Several models of tumor necrosis factor (TNF)/TNFreceptor 1 (TNF-R1)-dependent liver injury in mice were investigated with respect to caspase-3-like protease activation representing a pivotal mechanism of apoptotic cell death. Injection of TNF or T-cell-activating agents (i.e., agonistic anti-CD3 antibody or staphylococcal enterotoxin B [SEB]) into galactosamine (GalN)-sensitized mice caused TNF/TNF-R1-dependent liver injury. Intravenous concanavalin A (Con A) alone induced TNF-mediated hepatotoxicity dependent on both TNF-R1 and TNF-R2. Hepatic caspase-3-like proteases were activated in GalN/TNF, GalN/anti-CD3, or GalN/SEB-treated mice, but not in Con A-treated mice. Consistently, the broad-spectrum caspase inhibitor, benzoyloxycarbonyl-val-ala-asp-fluoromethylketone (zVADfmk), prevented TNF-mediated hepatotoxicity in all GalNdependent models, but failed to protect against Con A. Under transcriptional arrest, however, Con A induced TNF-R1-dependent, but not TNF-R2-dependent, activation of caspase-3-like proteases, and zVADfmk prevented animals from Con A-mediated liver injury under this condition. Histological analysis revealed distinct differences between Con A-and GalN/Con A-induced liver injury regarding apoptotic morphology of hepatocytes. We conclude that impaired transcription induces a switch of Con A hepatotoxicity toward a caspase-3-like protease-dependent pathway. The observation that the functional state of the transcriptional machinery decides whether TNF-driven hepatocyte apoptosis involves activation of caspase-3-like proteases or alternative signaling pathways in vivo might be of relevance for the immunopathology of the liver. (HEPATOL-OGY 1999;30:1241-1251.) Activation of T lymphocytes appears to be the initial event in the pathophysiology of a variety of autoimmune liver diseases (e.g., chronic active hepatitis) or viral hepatitis. 1 This lymphocyte activation and the ensuing interactions of these cells with macrophages leads to a systemic cytokine response. The continuous release of proinflammatory cytokines such as tumor necrosis factor (TNF) or interferon gamma (IFN-␥) into the circulation is currently held responsible for the onset of pathological symptoms and the clinical manifestation of a variety of immunologically mediated liver diseases. [2][3][4][5] Several animal models of cytokine-dependent liver tissue destruction allow the study of mechanisms of T-lymphocyte activation in relation to the extent and time course of subsequent hepatic injury. In two commonly used models, D-galactosamine (GalN)-sensitized mice are injected with T-cell-activating anti-CD3 monoclonal antibodies, 6 or with the superantigen, staphylococcal enterotoxin B (SEB). 7 These treatment regimens both result in severe liver injury 8,9 characterized by histological features of apoptosis as well as internucleosomal DNA fragmentation. 9 In contrast to these GalN models, naive mice, when injected with the T-cell mitogen, concanavalin A (Con A), develop an acute, partly apoptotic, hepatic injury that is subsequently ove...

Five main type H1 histones have been described in man (H1.1-H1.5) in addition to the testis specific type H1t and the replacement subtype H1 degrees, which is found mainly in highly differentiated cells. We have isolated this whole complement of H1 genes and have studied the expression of the seven human H1 subtype genes in several cell lines. The RNAase protection assay was used to discriminate between the very similar transcripts derived from the seven H1 subtype genes. With the exception of H1.2 and H1.4, we found substantial differences between the H1 mRNA levels in the different cell lines tested. No H1.1 mRNA was detected in most of the cell lines and just a low level of H1.1 mRNA was found in human testis. In contrast to the differential patterns of the other subtypes, H1.2 and H1.4 were in all cells expressed at a high level, indicating a basal function compared with the other H1 histones. Because differences in the timing of H1 protein subtype synthesis have been reported, we have analyzed the kinetics of accumulation of H1 subtypes in synchronized HeLa cells and observed that all H1 subtypes examined (H1 degrees, H1.2-H1.5) were expressed in a replication-dependent manner. The analysis showed a differential rise of mRNA levels during S-phase, from four-fold (H1 degrees) to 15-fold (H1.5). Our results may point at a specific function of each subtype and suggest that expression of the H1 histone subtype genes depends on common S-phase-depent factors as well as on individual regulatory systems. Thus, the data presented here provide a basis for further analysis of the regulation and function of the complex H1 gene and protein family.

