Knockout of myeloid cell leukemia-1 induces liver damage and increases apoptosis susceptibility of murine hepatocytes

Abstract

Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 protein family. It interacts with proapoptotic Bcl-2 family members, thereby inhibiting mitochondrial activation and induction of apoptosis. Mcl-1 is essential for embryonal development and the maintenance of B cells, T cells, and hematopoietic stem cells. We have recently shown that induction of Mcl-1 by growth factors rescues primary human hepatocytes from CD95-mediated apoptosis. This prompted us to further analyze the relevance of Mcl-1 for hepatocellular homeostasis. Therefore, we generated a hepatocyte-specific Mcl-1 knockout mouse (Mcl-1(flox/flox)-AlbCre). Deletion of Mcl-1 in hepatocytes results in liver cell damage caused by spontaneous induction of apoptosis. Livers of Mcl-1(flox/flox)-AlbCre mice are smaller compared to control littermates, due to higher apoptosis rates. As a compensatory mechanism, proliferation of hepatocytes is enhanced in the absence of Mcl-1. Importantly, hepatic pericellular fibrosis occurs in Mcl-1 negative livers in response to chronic liver damage. Furthermore, Mcl-1(flox/flox)-AlbCre mice are more susceptible to hepatocellular damage induced by agonistic anti-CD95 antibodies or concanavalin A.

Conclusion: The present study provides in vivo evidence that Mcl-1 is a crucial antiapoptotic factor for the liver, contributing to hepatocellular homeostasis and protecting hepatocytes from apoptosis induction.

Figure 1

Deletion of Mcl-1 in hepatocytes of Mcl-1^flox/flox-AlbCre mice. (A) DNA from ear biopsies (left) or liver specimens (right panel) was isolated. PCR was performed using primers for wildtype (wt), floxed, or deleted (Δ) Mcl-1, Cre, and actin as internal control. (B) Liver lysates of Mcl-1^flox/+, Mcl-1^flox/flox, Mcl-1^flox/+-AlbCre and Mcl-1^flox/flox-AlbCre mice at the age of 4 and 8 weeks, were analyzed by western blot for Mcl-1 expression and for α–Tubulin as loading control. (C) mRNA from total livers or from isolated hepatocytes of Mcl-1^flox/flox-AlbCre and control mice at the age of 4 or 8 weeks was isolated and transcribed into cDNA. Mcl-1 and GAPDH expression were analyzed by RealTime-PCR, and Mcl-1/GAPDH ratio was calculated. Both single (squares) and median values (bars) are presented. **: p<0.01; ***: p<0.001; n.s.: not significant.

Figure 2

Liver damage in Mcl-1^flox/flox-AlbCre mice. (A) 4 to 16 week old Mcl-1^flox/flox-AlbCre (Mcl-1^−/−) or control (Mcl-1^+/+) mice were analyzed for body weight and liver weight. (B) Macroscopic appearance of representative livers from 8 week old female Mcl-1^+/+ and Mcl-1^−/− mice. (C) Serum of Mcl-1^+/+ and Mcl-1^−/− mice between 4 and 16 weeks of age was collected. Serum AST and ALT levels were measured. Both single (squares) and median values (bars) are presented. Values beyond scaling are marked separately. *: p<0.05; ***: p<0.001; n.s: not significant.

Figure 3

Livers of Mcl-1^flox/flox-AlbCre mice show higher apoptosis rates. (A) Liver sections of 4 week old Mcl-1^+/+ and Mcl-1^−/− mice were stained for active caspase-3 and with eosin/hematoxylin. (B) Total liver lysates were analyzed for active caspase-3 activity using fluorometric substrates. (C) Liver sections were stained for DNA strand breaks using TUNEL-technology, and cell nuclei were counterstained using DAPI. The ratio of TUNEL-positive nuclei was calculated. Both single (squares) and median values (bars) are presented. The bar corresponds to 100μm. ** p<0.01, *** p<0.001. RLU: relative light units.

Figure 4

Livers of Mcl-1^flox/flox-AlbCre mice show higher proliferation rates. (A) Liver sections of 4 week old Mcl-1^+/+ and Mcl-1^−/− mice were stained for Ki67 and with eosin/hematoxylin. (B) Mcl-1^+/+ and Mcl-1^−/− mice received BrdU (0.8mg/ml; 60h) in their drinking water. Liver sections were stained for BrdU incorporation, and cell nuclei were counterstained using DAPI. The ratio of BrdU-positive nuclei was calculated. Both single (squares) and median values (bars) are presented. The bar corresponds to 100μm. ** p<0.01.

Figure 5

Fibrosis induction in Mcl-1^flox/flox-AlbCre mice. (A) mRNA from total liver lysates of 16 week old Mcl-1^+/+ and Mcl-1^−/− mice was isolated and transcribed into cDNA. Collagen-1 and GAPDH expression were analyzed by RealTime-PCR, and collagen/GAPDH ratio was calculated. Both single (squares) and median values (bars) are presented. **: p<0.01; n.s.: not significant. (B) Liver sections of 8 or 16 week old Mcl-1^+/+ and Mcl-1^−/− mice were stained with Sirius red for collagen deposition. The bar corresponds to 100μm.

Figure 6

Mcl-1^flox/flox-AlbCre mice are more susceptible towards CD95-mediated liver damage. 8 (A, C–E) or 24 week old (B) Mcl-1^+/+ and Mcl-1^−/− mice were treated with Jo2 (0.5mg/kg) i.p. After 3h, mice were sacrificed. (A, B) Blood was collected and serum AST and ALT levels were determined. Both single (squares) and median values (bars) are presented. (C) Liver sections were stained with eosin/hematoxylin and for active caspase-3. The bar corresponds to 100μm. (D) Total liver lysates were analyzed for caspase-3 activity. (E) Apoptosis rate and architectural damage were analyzed within H/E-stained sections and were clustered into 4 groups (0: no, 1: low, 2: moderate, 3: high apoptosis rate/architectural damage). * p<0.05; **: p<0.01; *** p<0.001. RLU: relative light units.

Figure 7

Mcl-1^flox/flox-AlbCre mice are more susceptible towards T cell-mediated liver damage. 8 week old Mcl-1^+/+ and Mcl-1^−/− mice were treated with ConA (25mg/kg) intravenously. After 4h, mice were sacrificed. Blood was collected and serum AST and ALT levels were determined. Both single (squares) and median values (bars) are presented. *: p<0.05.