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Am J Physiol Gastrointest Liver Physiol 292: G1578-G1585, 2007. First published March 1, 2007; doi:10.1152/ajpgi.00359.2006
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LIVER AND BILIARY TRACT

beta-Catenin is critical for early postnatal liver growth

Udayan Apte,1 Gang Zeng,1 Michael D. Thompson,1 Peggy Muller,1 Amanda Micsenyi,1 Benjamin Cieply,1 Klaus H. Kaestner,3 and Satdarshan P. S. Monga1,2

1Department of Pathology and 2Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh; and 3Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Submitted 3 August 2006 ; accepted in final form 23 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Wnt/beta-catenin pathway plays an important role in embryonic liver development, morphogenesis, and organogenesis. Here, we report on the activation of beta-catenin during early postnatal liver growth. Modulation of beta-catenin expression was studied in CD-1 mice livers over a time course of 0 to 30 postnatal days (PD) and 3 mo. Increases in total and active beta-catenin were observed in developing livers from PD 5 to 20. A concomitant increase in the beta-catenin-transcription factor (TCF) complex along with nuclear and cytoplasmic beta-catenin was also evident, which coincided with ongoing hepatocyte proliferation by PCNA immunohistochemistry. This activation of beta-catenin was multifactorial, including cyclical inhibition of glycogen synthase kinase-3beta, suppression of casein kinase-II{alpha}, and a transient increase in beta-catenin gene expression. Coprecipitation experiments revealed the formation of the beta-catenin-cadherin complex at PD 5, whereas adequate beta-catenin-c-Met complex at the hepatocyte membrane did not form until PD 20, which might be contributing to the free beta-catenin pool during early postnatal growth. Furthermore, beta-catenin liver-specific knockout mice exhibited smaller livers at PD 30, secondary to diminished hepatocyte proliferation. These data indicate that the activation of beta-catenin is critical for early postnatal liver growth and development.

glycogen synthase kinase-3beta; casein kinase II{alpha}; hepatocyte proliferation; transcription factor; Met

THE WNT/beta-CATENIN PATHWAY is involved in organogenesis, morphogenesis, and embryonic development (5, 15, 22). beta-Catenin activation has also been implicated in cancer of various tissues, where it is involved in uncontrolled cell division (9, 12, 33). Recently, we and others (17, 20, 21, 23, 30) have demonstrated that Wnt/beta-catenin signaling plays a crucial role in various aspects of liver biology including pathogenesis of hepatocellular carcinoma (HCC), liver regeneration following 2/3rd partial hepatectomy (PHX), and embryonic liver development. beta-Catenin is one of the earliest genes activated following PHX, within 5 min after the surgery, contributing to the initiation of cell proliferation following PHX (21). Antisense therapy against beta-catenin results in defective liver regeneration due to decreased cell division (30). We (32) have recently reported a delay in peak hepatocyte proliferation by 24 h in beta-catenin conditional-null mice. The majority of HCC cases exhibit beta-catenin mutations and/or cytoplasmic stabilization or nuclear translocation of beta-catenin (8). beta-Catenin transgenic mice exhibit hepatomegaly due to increased hepatocyte proliferation (31). beta-Catenin activation has also been observed during embryonic liver development both in vivo and in vitro and regulates events such as proliferation, survival, and differentiation.

beta-Catenin is located at the cell membrane in a complex with E-cadherin (25). In the cytoplasm, beta-catenin is a part of a multiprotein complex where it is phosphorylated by glycogen kinase synthase (GSK)-3beta and casein kinase (CK)-I and targeted for degradation. Similarly, CK-II{alpha} has been also shown to stimulate beta-catenin degradation (2, 14, 29). Upon the initiation of signaling via Wnt's binding to the membrane receptor Frizzled (Fz), GSK-3beta is downregulated, resulting in the cytoplasmic stabilization of beta-catenin. In addition, we (18) have demonstrated that beta-catenin forms a complex with c-Met, the HGF receptor, which is dissociated when HGF binds to Met, leading to an increase in free cytoplasmic beta-catenin. Similarly, beta-catenin plays an essential role in HGF-induced hepatocyte proliferation (1). beta-Catenin translocates to the nucleus, binds to transcription factor (TCF) to form a heterodimer, and stimulates the gene expression of target genes such as cyclin D1, c-Myc, glutamine synthatase, urokinase plasminogen activator receptor, and EGF receptor (32).

