Related Experiment Videos
Fas-mediated apoptosis in mouse hepatocytes involves the processing and activation of caspases
R A Jones1, V L Johnson, N R Buck
1School of Biological Sciences, University of Surrey, Guildford, England, UK.
Abstract:
The mechanism of Fas antigen-induced hepatocyte apoptosis was investigated. Using a monoclonal antibody directed against the Fas antigen, apoptosis was induced in freshly isolated murine hepatocytes within 90 minutes of antibody addition as assessed by plasma membrane bleb formation, chromatin condensation, and DNA fragmentation. Pretreatment of the cells with the caspase inhibitors, N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK), or Z-Asp-2,6-dichlorobenzoyloxymethylketone inhibited anti-Fas-mediated apoptosis. Likewise, the serine protease inhibitors, N-tosyl-L-phenyl chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI), prevented apoptosis, whereas N-tosyl-L-lysine chloromethyl ketone (TLCK), Ac-Leu-Leu-L-norleucinal, Ac-Leu-Leu-L-methional, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane were without effect. Examination of CED-3/caspase-3-related caspases revealed that pro-caspases-3 (CPP32) and -7 (Mch-3alpha) were rapidly processed after Fas antigen stimulation. Caspase-7 was further cleaved to form the catalytically active subunits. In contrast, the p17 subunit of caspase-3 was not detected, indicating slow formation or rapid degradation. The activation of CED-3-related caspases was further confirmed by an increase in the rate of Z-DEVD-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC) hydrolysis that was sensitive to Ac-DEVD-CHO and was inhibited by pretreatment of the cells with TPCK but not by DCI. In contrast, no increase in the rates of hydrolysis of Z-YVAD-AFC, a substrate for caspase-1, was detected. Investigation of the in situ proteolytic cleavage of the CED-3 related caspases substrate, poly(ADP-ribose) polymerase, revealed that this protein was not degraded in hepatocytes undergoing Fas-mediated apoptosis. Taken together, our results show that processing of caspases, in particular, caspases-7 and -3, occurs during Fas-induced apoptosis of mouse hepatocytes and suggest a role of these proteases as well as serine protease(s) in the apoptotic response.
Insights
Fas-induced hepatocyte apoptosis involves caspases-7 and -3 processing. Serine proteases also play a role in this apoptotic response in mouse liver cells.
Area of Science:
- Cell Biology
- Biochemistry
- Immunology
Background:
- Hepatocyte apoptosis is crucial for liver homeostasis.
- Fas antigen signaling is a key pathway inducing apoptosis in hepatocytes.
Purpose of the Study:
- To investigate the mechanism of Fas antigen-induced hepatocyte apoptosis.
- To identify the specific caspases and proteases involved in this process.
Main Methods:
- Induction of apoptosis in murine hepatocytes using anti-Fas monoclonal antibody.
- Assessment of apoptosis via plasma membrane blebbing, chromatin condensation, and DNA fragmentation.
- Inhibition studies using specific caspase and serine protease inhibitors.
Main Results:
- Fas-mediated apoptosis was confirmed by morphological and biochemical markers.
- Caspase inhibitors (Ac-DEVD-CHO, Z-VAD-FMK) and serine protease inhibitors (TPCK, DCI) blocked apoptosis.
- Pro-caspases-3 and -7 were processed upon Fas stimulation, with caspase-7 activation observed.
- Poly(ADP-ribose) polymerase was not degraded, suggesting specific apoptotic pathways.
Conclusions:
- Caspase processing, particularly of caspases-7 and -3, is essential for Fas-induced hepatocyte apoptosis.
- Serine proteases are implicated in the apoptotic response.
- This study elucidates key molecular players in Fas-mediated liver cell death.