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In man, the H1 class of histones consists of seven different isoforms, termed H1.1ϪH1.5, H1t and H1°. Analysis of the promoters of the respective genes reveals that all seven H1 gene promoters share conserved sequence elements: a TATA box at around position Ϫ25 (relative to the transcription start site), a CCAAT motif at about position Ϫ50 (except in the H1 promoter), an H1-box (AAACACA) around position Ϫ110 (except in the H1.1 promoter), and the highly conserved motif TGTGT/CTA (TG-box or CH1UE) at around nucleotide position Ϫ450 (except in the H1.1 promoter). Analysis of the H1.3 gene promoter was carried out with reporter gene assays (using the luciferase gene as a reporter gene) including stepwise deletion and site-directed mutagenesis. In addition, electrophoretic mobility-shift assays were carried out for the analysis of protein/DNA interactions at conserved promoter motifs. Mutation analysis indicates that the CH1UE motif is involved in mediating the S-phase-dependent expression of the H1.3 gene. Comparison of H1 promoters shows that the CCAAT-box is extended in each case by CA. Mutational analysis indicates that only the CCAATCA heptanucleotide, but not just the CCAAT sequence mediates the effect of this element in H1 gene promoters.Keywords : histone H1; histone gene promoter; (histone) gene expression ; gene regulation; luciferase activity.Histones are a major component in the eukaryotic nucleus and play an important role in forming the fundamental nucleosomal subunit structure of chromatin. They are subdivided into the group of core histones (H2A, H2B, H3 and H4), which form the histone octamer of the nucleosomal core and the group of H1-linker histones. These H1 histones seal two rounds of DNA at the surface of the core-histone octamer [1], influence the positioning of nucleosomes [2,3] and are essential for the formation of higher order chromatin structures [4]. In addition, H1 histones exert general inhibitory effects on transcription [5,6], but can also play a specific role in gene regulation [7Ϫ9].In mammals, the group of H1 histones consists of at least seven different subtypes. The H1 histones represent the most variable class of the otherwise highly conserved histone proteins. Based on both the timing of synthesis relative to the cell cycle and their tissue-specific occurrence, the H1 histones can be classified into three different groups of subtypes : (a) the main type-H1 histones, which are expressed in close relation to the S phase of the cell cycle ; (b) the replacement subtype H1, which is usually expressed in highly differentiated cells and (c) the subtype H1t which is expressed exclusively in the testis [10].

Proteolytic activation of caspases is a key step in the process of apoptosis. According to their primary structure, caspases can be divided into a group with a long prodomain and a group with a short prodomain. Whereas long prodomains play a role in autocatalytic processing, little is known about the function of the short prodomain, for example the prodomain of caspase-3. We constructed caspase-3 variants lacking the prodomain and overexpressed these in HeLa and yeast cells. We found that removal of the caspase-3 prodomain resulted in spontaneous proteolytic activation of the protein when expressed in HeLa cells. This processing was only partially autocatalytic, as demonstrated by a catalytically inactive caspase-3 mutant. Co-expression of the anti-apoptotic protein XIAP (X-chromosome-linked inhibitor of apoptosis protein) completely blocked the observed spontaneous activation, which excluded a direct involvement of caspase-8. Our findings indicate that the short prodomain of caspase-3 serves as a silencing component in mammalian cells by retaining this executioner caspase in an inactive state.