We (16) have previously reported beta-catenin activation during embryonic liver development from embryonic days (ED) 10–14 followed by a noteworthy decrease. After birth, the liver undergoes a period of intense proliferation resulting in an increase in liver size (10). In this report, we studied beta-catenin expression during early postnatal development starting from postnatal day (PD) 0 to PD 30. Furthermore, using liver-specific beta-catenin knockout mice (beta-catenin-null mice), we demonstrated that the activation of beta-catenin during PD 0–30 is crucial for a gain in postnatal hepatic size.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and tissue collection. Pregnant female CD-1 (ICR) and 3-mo-old male CD-1 (n = 5) mice were purchased from Charles River Laboratories (Wilmington, MA). Pups (n = 5) were obtained and killed after birth at PD 0, 5, 10, 15, 20, 25, and 30. All animal experiments were performed in strict accordance with the Institutional Animal Care and Use Committee at the University of Pittsburgh School of Medicine and National Institutes of Health guidelines.

Livers were obtained after animals had been killed, and one part of the liver was fixed in 10% neutral buffered formalin while the remaining part was frozen in liquid N2 and stored at –80°C for further analysis. Formalin-fixed livers were processed to obtain 4-µm-thick paraffin-embedded sections.

Generation of beta-catenin liver conditional knockout mice. beta-Catenin liver conditional knockout mice (beta-cateninloxP/loxP; Cre+/– mice) were generated using the Cre/loxP system (32). Briefly, mice carrying the beta-catenin gene flanked with loxP sites were bred with mice carrying the Cre recombinase enzyme gene governed by the liver-specific {alpha}-fetoprotein enhancer-albumin ({alpha}FP-Alb) promoter (1). beta-CateninloxP/loxP; Cre+/– mice were identified by PCR using tail DNA. The following primers were used: flox beta-catenin, forward 5'-AAAGTAGAGTGATGAAAGTTGTT-3' and reverse 5'-CACCATGTCTCTTGTCTATTC-3'; and Cre, forward 5'-ATGCCCAAGAAGAAGAGGAAGGT-3' and reverse 5'-GAAATCAGTGCGTTCGAAGCGTAGA-3'. Decreases in beta-catenin were confirmed by Western blot analysis. Thirty-day-old beta-cateninloxP/loxP; Cre+/– (n = 18) and wild-type (WT) mice (n = 17) were killed, and their livers were harvested and weighed to determined the liver weight-to-body weight ratio. A part of the liver was fixed in 10% neutral buffered formalin and was processed to obtain 4-µm-thick paraffin-embedded sections, which were used for immunohistochemistry.

Protein extraction and Western blot analysis. Whole cell lysates were prepared using RIPA buffer [9.1 mmol/l dibasic sodium phosphate, 1.7 mmol/l monobasic sodium phosphate, 150 mmol/l sodium chloride, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS (pH adjusted to 7.4)] containing fresh protease and phosphatase inhibitor cocktails (Sigma, St. Louis, MO) as previously described (21). The protein concentration was determined by the bicinconinic acid protein assay. SDS-PAGE was performed using 50 µg protein on 7.5% ready gels using the mini-PROTEIN 3-electrophoresis module assembly (Bio-Rad, Hercules, CA). Following a 1-h transfer at 100 V (constant) to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA), blots were incubated with primary and secondary antibodies with intermittent washing with washing buffer. The primary antibodies used in this study include mouse anti-beta-catenin anti-GSK-3beta, anti-cyclin D1, anti-Met, and anti-E-cadherin (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-active beta-catenin (1:1,000, Upstate Biotech, Waltham, MA). Horseradish peroxidase (HRP)-conjugated secondary antibodies (Chemicon, Temecula, CA) were used at 1:20,000 dilution. Blots were subjected to fresh Super-Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and visualized by autoradiography. Once scanned, densitometric analysis was performed using National Institutes of Health Imager software for quantitative assessment. The figures shown are representative of at least three blots.

