Tri-iodothyronine induces hepatocyte proliferation by protein kinase A-dependent β-catenin activation in rodents

Abstract

Thyroid hormone (T3), like many other ligands of the steroid/thyroid hormone nuclear receptor superfamily, is a strong inducer of liver cell proliferation in rats and mice. However, the molecular basis of its mitogenic activity, which is currently unknown, must be elucidated if its use in hepatic regenerative medicine is to be considered. F-344 rats or C57BL/6 mice were fed a diet containing T3 for 2-7 days. In rats, administration of T3 led to an increased cytoplasmic stabilization and nuclear translocation of β-catenin in pericentral hepatocytes with a concomitant increase in cyclin-D1 expression. T3 administration to wild-type (WT) mice resulted in increased hepatocyte proliferation; however, no mitogenic response in hepatocytes to T3 was evident in the hepatocyte-specific β-catenin knockout mice (KO). In fact, T3 induced β-catenin-TCF4 reporter activity both in vitro and in vivo. Livers from T3-treated mice demonstrated no changes in Ctnnb1 expression, activity of glycogen synthase kinase-3β, known to phosphorylate and eventually promote β-catenin degradation, or E-cadherin-β-catenin association. However, T3 treatment increased β-catenin phosphorylation at Ser675, an event downstream of protein kinase A (PKA). Administration of PKA inhibitor during T3 treatment of mice and rats as well as in cell culture abrogated Ser675-β-catenin and simultaneously decreased cyclin-D1 expression to block hepatocyte proliferation.

Conclusion: We have identified T3-induced hepatocyte mitogenic response to be mediated by PKA-dependent β-catenin activation. Thus, T3 may be of therapeutic relevance to stimulate β-catenin signaling to in turn induce regeneration in selected cases of hepatic insufficiency.

Figure 1. Activation of β-catenin signaling in rat livers after T3-feeding

Immunostaining shows β-catenin localizing to the hepatocyte membrane in the control livers, while it accumulates in the hepatocyte cytoplasm in T3-fed rats at 2 days. A progressive shift of β-catenin stabilization from zone I towards zone II along with nuclear translocation of β-catenin-positive is observed at day 4 of T3 feeding. A concomitant increase in the number of GS-positive cells around central vein is observed at 4 days after T3. Progressive Cyclin-D1 nuclear staining is evident in zone I at 2 days, and in zone I and II at 4 days of T3 feeding as compared to the control livers. Four rats per group were used for this study.

Figure 2. Lack of β-catenin in hepatocytes impairs hepatocyte proliferation and Cyclin-D1 expression in response to T3 diet

A. Representative microphotographs showing immunohistochemical staining for BrdU in the WT and KO livers after T3-treatment for 4 days (200×). Several BrdU-positive hepatocyte nuclei are observed in livers of WT mice fed T3 for 4 days. KO after T3 treatment lack BrdU staining in the hepatocytes although BrdU-positive non-parenchymal cells are evident, similar to WT livers. At least three WT and KO were used throughout this study. B. A significant (*p<0.05) decrease in BrdU LI of hepatocytes in KO versus WT mice after T3 treatment. While T3 stimulated BrdU incorporation in the WT hepatocytes, no significant increase was evident in KO hepatocytes. At least 5000 hepatocyte nuclei per liver were scored. The LI is expressed as number of BrdU-positive hepatocyte nuclei/100 nuclei. Results are expressed as mean ± standard error (S.E.) of 3 or more mice per group. C. LI of pancreatic acinar cells in WT and KO mice shows no significant difference between the two groups following treatment with T3-supplemented diet (4 mg/kg) for 7 days. T3 stimulated BrdU incorporation in pancreatic acinar cells comparably and significantly (*p<0.05) in WT and KO. At least 2000 acinar cell nuclei per pancreas were scored. LI was expressed as number of BrdU-positive acinar cell nuclei/100 nuclei and results expressed as mean ± standard error (S.E.) of 3 or more mice per group. D. Representative photomicrographs showing labeling of BrdU in the pancreas of WT and KO mice treated with T3 for 4 days (200×). E. Representative microphotograph of Cyclin-D1 staining in the WT and KO livers after 4 days of T3-feeding (200×). Absence of Cyclin-D1 immunoreactivity in KO hepatocytes nuclei is clearly evident despite T3 feeding as compared to the WT. F. qRT-PCR analysis of TRα and TRβ expression in livers of untreated WT and KO mice. 18S was used as endogenous control. Error bars represent the standard error (S.E) of TaqMan RT-PCR performed in triplicates.

