Gary A. Bulla

GP73, a novel Golgi-localized protein upregulated by viral infection.

Abstract We report the isolation and characterization of GP73, a novel 73kDa human Golgi protein. The GP73 cDNA was cloned by differential screening of a cDNA library derived from the liver of a patient with adult giant-cell hepatitis (GCH), a rare form of hepatitis with presumed viral etiology. In vitro transcription–translation studies indicate that GP73 is an integral membrane protein, and immunolocalization experiments using epitope-tagged GP73 demonstrate that the protein is localized to the Golgi apparatus. Northern blot analysis of RNA from multiple human tissues reveals a single GP73 mRNA transcript with a size of approximately 3.0kb. Immunohistochemical studies using rabbit polyclonal antisera directed against recombinant GP73 demonstrate that the protein is preferentially expressed by epithelial cells in many human tissues. In normal livers, GP73 is consistently present in biliary epithelial cells, whereas hepatocytes show little or no signal. In contrast, livers of patients with GCH display strong GP73 immunoreactivity in multinucleated hepatocytes. GP73 mRNA and protein are expressed in highly differentiated HepG2 hepatoma cells after infection with adenovirus in vitro. We conclude that GP73 represents a novel, epithelial cell-specific integral membrane Golgi protein that can be upregulated in response to viral infection.

Modulation of lipopolysaccharide-mediated activation in rat Kupffer cells by antioxidants

Activation of Kupffer cells, the resident macrophage population of the liver, has been implicated in the pathogenesis of several types of liver injury. The aim of this study was to investigate whether the antioxidants N-acetylcysteine (NAC) and alpha-tocopherol succinate (alpha-TOC) suppress lipopolysaccharide (LPS)-induced activation of rat Kupffer cells. LPS activated NF-kappaB in Kupffer cells, and this response was inhibited by NAC and alpha-TOC. NAC and alpha-TOC also markedly suppressed LPS-induced tumor necrosis factor-alpha (TNF-alpha) mRNA levels and secretion. We further show that LPS was unable to increase TNF-alpha mRNA in drug-treated cells even when stimulation occurred after NAC or alpha-TOC were removed. These results indicate that antioxidants persistently suppress LPS activation in Kupffer cells, and suggest that the mechanism responsible for this involves more than mere quenching of free radical production. The demonstration that NAC and alpha-TOC have inhibitory effects on LPS-mediated Kupffer cell activation suggests that these compounds may have a beneficial effect in liver injury involving oxidative stress and endotoxemia.

Negative regulation of the hepatitis B virus pre-S1 promoter by internal DNA sequences

Expression of the surface antigen gene (S gene) of hepatitis B virus is directed by two distinct promoter elements with markedly different activities. The upstream (pre-S1) promoter produces a 2.4-kb transcript at very low levels while the downstream (pre-S2) promoter produces an approximately 2.1-kb transcript in relative abundance. We have constructed a series of internal deletion mutants to analyze differential regulation of the two S gene promoters. We show here that expression directed by the pre-S1 promoter is negatively regulated by DNA sequences containing the downstream pre-S2 promoter region. Nuclear run-on analysis indicates this down-regulation to be at the level of transcription. Furthermore, promoter repression does not appear to be due to products of the S gene region. Deletion mutagenesis studies have permitted the localization of a 61-bp region that may be involved in the apparent down-regulation of the pre-S1 promoter. These results suggest the use of an unusual regulatory mechanism by a dipromoter gene in which an active internal promoter may preclude efficient use of an upstream promoter.

Direct selection of hepatoma cell variants deficient in α1-antitrypsin gene expression

Expression plasmids containing the human α1-antitrypsin (α1 AT) promoter fused to either adenine phosphoribosyltransferase (aprt) or xanthine-guanine phosphoribosyltransferase (gpt) coding sequences were sequentially introduced into APRT− HPRT− rat hepatoma cells. Stable transfectants expressing both transgenes were isolated and characterized. Nonexpressing variants were subsequently obtained by selecting against expression of one or both transgenes. Variants isolated by selecting against expression of either transgene alone generally displayed deficiency phenotypes incis, as only three of 20 clones tested were affected for expression of α1AT mRNA. In contrast, double selection yielded predominantlytrans effects: 12 of 14 lines tested showed impaired ability to express their chromosomal α1AT genes. Furthermore, expression of several other liver genes, including the gene encoding the HNF-1trans-activator, was repressed in many of the variant lines. Thus, double selection using chimeric transgenes is a useful approach for generating variant cell lines deficient in expression of specific mammalian genes.

