Mice lacking thyroid hormone receptor Beta show enhanced apoptosis and delayed liver commitment for proliferation after partial hepatectomy

Abstract

Background: The role of thyroid hormones and their receptors (TR) during liver regeneration after partial hepatectomy (PH) was studied using genetic and pharmacologic approaches. Roles in liver regeneration have been suggested for T3, but there is no clear evidence distinguishing the contribution of increased amounts of T3 from the modulation by unoccupied TRs.

Methodology/principal findings: Mice lacking TRalpha1/TRbeta or TRbeta alone fully regenerated liver mass after PH, but showed delayed commitment to the initial round of hepatocyte proliferation and transient but intense apoptosis at 48h post-PH, affecting approximately 30% of the remaining hepatocytes. Pharmacologically induced hypothyroidism yielded similar results. Loss of TR activity was associated with enhanced nitrosative stress in the liver remnant, due to an increase in the activity of the nitric oxide synthase (NOS) 2 and 3, caused by a transient decrease in the concentration of asymmetric dimethylarginine (ADMA), a potent NOS inhibitor. This decrease in the ADMA levels was due to the presence of a higher activity of dimethylarginineaminohydrolase-1 (DDAH-1) in the regenerating liver of animals lacking TRalpha1/TRbeta or TRbeta. DDAH-1 expression and activity was paralleled by the activity of FXR, a transcription factor involved in liver regeneration and up-regulated in the absence of TR.

Conclusions/significance: We report that TRs are not required for liver regeneration; however, hypothyroid mice and TRbeta- or TRalpha1/TRbeta-deficient mice exhibit a delay in the restoration of liver mass, suggesting a specific role for TRbeta in liver regeneration. Altered regenerative responses are related with a delay in the expression of cyclins D1 and E, and the occurrence of liver apoptosis in the absence of activated TRbeta that can be prevented by administration of NOS inhibitors. Taken together, these results indicate that TRbeta contributes significantly to the rapid initial round of hepatocyte proliferation following PH, and improves the survival of the regenerating liver at later times.

Figure 1. TRβ is transiently downregulated after PH and liver regeneration is delayed in TRα1/TRβ double KO mice.

(A,B) WT and TRα1/TRβ KO mice (‘KO’) were submitted to 70% PH, and the protein levels of TRα1 (55 kDa) and TRβ (47 kDa) were determined by Western blot, and expressed as percentage of the normalized band intensities (using β-actin as control) vs. sham operated animals at 0 h. (C) mRNA levels of TRα1 and TRβ were determined by quantitative real time RT-PCR. (D) The serum levels of T3 and T4 after PH were measured and expressed as percentage vs. sham operated animals at 0 h. The basal values were 7.4±0.5 and 36.5±4.2 µg/dl for T4 in WT and KO, respectively; 78±5 and 2965±307 ng/dl for T3 in WT and KO, respectively. (E) Liver mass recovery after PH was determined in these animals and in WT mice treated with MMI to pharmacologically induce hypothyroidism, and in one group of animals thyroid hormone was restituted by administration of T4. (F) Acute liver injury after PH was evaluated by measuring serum AST levels. Results show means ± SD of 6 to 8 animals per condition (B,C,E,F), 4 animals (D) or a representative blot of three (A). #P<0.01 vs. the corresponding condition at 0h (B,D); *P<0.05, **P<0.01 vs. WT condition or T4-untreated WT animals (E,F).

Figure 2. TRα1/TRβ deficiency results in delayed hepatocyte proliferation after PH.

(A) Effect of TRα1/TRβ deficiency on survival rates after PH (n = 19–32); animal death occurred in the first 24h post-PH. (B) Percentage of Ki67-positive cells in liver sections. (C) Hepatocyte ploidy distribution determined in preparations of liver disaggregated cells in a Medimachine. (D) Time-course of PCNA, cyclins E and D1, C/EBPα and C/EBPβ protein levels determined in liver extracts after PH. Results show means ± SD of 6 animals per condition (B,C) or a representative blot of three (D). *P<0.05, **P<0.01 vs. WT condition.

Figure 3. TRβ inhibits apoptosis in regenerating liver.