Figure 3. T3 induces β-catenin activation in vitro and in vivo

A. TOPflash reporter assay shows an increase in luciferase activity 48 hours after T3 treatment of primary rat hepatocytes. A vector containing renilla luciferase was used as an internal control for transfection efficiency, and results are expressed as relative firefly/renilla luciferase activity. The results presented are the mean ± standard error (S.E.) for three experiments; *p < 0.05. B. TOPflash reporter assay shows an increase in luciferase activity 48 hours after T3 treatment of primary murine hepatocytes. A vector containing renilla luciferase was used as an internal control for transfection efficiency, and results are expressed as relative firefly/renilla luciferase activity. The results presented are the mean ± standard error (S.E.) for three experiments; *p < 0.05. C. TOPGAL mice fed T3 diet for 4 and 7 days shows increased β-galactosidase expression by indirect immunohistochemistry. At baseline β-galactosidase expression was detected in pericentral hepatocytes only, whereas T3 feeding led to widening of the expression to several hepatocytes layers around the central vein demonstrating an increase in the activity of β-catenin-TCF complex. A total of three TOPGAL mice were used for this study.

Figure 4. T3 induced β-catenin activation via Ser675-phosphorylation

A. qRT-PCR analysis of β-catenin expression (normalized to cyclophilin A) in C57BL6 mice treated with T3 for 4 days shows no change. Error bars represent the standard error (S.E.) of TaqMan RT-PCR performed in triplicates. B. Representative western blots show T3 feeding for 4 days does not lead to a notable increase in total β-catenin when compared to basal diet fed mice. However Cyclin-D1 levels are consistently increased. GSK3β-Ser9 levels remain unaffected at 4 days of T3 treatment. Gapdh verifies equal loading. Each lane represents an individual sample. C. Immunoprecipitation studies from three representative livers show no change in association of β-catenin and E-cadherin in the livers of 4 days T3- versus basal diet-fed C57BL/6 mice. D. Representative western blot shows a noteworthy increase in pSer675-β-catenin levels in 4 days T3-fed as compared to control diet-fed C57BL/6 mice. Gapdh verified equal loading.

Figure 5. Blockade of Protein Kinase A impairs T3's effect on β-catenin, Cyclin-D1 and hepatocyte proliferation in mice

A. A representative western blot using pooled samples from three wells per condition (left) shows increased levels of pSer675-β-catenin and pSer133-CREB in primary mouse hepatocytes after 30 minutes of T3 treatment. Inclusion of PKA inhibitor H89 (1 μM) in the media 30 minutes prior to the addition of T3 (100 nM) showed a notable decrease in pSer675-β-catenin and pSer133-CREB levels. Densitometry on the representative WB (right) shows an increase in pSer675-β-catenin and pSer133-CREB after T3 treatment, which was blocked by H89 treatment. (I.O.D. – integrated optical density). B. A representative western blot shows a decrease in the hepatic levels of pSer675-β-catenin and Cyclin-D1 when H89 was injected twice IP in 3-day T3 fed mice as compared to 3 day T3 only group. Gapdh verifies equal loading. Each lane represents an individual sample. C. A representative micrograph (200×) illustrates a decrease in the number of Cyclin-D1-positive hepatocytes when H89 was injected twice to the 3-day T3-fed mice as compared to T3 only group. Three or more mice per group were used for this study. D. Quantification of the Cyclin-D1-positive hepatocytes shows a significant decrease in positive cells in H89+T3 as compared to T3 only group (*p<0.05). E. A representative micrograph (200×) illustrates a noteworthy increase in BrdU uptake by the hepatocytes in mice after 5 days of T3 feeding, which was dramatically decreased in animals simultaneously administered H89 IP every 24 hours. Three or more mice per group were used for this study. F. Quantification of BrdU positive hepatocytes shows a significant (*p<0.05) decrease in the LI in T3+H89 group as compared to T3 only.

Figure 6. Blockade of Protein Kinase A impairs T3's effect on β-catenin, Cyclin-D1 and hepatocyte proliferation in rats

A. Representative microphotographs (200×) illustrate the effect of H89 on T3-induced rat hepatocyte proliferation by BrdU immunohistochemistry. H89 was given 1 hour prior to a single dose of T3 (20 μg/100 g) and the rats were sacrificed 24 hours later. Minimum four rats per group were used for this entire study. B. Quantification of BrdU positive hepatocytes in A shows a significant (*p<0.05) increase in the LI after T3 treatment, which was significantly abrogated in the presence of H89 (*p<0.05). C. Representative microphotographs (200×) show increased nuclear Cyclin-D1 expression in hepatocyte following a single injection of T3, which was decreased in the group that simultaneously received H89 as well. D. Quantification of Cyclin-D1-positive hepatocytes to calculate LI shows a significant increase after T3 treatment (*p<0.05), which was reduced significantly in the H89 pretreatment group (*p<0.05). E. A representative western blot shows the effect of H89 on T3-induced Ser675-β-catenin levels in rat liver. Gapdh depicts protein loading. F. Densitometric analysis of E (Ser675-β-catenin normalized to Gapdh) using the ImageJ software shows a significant (*p<0.05) decrease in Ser675-β-catenin levels in T3+H89 as compared to T3 only group. (I.O.D. – integrated optical density).