Characterization of the regulation and function of zinc-dependent histone deacetylases during rodent liver regeneration

Abstract

The studies reported here were undertaken to define the regulation and functional importance of zinc-dependent histone deacetylase (Zn-HDAC) activity during liver regeneration using the mouse partial hepatectomy (PH) model. The results showed that hepatic HDAC activity was significantly increased in nuclear and cytoplasmic fractions following PH. Further analyses showed isoform-specific effects of PH on HDAC messenger RNA (mRNA) and protein expression, with increased expression of the class I HDACs, 1 and 8, and class II HDAC4 in regenerating liver. Hepatic expression of (class II) HDAC5 was unchanged after PH; however, HDAC5 exhibited transient nuclear accumulation in regenerating liver. These changes in hepatic HDAC expression, subcellular localization, and activity coincided with diminished histone acetylation in regenerating liver. The significance of these events was investigated by determining the effects of suberoylanilide hydroxyamic acid (SAHA, a specific inhibitor of Zn-HDAC activity) on hepatic regeneration. The results showed that SAHA treatment suppressed the effects of PH on histone deacetylation and hepatocellular bromodeoxyuridine (BrdU) incorporation. Further examination showed that SAHA blunted hepatic expression and activation of cell cycle signals downstream of induction of cyclin D1 expression in mice subjected to PH.

Conclusion: The data reported here demonstrate isoform-specific regulation of Zn-HDAC expression, subcellular localization, and activity in regenerating liver. These studies also indicate that HDAC activity promotes liver regeneration by regulating hepatocellular cell cycle progression at a step downstream of cyclin D1 induction.

Figure 1. Hepatic Zn-HDAC Activity and mRNA Expression after PH

(A-C) Hepatic Zn-HDAC activity (relative units) at serial times after PH or sham surgery in (A) whole liver lysates (*p<0.01 vs. 12 hours PH; ^p≤0.001 vs. 0 and 12-24 hours PH; ⁺p<0.01 vs. 0 and 12-24 hours PH and p<0.03 vs. 72 hours sham; hepatic HDAC enzyme specific activity in 0 hour replicates: 149±25 pmol/mg/min); (B) cytoplasmic lysate preparations (*p<0.04 vs. 0 hours; specific activity in 0 hour replicates: 217±7 pmol/mg/min); and (C) nuclear lysate preparations (p<0.04 between groups; specific activity in 0 hour replicates: 440±24 pmol/mg/min). TSA-resistant activity in all samples is also shown. (D-F) Hepatic mRNA expression of (D) HDAC1 (*p<0.001 vs. 0 and 36 hours sham), (E) HDAC8 (*p<0.02 vs. sham), and (F) HDAC4 (*p<0.03 vs. sham) at serial times after PH or sham surgery. Data standardized to expression of β2-microglobulin.

Figure 2. Hepatic Zn-HDAC Protein Expression during Liver Regeneration

(A) Representative protein immunoblot analyses for class I (, , , and 8) and class II (, , and 7) HDACs in pooled replicate lysates of quiescent (0 hour, 0h), regenerating (12 hours (12h) to 7 day (7d)), and sham-operated (12h to 7d) livers. HeLa cell nuclear extract is shown as positive control and β-actin and GAPDH expression as loading controls. (B) Quantification of HDAC protein levels standardized to GAPDH.

Figure 3. Subcellular Compartment-Specific Hepatic Zn-HDAC Expression during Liver Regeneration

(A) Representative protein immunoblot analyses for class I and class II HDACs in pooled replicate nuclear and cytoplasmic lysates of quiescent (0h) and regenerating (12h to 7d) livers. β-actin and GAPDH expression shown as loading controls. (B) Quantification of HDAC protein levels standardized to GAPDH (cytoplasmic) or β-actin (nuclear).

Figure 4. Hepatic Acetyl-Protein Abundance after PH

Representative immunoblot analyses for (A) acetyl-lysine-containing proteins in pooled replicate whole liver lysates and (B) acetyl-lysine-containing histones, histone H3 acetylated on Lys9 (Ac-H3K9), and total histone H3 in pooled replicate acid extracts of whole liver harvested at serial times after PH or sham surgery. (C) Quantification of acetyl-histones standardized to total histone H3.

Figure 5. The Effect of SAHA on Histone Acetylation and Hepatocellular Proliferation after PH

(A) Representative immunoblot analyses of acetyl-lysine containing histone H3 (Ac-H3) and H4 (Ac-H4) and total H3 in pooled replicate acid extracts of whole liver from vehicle- and SAHA-treated mice harvested 12-48 hours after PH. (B) Quantification of acetyl-histone standardized to total histone H3. (C) Immunohistochemical analysis (36 hours after PH; 100 micron bar shown) and (D) quantification (24-48 hours after PH) of hepatocellular BrdU incorporation in vehicle- and SAHA-treated mice (*p≤0.05 vs. vehicle). (E) Hematoxylin and eosin staining and (F) quantification of hepatocellular mitotic frequency (per 10 high-powered fields) 48 hours after PH in vehicle- and SAHA-treated mice.

Figure 6. The Effect of SAHA on Hepatic Expression of Cell Cycle Regulators after PH

Hepatic mRNA expression of (A) cyclin D1, (B) cyclin E1, (C) cyclin A2 (*p<0.001 vs. vehicle), and (D) cyclin B1 (*p<0.001 vs. vehicle) in un-operated (control) and vehicle- or SAHA-treated mice 12-36 hours after PH. Data standardized to expression of β2-microglobulin. (E) Representative immunoblot analyses of cyclin D1 and cyclin B1 in pooled replicate whole liver lysates from vehicle- and SAHA-treated mice 12-48 hours after PH. GAPDH shown as loading control. (F) Quantification of cyclin expression standardized to GAPDH.

Figure 7. The Effect of SAHA on Hepatic p19^INK4dExpression and Promoter Acetylation in Regenerating Liver

Hepatic mRNA expression of p19^INK4d (A) in un-operated (control) and vehicle- or SAHA-treated mice 12-36 hours after PH and (B) in untreated mice at serial times after PH or sham surgery (*p<0.001 vs. 0 hour and sham). Data standardized to expression of β2-microglobulin. (C) Representative immunoblot expression analyses and (D) quantification of hepatic p19^INK4d expression after PH or sham surgery in pooled replicate nuclear and cytoplasmic fractions from untreated mice or 12 hours after PH in vehicle- and SAHA-treated mice. Ac-H3K9 (or normal rabbit IgG as control) ChIP-PCR of (E) p19^INK4d (*p=0.01 vs. vehicle) and (F) cyclin A2 in liver from vehicle- and SAHA-treated mice.

Figure 8. A Model of the Effect of SAHA on Hepatocellular Cell Cycle Progression during Liver Regeneration

PH induces quiescent (G₀) hepatocytes to re-enter the cell cycle (G₁). As reported here, PH also induces hepatic Zn-HDAC activity and SAHA, which inhibits such activity, impairs PH-stimulated hepatocellular cell cycle progression downstream of cyclin D1 induction and augments hepatic p19^INK4d expression. These data suggest that Zn-HDAC activity promotes hepatocellular G₁ cell cycle progression during normal liver regeneration and that p19^INK4d mediates the inhibitory effect of SAHA on such regeneration. (Solid lines indicate positive (→ and negative () regulation demonstrated by data reported here. Dashed lines represent hypothesized regulation based on these data and discussed in the text.)