Immunoprecipitation. Five hundred microgams of whole cell lysate (in the presence of protease and phosphatase inhibitors) were precleared in a 1-ml volume using appropriate control IgG (normal goat) together with 20 µg protein A/G agarose for 30 min to 1 h at 4°C (Santa Cruz Biotechnology). The supernatant obtained after centrifugation (1,000 g) at 4°C was incubated with 20 µl (40 µg) of agarose-conjugated goat anti-beta-catenin antibody (Santa Cruz Biotechnology) for 1 h or overnight at 4°C. Pellets were collected by centrifugation (1,000 g) and washed four times for 5 min each with RIPA buffer at 4°C. Pellets were then resuspended in an equal volume of standard electrophoresis loading buffer with SDS and fresh beta-mercaptoethanol and boiled for 5 min; 30 µl of the samples were resolved on ready gels and transferred as described above. The antibodies used for blotting as well as HRP-conjugated secondary antibodies have been described above.

Real-time PCR analysis. Frozen livers (n = 5) were used to isolate mRNA using the Qiagen RNeasy kit (Qiagen, San Diego, CA) according to the manufacturer's protocol. Following DNase treatment, mRNA was converted to cDNA using the Moloney murine leukemia virus reverse (M-MuLV) transcriptase enzyme (Invitrogen, Carlsbad, CA) in a RT mastermix containing random primers, 5x RT buffer, dNTP mix, RiboLock, and M-MuLV reverse transcriptase enzyme. Actin was used as an internal control. beta-Catenin mRNA was estimated using the mouse TaqMan Gene Expression Assay (Assay ID: Mm00483033_m1, Applied Biosystems) with beta-actin as an internal control in a ABI PRISM 7000 machine according to the manufacturer's protocol.

Nuclear protein isolation and EMSA for beta-catenin activation. Liver tissue (200 mg, n = 3) was finely chopped and then homogenized in a glass Dounce homogenizer (50–100 strokes) in 1 ml hypotonic buffer [10 mmol/l HEPES (pH 7.9), 10 mmol/l NaH2PO4, 1.5 mmol/l MgCl2, 1 mmol/l DTT, 0.5 mmol/l spermidine, and 1 mol/l NaF, with protease and phosphatase inhibitor cocktails (P8340, P5726, and P2850, Sigma) used at 1:100 dilution]. Homogenization was monitored under a microscope using trypan blue dye to control for the release of nuclei. Homogenates were centrifuged for 5 min at 800 g. Pelleted nuclei were washed and repelleted two times in 2 ml hypotonic buffer. Nuclear proteins were extracted in 25–50 µl hypertonic buffer [30 mmol/l HEPES (pH 7.9), 25% glycerol, 450 mmol/l NaCl, 12 mmol/l MgCl2, 1 mmol/l DTT, and 0.1 mmol/l EDTA, with protease and phosphatase inhibitor cocktails (P8340, P5726, and P2850, Sigma) used at 1:200 dilution] for 30–45 min at 4°C with continuous agitation. Extracts were centrifuged at 30,000 g for 30 min. Supernatants were collected, and the protein concentration was determined by a bicinchoninic acid assay with BSA as a standard. EMSA was performed by incubating 5 µg of nuclear protein extracts with a 32P end-labeled oligonucleotide containing the core TCF/lymphoid enhancement factor (LEF) binding site (5'-CTCTGCCGGGCTTTGATCTTTGCTTAACAACA-3') at room temperature for 20 min (31). The binding reaction conditions were 15 mmol/l HEPES (pH 7.9), 75 mmol/l NaCl, 6 mmol/l MgCl2, 0.025 mmol/l EDTA, 2.5 mmol/l Tris (pH 7.6), 12.5% glycerol, and 1 mmol/l DTT, with protease and phosphatase inhibitor cocktails (P8340, P5726, and P2850, Sigma) at 1:400 dilution. Reaction products were analyzed on a 5% nondenaturing polyacrylamide gel with 0.5% Tris-borate-EDTA buffer. The specificity of the DNA binding complex was determined by the addition of 20x cold TCF/LEF oligonucleotide as a competitor. Gels were dried and exposed to BioMax MR film (Eastman Kodak, Rochester, NY).