(A) TUNEL staining of cells undergoing apoptosis in regenerating liver (green). Liver sections were obtained at the indicated times after PH. The mean (n = 5 sections) of TUNEL positive cells per 100 nuclei (blue; TO-PRO-3 staining) is given at 48 h. (B) Caspase 3 activity was determined fluorometrically in liver extracts obtained at the indicated times after PH, and expressed vs. the activity of sham samples at 0h. (C) Alternatively, liver sections were stained with eosin/hematoxylin or Nile red to visualize lipid bodies. (D,E) Intrahepatic levels of cholesterol and triglycerides determined in liver extracts obtained at the indicated times after PH. Results show means ± SD of 6 animals per condition (B,D,E) or sections from a representative experiment of three (A,C). *P<0.05 vs. the WT condition.

Figure 4. Hydrodynamic transfection of TRβ restores liver regeneration index and impairs caspase activation in regenerating liver of TR KO mice.

Before PH, animals (WT, TRβ single KO and TRα1/TRβ double KO mice, referred as ‘KO’) were transfected hydrodynamically with GFP and TRβ expression vectors, or the TRβ-void vector pCX. The expression of GFP and the levels of TRβ (A), the liver mass regeneration index (B) and the levels of procaspase 3 and caspases 3 and 9 (C) were determined 48 h after PH. (D,E) Activities of caspase 9 and caspase 3 using specific peptide substrates were determined in liver extracts obtained 48 h post-PH. Results show the mean ± SD of 5 animals per condition (B,D,E) or a representative section or Western blot out of three (A,C). *P<0.01 vs. the WT condition; #P<0.01 vs. animals of the same genotype transfected with control TRβ-void vector (pCX).

Figure 5. Metabolic and enzymatic markers of oxidative and nitrosative stress in regenerating liver after PH.

(A) Content of 8-oxo-deoxyguanosine (8-oxo-dG), malondialdehyde (MDA), GSH and GSSG and the activity of glutathioneperoxidase (GPx) were determined in samples of liver obtained at the indicated times after PH. (B) Nitrosative stress in the remnant liver tissue was determined by ELISA of nitro-tyrosine. (C) Western blots of the protein levels of nitric oxide synthase 2 and 3 (NOS-2, -3) and the phosphorylation state of NOS-3 (at S473), and glutathione-S-transferase (GST). The levels of β-actin were used as control of lane charge (C). Results show means ± SD of 5 animals (A,B) or a representative experiment of 3 (C). *P<0.05, **P<0.001 vs. the WT condition (B).

Figure 6. ADMA levels decrease after PH of MMI-treated WT mice or TRβ KO and TRα+TRβ double KO mice.

(A) ADMA was determined in liver extracts at the indicated times after PH. (B) Enzymatic activity and protein and mRNA levels of DDAH I measured in liver extracts at the indicated times. (C) Total protein levels of FXR (59 kDa) were determined in liver extracts using mitochondrial porin (30 kDa) as control. (D) Hepatic levels of FXR and mBSEP mRNA after PH, determined by real-time q-PCR. Results show means ± SD of 4 animals (A–D) or a representative experiment of 3 (C). *P<0.05, **P<0.001 vs. time-controlled WT. #P<0.01 vs. the condition at time 0.

Figure 7. Inhibition of NOS-2 impairs apoptosis in the remnant liver of TRα1/TRβ double KO mice.

Animals were submitted to PH as described in Fig. 1 and 1400W (20 mg/kg) was administered intraperitoneally 8 h and 32 h after PH, and analyses were done at 48 h. (A) Apoptosis was determined by TUNEL staining of liver sections, caspase 3 activity was measured, and hepatic levels of nitrotyrosine were determined by ELISA. (B) ADMA was measured in liver at 48 h post-PH. (C) Western blots showing the levels of Bcl-2 (26 kDa), Bax (21 kDa), intact Bid (26 kDa), IAP-1 (72 kDa), x-IAP (57 kDa), PCNA and cyclin E in liver extracts obtained at 48 h post-PH. Results show means ± SD of 3 animals (A,B) or a representative experiment out of 3 (C). *P<0.05, **P<0.001 vs. the condition in the absence of 1400W.

Figure 8. Possible mechanisms leading to apoptosis in the regenerating liver of TRα1/TRβ double KO mice after PH.

Loss of signaling via TRβ, through TR gene deletion or MMI treatment, promotes a transient increase in FXR levels and activity favoring an increase in DDAH-1 activity that reduces ADMA levels –a NOS inhibitor- and promotes the synthesis of higher levels of NO. This enhancement in the activity of hepatic NOS induces nitrosative stress and promotes apoptosis. Inhibition of NOS-2 activity with 1400W, a selective inhibitor, abrogates nitrosative stress in liver and partially prevents apoptosis at 48 h after PH in TRβ-targeted mice (Fig. 7).