adult liver. (B) β-Catenin is seen in hepatocytes and Kupffer cells. (C) Immunofluorescence staining reveals localizing of β-catenin (green) on the membranes and cytoplasm (immediately next to the membrane) in the rat liver uniformly. Some hepatocytes show nuclear localization (arrowheads). Inset reveals nuclear localization as depicted by the yellow color by overlay (arrowhead) of Cy3 (red) for β-catenin and Sytox Green for the nuclei. Bar = 10 μm. (D) E-cadherin (red) localizes to the membrane (arrowheads) and the cytoplasm (immediate proximity to the membrane) of the hepatocytes in a normal rat liver. Bar = 10 μm.

Fig. 2

Changes in wnt signaling pathway components during liver regeneration after 70% partial hepatectomy. (A) Elevation in β-catenin protein at 1 to 5 minutes, followed by a significant decrease at 15 minutes onward, until 48 hours in partial-hepatectomy livers. No apparent change in sham-operated animals with comparable film-exposure times. The bottom 2 panels show Northern blot analysis. An increase in β-catenin expression is seen at 6 hours through 72 hours after partial hepatectomy. The lowest panel shows a minimal β-catenin expression in sham operation. (B) Representative Western blots demonstrate decreased wnt-1 protein (upper band in this blot corresponds to the 45-kd size) at 5 minutes through 12 hours that returns to normal level at 18 hours. No changes are seen in sham-operated animals. Minimal levels of APC protein at the 0 time point begins to increase at 5 minutes and is elevated until 18 hours, followed by a gradual decrease to almost physiologic levels at 72 hours after partial hepatectomy. No changes are seen in sham-operated animals. No significant changes in GSK3β or TCF-4 protein levels are observed. A representative antiactin antibody–probed Western blot confirms the equal loading of proteins from various time points after partial hepatectomy and sham samples.

Fig. 3

Increase in serine phosphorylation of β-catenin and axin indicates activation of β-catenin degradation pathway after 5 minutes of partial hepatectomy. (A) A decrease in serine phosphorylation is evident at 1 minute after hepatectomy, followed by an increase after 5 minutes (open arrow) as compared with the sham-operated animal at the same time in lowermost panel of (A). The middle panel of (A) shows the stripped hepatectomy blot re-probed for β-catenin. (B) Stoichiometric analysis of serine phosphorylation of β-catenin (ratio of serine-phosphorylated β-catenin to total β-catenin protein at each time point). Also plotted is the total β-catenin protein alone at each time point. A 0.25-fold decrease in serine-phosphorylated β-catenin is visible during the first minute of hepatectomy that corresponds to about 2-fold increase in β-catenin protein that continues to a 2.5-fold increase at 5 minutes. This is followed by an approximately 1.5-fold increase in serine phosphorylation of β-catenin, following which the protein decreases about 70%. A second peak of serine phosphorylation is seen at 6 hours after hepatectomy that causes further decrease in residual protein. (C) An appreciable increase in the serine-phosphorylated axin is evident at 5 minutes (open arrow) through 18 hours after hepatectomy.

Fig. 4

Immunostaining demonstrates increased nuclear translocation of residual β-catenin at 5 minutes after hepatectomy (bar = 10 μm). (A) β-Catenin (red) localizes to the membrane and the cytoplasm in close proximity to the membrane at the 0 time point. Only a few hepatocytes (<1%) show yellow nuclei showing presence of β-catenin (arrowheads). (B) β-Catenin at 1 minute shows similar localization. (C) Almost all hepatocytes exhibit nuclear localization of β-catenin at 5 minutes (arrowheads). Membrane staining is intact, but cytoplasmic staining decreases. (D) The above redistribution is maintained at 30 minutes (arrowhead). (E) Increased nuclear and membrane labeling for β-catenin is seen at 6 hours. Most nuclei of the hepatocytes at this stage are positive for β-catenin (arrowheads). (F) Decreased nuclear staining for β-catenin is evident at 48 hours. Staining is mainly membranous and cytoplasmic with very few hepatocytes showing nuclear β-catenin (arrowheads).

Fig. 5

Modulations in β-catenin and E-cadherin interactions favor negative regulation of cell-cell adhesion at 6 hours and more during liver regeneration. (A) E-cadherin protein levels decrease at 1 minute, followed by an increase at 5 minutes. A substantial elevation is seen at 15 minutes through 6 hours after partial hepatectomy, followed by a gradual decrease at 12 hours through 48 to 72 hours. (B) Levels of tyrosine-phosphorylated β-catenin increase at 6 hours through 48 hours during liver regeneration as seen in the upper panel. The lower panel of (B) shows re-probing for β-catenin for stoichiometric analysis. (C) Stoichiometry of tyrosine phosphorylation during liver regeneration. Ratio of tyrosine phosphorylated to total β-catenin protein was plotted for each time point. A 3-fold increase is seen at 6 hours that is still maintained through 48 hours. There was a single-fold decrease at 18 hours, but it was still about 2-fold higher than the 0 time point.

Fig. 6

Meaningful variations in wnt signaling pathway during liver regeneration and working hypotheses in regulated growth. (A) Graphic analysis of quantitative changes in wnt pathway components during liver regeneration is shown. There is an initial 2.5-fold increase in β-catenin protein, followed by an almost 3-fold decrease. There is also a 0.5-fold decrease in wnt-1 protein as the degradation pathway for β-catenin is activated. Following this, there is a 7-fold increase in APC protein apart from elevated serine-phosphorylated β-catenin and axin. TCF-4 levels remain mostly unaltered during these events. This graph also depicts an inverse quantitative relationship between E-cadherin and β-catenin proteins. An initial increase in β-catenin protein coincides with a decrease in E-cadherin levels, followed by an increase in E-cadherin protein as β-catenin decreases. (B) A model of regulated growth is hypothesized. Modulation of wnt pathway by altered β-catenin levels (decrease in the wnt-1 protein levels following an increase in the β-catenin protein) favors a possibility of a feedback-inhibitory loop in this pathway. Excess β-catenin protein [1] may activate this feedback loop, causing a decrease in wnt-1 protein levels [2]. This may lead to activation of the degradation pathway for β-catenin through ubiquitination, thus limiting and regulating availability of residual β-catenin for nuclear translocation [3] to control the target gene expression and cell proliferation. Aberrations in this feedback inhibition or checkpoint may lead to growth deregulation.