Histology and immunohistochemistry. Paraffin liver sections were subjected to immunohistochemical analysis for PCNA (Dako, Carpinteria, CA) and beta-catenin (BD Biosciences, San Jose, CA) using the indirect immunoperoxidase technique as previously described (16). Briefly, 4-µm-thick paraffin sections were passed through xylene and graded alcohol and rinsed in PBS. Endogenous peroxide was inactivated using 3% hydrogen peroxide (Sigma). Slides were microwaved in citrate buffer for 20 min, followed by blocking in the blue blocker (Shandon Lipshaw, Pittsburgh, PA) and an overnight incubation at 4°C in either anti-PCNA (1:200) or anti-beta-catenin (1:50) in PBS. Following washes, sections were incubated in HRP-conjugated secondary antibody (Chemicon) for 1 h at room temperature, and the signal was detected using an ABC Elite kit (Vector Laboratories, Burlingame, CA) following the manufacturer's instructions. For PCNA immunohistochemistry, sections were stained with DAB reagent, and for beta-catenin staining, sections were incubated with the AEC chromogen at room temperature and the development of color was observed under a microscope. Sections were counterstained with Harris hematoxylin solution (Sigma) and passed through the dehydration process followed by coverslipping and mounting using DPX (Fluka Labs, St. Louis, MO). For negative control, the sections were incubated with secondary antibodies only.

Statistical analysis. Averages and SDs were calculated in Microsoft Excel. To determine whether the difference between the groups was statistically significant, Student's t-test was performed, and P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Histological changes in the liver during postnatal development. Hematoxylin-eosin-stained liver sections were used to determine histological changes that occurred in the mouse liver during the first 30 days after birth (Fig. 1). Islands of hematopoietic cells were evident in the liver at PD 0 and 5 (Fig. 1, A and B) while extensive proliferation of biliary cells was observed along with an increase in portal triads during PD 10 and 15 (Fig. 1, C and D) and PD 20 (not shown). Several mitotic figures were observed in hepatocytes from PD 5 to 20 (shown PD 20, Fig. 1E), with only a few mitotic figures observed at PD 25 (Fig. 1F). While the hepatic architecture began to resemble the adult liver after PD 20, other characteristics such as low mitotic figures became more apparent at PD 30 (Fig. 1G), when it was more similar to the adult liver as seen at 3 mo (Fig. 1H).


Figure 1
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  		Fig. 1. Histological changes during postnatal liver development. A–H: representative photomicrographs of hematoxylin-eosin-stained liver sections showing the hematopoietic component (arrows) in the liver during postnatal day (PD) 0 (A) and PD 5 (B), proliferation of biliary epithelium (arrowheads) at PD 10 (C) and PD 15 (D), mitotic figures at PD 20 (E) and PD 25 (F), and differentiated mature hepatocytes at PD 30 (G) and 3 mo (H) in the pericentral area of the liver. Original magnification: x400.

 
Cell proliferation during postnatal liver development. PCNA immunohistochemistry was used to study cell proliferation during postnatal liver development (Fig. 2). Increases in cell proliferation, as indicated by large numbers of PCNA-positive resident hepatocytes identified in the S phase, were evident from PD 5 to 20, with no to a few PCNA-positive cells observed at PD 0, 25, or 30 or 3 mo.


Figure 2
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  		Fig. 2. Hepatocyte proliferation during postnatal liver development by PCNA immunohistochemistry. A: only a few PCNA-positive hepatocytes were observed at PD 0. B–E: several resident hepatocytes were strongly PCNA positive at PD 5, 10, 15 and 20. F: dramatic decreases in the numbers of PCNA-positive hepatocytes as well as its staining intensity were evident at PD 25. G: further decreases continued at PD 30. H: almost all hepatocytes were PCNA negative at 3 mo. Arrows, cells in the S phase; arrowheads, PCNA-negative cells. Original magnification: x400.

 
beta-Catenin expression in postnatal liver development. Since beta-catenin correlated with cell proliferation during prenatal liver development and liver regeneration, we examined the expression of beta-catenin during postnatal liver development. Its protein expression was studied by Western blot analysis (Fig. 3A) and immunohistochemistry (Fig. 4). beta-Catenin activation was further confirmed by EMSA to identify the beta-catenin-TCF complex during postnatal liver development (Fig. 3C). Western blot analysis (Fig. 3A) showed that total beta-catenin protein levels were low at birth (PD 0), but were induced at PD 5, remained elevated until PD 20, and decreased to near adult levels (3 mo) during PD 25 to 30. Activated beta-catenin (hypophosphorylated at Ser37/Thr41) increased at PD 5 compared with birth and was further induced at PD 10, 15, and 20, returning to near adult levels at PD 25. Levels of cyclin D1, a known target of beta-catenin and a protein known to be involved in cell division, showed a twofold increase at PD 5 compared with PD 0 levels and until PD 30, with low levels observed in 3-mo-old livers. EMSA performed using a probe containing a TCF-binding sequence from the cyclin D1 promoter indicated significant DNA binding at PD 10, 15, and 20. These data further corroborate the beta-catenin activation observed in developing livers at these stages.


