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. An established phenomenon is that of plated hepatocytes becoming refractory to the Fas ligand, reversible when the protein synthesis inhibitor, cycloheximide is administered. In order to elucidate by which mechanism this is regulated, various signalling protein inhibitors where used. The role of the MAPK as a negative regulator of Fas-mediated apoptosis was investigated using the MEK1 inhibitor, PD089059. Inhibition of MEK1 in conjunction with anti-Fas antibody induced apoptosis in cultured hepatocytes with similar kinetics to cycloheximide in combination with anti-Fas antibody. Both PD089059 and Wortmannin induced apoptosis in plated hepatocyte cultures in the absence of anti-Fas antibody. This study showed that the refractory nature of culture hepatocytes was due to the regulatory ability of constitutively synthesised proteins combined with one or both PI-3Kinase and MAPK activities rather than an adherence process. Pre-treatment of the freshly isolated hepatocytes in cell suspension with the caspase peptide 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-dichlorobenzoyloxymethyl-ketone inhibited anti-Fas-mediated apoptosis. Likewise, the serine protease inhibitors, N-tosyl-L-phenyl chloromethyl ketone and 3,4-dichloroisocoumarin prevented apoptosis, whereas N-tosyl-Z-lysine chloromethyl ketone, 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-3a) 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 pre-treatment of the cells with N-tosyl-Z-phenyl chloromethyl ketone but not by 3,4-dichloroisocoumarin. 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, this study shows that processing of caspases, in particular, caspases-7 and -3 occurs during Fas-induced apoptosis of mouse hepatocytes and suggests a role of these proteases as well as serine protease(s) in the apoptotic response. A cell-free system using isolated nuclei from liver and thymocytes together with hepatocyte and thymocyte apoptotic extracts was established to examine why liver PARP did not undergo proteolysis during apoptosis. The distinct morphological changes characteristic of apoptosis; chromatin condensation and DNA fragmentation, were seen in the nuclei incubated with apoptotic cell extract. In spite of these morphological and nuclear changes, apoptotic hepatocyte extract did not cleave PARP in thymocyte or liver nuclei but processed purified PARP to its 85 kDa fragment. However, incubation of apoptotic thymocyte extract with thymocyte nuclei resulted in rapid PARP cleavage, but failed to cleave liver PARP. Sonication of liver nuclei to liberate PARP did not render PARP cleavable by either extracts from apoptotic hepatocytes or thymocyte or even an excess of recombinant human caspase-3 activity. Take together this study showed that liver PARP is resistant to proteolytic cleavage by caspases and that the caspase-3/PARP machinery is not requisite for apoptosis in Fas-mediated apoptosis in mouse hepatocytes. The above findings showed that the refractory nature of cultured hepatocytes towards Fas receptor mediated apoptosis is owing to the activation of PI-3Kinases and MAPK. Further, the mechanism of Fas-induced hepatocyte apoptosis involves caspases, in particular caspase-7 and possibly caspase-3, as well as a TPCK/DCI sensitive serine-type proteases. The DNA-repair enzyme, PARP present in mouse liver is precluded from proteolytic cleavage by caspase-3 and related caspases which indicates that PARP proteolysis is not required for DNA fragmentation and the development of morphological features of apoptotic liver nuclei.