Caspase-3/7-mediated Cleavage of β2-spectrin is Required for Acetaminophen-induced Liver Damage

Abstract

The ubiquitously expressed β2-spectrin (β2SP, SPTBN1) is the most common non-erythrocytic member of the β-spectrin gene family. Loss of β2-spectrin leads to defects in liver development, and its haploinsufficiency spontaneously leads to chronic liver disease and the eventual development of hepatocellular cancer. However, the specific role of β2-spectrin in liver homeostasis remains to be elucidated. Here, we reported that β2-spectrin was cleaved by caspase-3/7 upon treatment with acetaminophen which is the main cause of acute liver injury. Blockage of β2-spectrin cleavage robustly attenuated β2-spectrin-specific functions, including regulation of the cell cycle, apoptosis, and transcription. Cleaved fragments of β2-spectrin were physiologically active, and the N- and C-terminal fragments retained discrete interaction partners and activity in transcriptional regulation and apoptosis, respectively. Cleavage of β2-spectrin facilitated the redistribution of the resulting fragments under conditions of liver damage induced by acetaminophen. In contrast, downregulation of β2-spectrin led to resistance to acetaminophen-induced cytotoxicity, and its insufficiency in the liver promoted suppression of acetaminophen-induced liver damage and enhancement of liver regeneration.

Conclusions: β2-Spectrin, a TGF-β mediator and signaling molecule, is cleaved and activated by caspase-3/7, consequently enhancing apoptosis and transcriptional control to determine cell fate upon liver damage. These findings have extended our knowledge on the spectrum of β2-spectrin functions from a scaffolding protein to a target and transmitter of TGF-β in liver damage.

Keywords: TGF-β; acetaminophen; caspase-3/7; liver damage; β2-spectrin.

Fig 1

Cleavage of β2-spectrin is induced by acetaminophen in a TGF-β-dependent manner. (A) Various liver-damaging conditions and reagents were tested in exogenous β2-spectrin (β2SP)-expressing Huh7 cells. The arrow indicates an acetaminophen-induced fast migrating immunoreactive signal against the β2-spectrin antibody. (B, C) C57BL/6 mice were treated with acetaminophen (200 mg/kg, ip), and their livers collected at 1 and 3 days post-injection. The patterns of β2-spectrin expression were analyzed using Western blot, and the amounts measured as described in Materials and Methods. Relative amounts of full-length (β2SP-FL) and fast migrating β2-spectrin (β2SP-FM) are presented on the histogram. (D) The patterns of TGF-β signaling and apoptosis proteins were analyzed in Huh7 cells treated with various concentrations of acetaminophen at the indicated days. (E) C-terminal V5/His-tagged β2-spectrin- transfected Huh7 cells were incubated in the absence or presence of TGF-β (100 pM) in for the indicated times, and analyzed via Western blot using antibodies against the V5 and His epitopes. β2-Spectrin-transfected Huh7 cells treated with 10 mM acetaminophen for 16 h were applied as the positive control for cleavage of β2-spectrin. The numbers indicate relative amounts of β2SP-FM compared untreated. β-actin was used as the loading control.

Fig 2

Acetaminophen-induced β2-spectrin cleavage is mediated by caspase-3/7. (A) COS7 cells were transfected with N-terminal FLAG- (F) or C-terminal V5 (V)-tagged β2-spectrin constructs. Two days later, cells were harvested and subjected to Western blot by reprobing using antibodies against FLAG and V5 epitopes in identical membranes. (B) Purified human recombinant β2-spectrin (2 μg) was incubated with 1 unit of purified recombinant caspase-3, calpain-1 or calpain-2 at 37°C for 2 h. The reaction was terminated by adding Laemmli sampling buffer for SDS-PAGE, followed by Coomassie staining. The arrows indicate the fragment of β2-spectrin migrating similarly with acetaminophen treatment. (C) Acetaminophen (3 mM)-treated Huh7 cells were incubated in the absence or presence of increasing amounts of Ac-DEVD-CHO, a caspase-3 inhibitor, or Z-VAD-FMK, a pan-caspase inhibitor. The resulting cell lysates were subjected to SDS-PAGE, followed by Western blot using the β2-spectrin antibody with β-actin as the loading control. (D) Purified human recombinant β2-spectrin was incubated with various caspases, and further analyzed with SDS-PAGE and Coomassie staining.