Figure 3
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  		Fig. 3. beta-Catenin expression and activation during postnatal liver development. A: Western blot (WB) analysis of total and activated (dephosphorylated) beta-catenin showing an increase in their respective proteins from PD 5 to 30 and more prominently from PD 5 to 20. A concomitant increase in total cyclin D1 was observed at PD 5–30. B: densitometric analysis of total and activated beta-catenin expression during postnatal development. Three representative blots were scanned, and densitometry was performed using National Institutes of Health Image software. C: gel shift assay of the beta-catenin-transcription factor (TCF) complex using nuclear lysates from pooled developing livers showed the beta-catenin-TCF complex from PD5 onward, with extensive binding from PD 10 to 20.

 

Figure 4
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  		Fig. 4. Immunohistochemical localization of beta-catenin during postnatal liver development. A–F: beta-catenin immunohistochemistry showed predominantly membranous beta-catenin (double arrows) around small hepatocyte clusters at PD 5 (A); nuclear (arrowhead) and cytoplasmic (arrow) beta-catenin at PD 10 [at x40 (B) or x60 (C)]; nuclear (arrowhead) and cytoplasmic (arrow) beta-catenin at PD 15 [at x40 (D) or x60 (E)]; and membranous beta-catenin (double arrows) at PD 25 [at x40 (F)].

 
Immunohistochemistry for beta-catenin identified predominantly membranous beta-catenin at PD 5 around small hepatocyte clusters (Fig. 4A), followed by nuclear and cytoplasmic beta-catenin at PD 10–20 (shown at PD 10 in Fig. 4, B and C; and PD 15 in Fig. 4, D and E), with most beta-catenin localized to the hepatocyte membrane after PD 25 (Fig. 4F). This also complements the observed beta-catenin activation and its complex with TCF at these stages of liver development.

Mechanism of beta-catenin activation during postnatal liver development. Real-time PCR was employed to estimate beta-catenin mRNA levels (Fig. 5A), which revealed comparable levels of beta-catenin gene expression at all stages except PD 15, where an ~2.5-fold increase was identified.


Figure 5
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  		Fig. 5. Mechanism of beta-catenin activation and its other interactions during postnatal liver development. A: real-time PCR analysis of beta-catenin mRNA indicating the induction of beta-catenin gene expression at PD 15. B: WB analysis of glycogen synthase kinase (GSK)-3beta, Ser9-phosphorylated (inactive) GSK-3beta, and casein kinase (CK)-II{alpha} at various time points during postnatal liver development. C: coprecipitation experiments showing beta-catenin associations to E-cadherin and Met during postnatal liver development at PD 5 and 10, respectively; both these proteins were independently present at even earlier stages. IP, immunoprecipitation.

 
To further elucidate the mechanism of beta-catenin activation during postnatal liver development, especially via the canonical Wnt pathway, GSK-3beta levels (the main negative regulator of beta-catenin) were estimated by Western blot analysis (Fig. 5B). No dramatic changes in total GSK-3beta were observed during various stages of postnatal liver development. Western blots for the inactive or Ser9-phosphorylated GSK-3beta revealed a cyclical pattern of activation and inactivation between PD 0 and 20 (Fig. 5B), with a decrease at PD 5 compared with birth but an increase at PD 10 compared with PD 5. Similarly, decreased Ser9-phosphorylated GSK-3beta was observed at PD 15, which increased at PD 20, following which the levels remained relatively unaltered. Western blots of CK-II{alpha}, another modulator of beta-catenin, identified a dramatic decrease from PD 5 to 30 (Fig. 5B). Taken together, all these results, along with the observed increase in activated beta-catenin (the antibody recognized only Ser37/Thr41-hyposphosphorylated beta-catenin), indicated successful transcriptional and posttranslational modification of beta-catenin favoring its stabilization and activation during early postnatal liver growth.