SELECTIVE LOSS OF THE HEPATIC PHENOTYPE DUE TO THE ABSENCE OF A TRANSCRIPTIONAL ACTIVATION PATHWAY

Liver-enrichedtrans-acting factors hepatocyte nuclear factor-1α (HNF1 α) and-4 (HNF4) are components of a transcriptional activation pathway that is thought to play a major role in hepatic gene activation. We previously described the isolation and characterization of distinct classes of hepatoma variants which lack the HNF4→HNF1α pathway (1). In order to determine the influence of the HNF4→HNF1α pathway on hepatic gene expression, genetic rescue experiments were done using hepatoma variant line H11 as a model system. Results suggest that this pathway is required for basal expression of a number of endogenous hepatocyte-specific genes. Complementation groups were established by fusion of H11 cells with other variant lines. Lastly, introduction of human chromosome 20 (containing the HNF4 locus) or randomly-marked human chromosomes into H11 cells failed to rescue the hepatic phenotype, suggesting that what appears to be a ‘simple’ defect may involve multiple genetic loci.

Tissue-specific extinguisher loci in the murine genome: a screening study based on a rat/mouse microcell hybrid panel.

Extinction of tissue-specific traits in intertypic somatic cell hybrids is a well-known phenomenon. In the past few years, microcell hybrids have been used in attempts to dissect this phenotype genetically, and tissue-specific extinguisher loci have been mapped to two different mouse chromosomes. When transferred from fibroblasts into hepatoma cells by microcell fusion, these loci down-regulate expression of specific liver genes intrans. However, other liver genes that are extinguished in genotypically complete hybrids seem not to be extinguished in monochromosomal hybrids. To assess the generality of monochromosomal extinction phenotypes, we assembled a collection of rat hepatoma/mouse fibroblast microcell hybrids that represent most of the mouse chromosome complement, and we screened them for expression of a large number of liver-specific genes. Phosphoenolpyruvate carboxykinase gene expression was down-regulated in hybrids containing mouse chromosome 7 or mouse chromosome 11, but other extinction phenotypes were not readily apparent. These results indicate that extinction of many liver genes may be a polygenic trait.

Tissue-specificity of apoptosis in hepatoma-derived cell lines

Apoptosis is known to play a critical role in development and homeostasis in metazoans. Although apoptotic responses vary widely among cell types, the underlying mechanisms responsible for these differences are not known. In order to understand the molecular basis for these differences, we have studied a cell culture model comparing hepatoma cells to dedifferentiated cell lines derived from them. We recently reported evidence suggesting that a common regulatory locus affects both liver-specific function and sensitivity to lipopolysaccharide (LPS)-mediated apoptosis. Here, we show that dedifferentiated hepatoma cells undergo apoptosis in response to multiple compounds, including sorbitol (to induce hyperosmotic shock), TNFα and the microtubule damaging agent vinblastin. In contrast, the hepatoma parental cells fail to undergo apoptosis in response to any of the compounds tested. Further analysis of LPS-mediated cell death found that antioxidants N-acetylcysteine and α-tocopherol partially prevented apoptosis. Lastly, evidence is presented showing that LPS-mediated cell death of the hepatoma variant cell lines is caspase-dependent. These results suggest that pathways dictating hepatic phenotype also affect general cellular survival mechanisms in response to multiple agents. The dedifferentiated cells provide a model to examine the influence of cell-type specific expression on apoptotic signaling.

Genome-wide analysis of hepatic gene silencing in mammalian cell hybrids

Silencing of tissue-specific gene expression in mammalian somatic cell hybrids is a well-documented epigenetic phenomenon which is both profound (involving a large number of genes) and enigmatic. Our aim was to utilize whole-genome microarray analyses to determine the true extent of gene silencing on a genomic level. By comparing gene expression profiles of hepatoma×fibroblast cell hybrids with those of parental cells, we have identified over 300 liver-enriched genes that are repressed at least 5-fold in the cell hybrids, the majority of which are repressed at least 10-fold. Also, we identify nearly 200 fibroblast-enriched genes that are repressed at least 5-fold. Silenced hepatic genes include several that encode transcription factors and proteins involved in signal transduction pathways. These data suggest that extensive reprogramming occurs in cell hybrids, leading to a nearly global (although not complete) loss of tissue-specific gene expression.

Defective NF-kappaB signaling in dedifferentiated hepatoma cells.

Dedifferentiated rat hepatoma cells contain defects that result in the loss of hepatic gene expression, including the liver-enriched HNF4/HNF1α pathway. We examined induction of NF-κB, a key mediator of the inflammatory response, in hepatoma and dedifferentiated hepatoma cells. We show that exposure of dedifferentiated hepatoma cells, but not rat and human hepatoma cell lines, to proinflammatory cytokines or lipopolysaccharide resulted in rapid and sustained NF-κB induction. IκB-β levels, but not NF-κB subunit p65 or IκB-α levels, were elevated compared with those for parental hepatoma cells. Interestingly, LPS-mediated activation of NF-κB was found to be independent of degradation of IκB-α or IκB-β. Thus, these results suggest that loci responsible for maintaining hepatic gene expression also influence cellular responses to inflammatory agents.