Fig 3

Prevention of β2-spectrin cleavage suppresses its activities (A) Patterns of cell cycle and apoptosis regulatory proteins following transfection of wild-type (W) or uncleaved mutant (M) β2-spectrin in SNU-761 cells are shown. Expression of exogenous β2-spectrin was monitored using the V5 antibody, with β-actin as the loading control. (B) Following transient transfection with 3TP-Lux construct containing wild-type or mutant forms of β2-spectrin, Huh7 cells were incubated for 24 h in the absence or presence of 100 pM TGF-β and TGF-β-dependent transcriptional activity measured using the luciferase activity assay, as described in Materials and Methods. Data are expressed as mean values ± SE of three independent experiments. (C) Acetaminophen-induced cytotoxicities of wild-type and mutant β2-spectrin-transfected Huh7 cells were estimated with the MTT assay. Each number represents the mean ± SE of quadruplicate determinations. Statistically significant differences (P<0.05) are indicated by asterisks.

Fig 4

Cleaved fragments of β2-spectrin exhibit individual functions. (A) Patterns of cell cycle and apoptosis regulatory proteins induced following transfection of various forms of β2-spectrin in Huh7 cells. β-Actin was used as the loading control. (B) 293T cells were transfected with FLAG-Smad3 and various forms of V5-β2-spectrin or empty vector. Cell lysates were immunoprecipitated with anti-V5 antibody-conjugated agarose and subjected to Western blotting using anti-V5 and anti-FLAG mouse IgG. The inputs represent 5% of protein extracts without immunoprecipitation. (C) Following transient transfection with various forms of β2-spectrin and the 3TP-Lux construct, Huh7 cells were incubated for 24 h in the absence or presence of 100 pM TGF-β, and TGF-β-dependent transcriptional activity measured using the luciferase assay, as described in Materials and Methods. Data are expressed as mean ± SD of three independent experiments. (D) V5-β2-spectrin and HA-53BP2-transfected 293T cells were immunoprecipitated with anti-V5 antibody-conjugated agarose, and analyzed with Western blotting using the indicated antibodies. (E) Representative histograms showing DNA content of Huh7 cells. Full-length or cleaved β2-spectrin-transfected Huh7 cells were treated with TGF-β (100 pM) for 1 day and analyzed via flow cytometry with PI staining. (F) Percentages of cell distribution in the sub-G1 phase are shown on the histogram. Data are expressed as mean ± SD of three independent experiments. *P<0.01.

Fig 5

Differences in β2-spectrin fragment localization. (A) HeLa and U-2 OS cells were transfected with V5-tagged full-length, N-terminal and C-terminal β2-spectrin, and immunostained with an antibody against the V5-epitope. (B) Huh7 cells were transfected with various forms of V5-tagged β2-spectrin. Total cell lysates were additionally separated into cytosol and nuclear fractions, and subjected to immunoblot analysis using an anti-V5 antibody to determine the subcellular localization of fragments. PARP and α-tubulin were used as the nuclear and cytosol localization markers, respectively. (C) Four-month old male C57BL/6 mice were injected with acetaminophen (500 mg/kg, ip), and their livers were collected at 24 h day later. The left panels show the liver sections stained with the antibody raised against C-terminus (2101-2189) of β2-spectrin. Right panels are magnified images of the boxed areas. Scale bar: 200 μm.

Fig 6

Impairment of acetaminophen-induced damage response upon β2-spectrin downregulation. (A) Following transduction with β2-spectrin shRNA or empty construct containing lentivirus, HepG2 cells were incubated in the absence or presence of 5 mM acetaminophen for 48 h. The arrows indicate surviving colonies in the presence of acetaminophen upon downregulation of β2-spectrin shRNA. (B) Survival of HepG2 cells at the indicated concentrations of acetaminophen was estimated with the MTT assay after transduction with empty or β2-spectrin shRNA-containing lentivirus. The numbers represent mean values ± SD, with significant differences indicated by asterisks. **P<0.01; *P<0.05. (C, D) Four-month old male mice with spnb2^+/+, and spnb2^+/- were injected with acetaminophen (200 mg/kg, ip), and livers collected 9 days later. The left panels show the morphology of liver sections stained with H&E. Proliferation was evaluated immunohistochemically in wild-type and mutant livers using an antibody against Ki-67 (right panels). Relative amounts of Ki-67-positive nuclear and integrated signal intensities are shown on the histogram.