beta-Catenin interactions with E-cadherin and c-Met at the membrane. Next, we performed immunoprecipitation experiments to estimate the levels of beta-catenin-E-cadherin and the beta-catenin-Met complex (Fig. 5C). Immunoprecipitation experiments identified a complex of beta-catenin and E-cadherin at PD 5, which increased gradually, and peaking at PD 15, followed by a steady decrease to adult levels after PD 25 (Fig. 5C). Total E-cadherin was clearly detectable at PD 0 and onward, with a small decrease to adult levels at PD 25 onward. Immunoprecipitation experiments also identified the beta-catenin complex with Met at PD 10, which was more pronounced at PD 20. This happened despite the presence of both beta-catenin as well as Met proteins during earlier stages, especially at PD 5 and 10, suggesting ongoing HGF-mediated signaling. As tyrosine phosphorylation of beta-catenin secondary to HGF/Met signaling impedes the formation of the Met-beta-catenin complex, HGF-mediated signaling might be ongoing during early postnatal liver growth, further adding to the beta-catenin activation at these stages.

Decreases in cell proliferation and liver size in beta-catenin-null mice. To confirm the functional relevance of the ongoing Wnt/beta-catenin signaling during postnatal liver development, we used liver-specific beta-catenin-null (beta-cateninloxP/loxP; Cre+/–) mice and their WT (beta-cateninloxP/loxP; Cre–/–) littermates. The successful deletion of exons 2–6 was confirmed by PCR as described elsewhere, and mice with that genotype demonstrated a dramatic beta-catenin decrease by Western blot analysis (Fig. 6A). The remnant beta-catenin was a function of sustained beta-catenin in nonparenchymal cells since {alpha}FP-Alb-Cre is expressed in hepatocytes only. beta-Catenin and PCNA immunohistochemistry exhibited a drastic decrease in both in the beta-catenin-null mice compared with their WT littermates (Fig. 6B). This resulted in a significant decrease in liver size at PD 30 in beta-catenin-null mice compared with littermate controls (Fig. 6C), as indicated by a decrease in their liver weight-to-body weight ratio by 28% in males (P = 0.0007) and by 17.5% in females (P = 0.0004).


Figure 6
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  		Fig. 6. Impaired postnatal liver growth in beta-catenin-null (beta-cateninloxP/loxP; Cre+/–) mice. A: beta-catenin-null [knockout (KO)] mice were generated as described in MATERIALS AND METHODS. Top, schematic representation of the Cre/loxP strategy used to generate beta-catenin-null mice; bottom, PCR analysis to identify beta-catenin-null mice. beta-Catenin decreases were confirmed by WB analysis. B: representative photomicrographs of beta-catenin immunohistochemistry in wild-type (WT; 1) and beta-catenin-null (3) mice and PCNA analysis in WT (2) and beta-catenin-null (4) mice at PD 15. D: there was a 15–25% decrease in the liver weight-to-body weight ratio in beta-catenin-null male (M) and female (F) mice at PD 30 compared with their littermate controls (P < 0.001).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The role of the Wnt/beta-catenin pathway in development, especially in organogenesis and morphogenesis, has been well documented (5, 15, 34). Wnt/beta-catenin signaling is critical for a variety of processes during development such as segmentation, central nervous system patterning, and asymmetric cell division (15). Extensive evidence exists on the proproliferative properties of beta-catenin in various tissues such as the colon, breast, and liver (4, 12, 13, 31, 32). Here, we extended our previous observations that reported a role of beta-catenin in embryonic liver development to also being involved in early liver growth during postnatal development.

We observed a surge of cell proliferation in the liver during PD 5–20, which contributed to an increase in liver weight. Immunohistochemistry, Western blot analysis, and gel shift assay data identified a sizeable increase in total beta-catenin protein along with nuclear translocation during PD 5–20 coinciding with liver cell proliferation, indicating the involvement of beta-catenin in ongoing cell proliferation. Most importantly, these data are corroborated by the observations in beta-catenin-null mice, which exhibited a significant decrease in cell proliferation during the liver growth spurt at PD 15. As a result, beta-catenin-null mice exhibited a deficit in liver weight at 1 mo of age. In fact, our observations indicate that beta-catenin-null mice have smaller livers throughout their life span due to lower basal hepatocyte proliferation (32). Thus, we have identified an important role of beta-catenin in the stimulation of cell proliferation during the postnatal hepatic growth spurt that dictates normal liver size and the lack thereof, as exhibited by a decrease in the liver weight-to-body weight ratio.

The mechanism of beta-catenin activation seems to be multifactorial. We observed an increase in beta-catenin gene transcription by real-time PCR at PD 15. Additional time points might have identified other such surges. However, de novo synthesis was clearly identifiable at PD 15. An interesting cyclic pattern in inactivation and activation of GSK-3beta, the main negative modulator of beta-catenin in the canonical pathway, was observed. GSK-3beta inactivation was observed at PD 10 and 20, and GSK-3beta activation was observed at PD 5 and 15. These data might suggest various feedback loops within Wnt/beta-catenin signaling as well as cross-talk of GSK-3beta with other pathways. Indeed, feedback loops such as TCF-1 being a downstream target of the beta-catenin pathway (24) and interactions of GSK-3beta with relevant hepatotrophic factors such as insulin have been reported previously (7, 26). Another kinase known to regulate beta-catenin by phosphorylation, CK-II{alpha}, was found to be downregulated during much of the postnatal period. Although much is known about the regulation of beta-catenin by CK-I{alpha}, a member of the multiprotein complex along with adenomatuous polyposis coli gene product (APC), axin, and GSK-3beta, recent evidence suggests that CK-II{alpha} can also phosphorylate beta-catenin, resulting in its degradation in several cell types including breast cancer cells (2, 14, 29). We observed decreases in CK-II{alpha} levels during PD 5–30 with a small increase at PD 10, indicating that CK-II{alpha} may be involved initially in the activation of beta-catenin and later in the inactivation of beta-catenin.

beta-Catenin forms complexes with E-cadherin as well as c-Met at the hepatocyte membrane. While the beta-catenin-E-cadherin complex is known to regulate cell-cell adhesion, the beta-catenin-Met complex is known to be involved in mitogenic signaling via HGF (1, 19). The beta-catenin-E-cadherin complex was detected fairly early due postnatal liver development. However, the beta-catenin-Met complex was not formed at high levels until PD 20 despite the presence of significant amounts of both proteins. We have previously demonstrated that the beta-catenin-Met complex is sensitive to HGF and is dissociated upon HGF binding to Met due to tyrosine phosphorylation. In addition, it is known that HGF signaling is ongoing during early liver development (11). These data suggest the possibility of HGF-mediated dissociation of the beta-catenin-Met complex or HGF-mediated retardation of Met-beta-catenin complex formation secondary to heightened HGF/Met signaling, thus adding to the free pool of beta-catenin for nuclear translocation. This also suggests that the formation of the Met-beta-catenin complex, also observed as membranous localization of beta-catenin, might be a part of the hepatocyte differentiation process reported previously (16, 19, 27).

Together, these data confirm that beta-catenin activation is essential for normal postnatal growth and development of the liver. Our results indicate that the primary role of beta-catenin activation, especially during PD 5–20, seems to be stimulation of hepatocyte proliferation resulting in an increase in liver size to adult levels. Recent studies have demonstrated that, apart from being promitogenic, beta-catenin is responsible for maintaining normal expression of genes involved in physiological processes such as drug metabolism and detoxification [cytochrome P-450 (CYP)2E1 and CYP1A2] and nitrogen metabolism (glutamine synthatase) (3, 6, 28, 32). A role of beta-catenin has also been shown in the metabolic zonality of the liver (3). It remains to be seen whether beta-catenin activation during the postnatal period is essential for the induction of these target genes involved in normal liver physiology and bringing their expression to adult levels. While further studies are needed to test whether beta-catenin is an overall "maturation factor" for the liver, the data presented here confirm the critical role of beta-catenin in early postnatal liver growth.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by American Cancer Society Grant RSG-03-141-01-CNE (to S. P. S. Monga) and by National Institute of Diabetes and Digestive and Kidney Diseases Grant 1-R01-DK-62277 (to S. P. S. Monga). Additional support was available from the Rango's Fund for Enhancement of Pathology Research and Cleveland Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. P. S. Monga, Depts. of Pathology and Medicine, Univ. of Pittsburgh School of Medicine, 200 Lothrop St., S-421 BST, Pittsburgh, PA 15216 (e-mail: smonga+{at}pitt.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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