Gene transcription in hepatocytes during the acute phase of a systemic inflammation: from transcription factors to target genes.

During an acute, systemic inflammation, the liver is triggered by blood-borne pro-inflammatory cytokines such as Tumor Necrosis Factor alpha, Interleukin-1beta and Interleukin-6. The end result is an up- or down-regulated synthesis and/or activation of liver-enriched transcription factors that in turn regulate many target genes coding for resident or secreted acute phase proteins. In this review, various classifications of these acute phase proteins are presented. Major inflammation-driven changes in the synthesis and/or activity of the hepatic transcription factors are illustrated. Some of their up- or down-regulated target genes are used as paradigms of the various transcriptional mechanisms that take place on gene promoters during an acute, systemic inflammation. Finally, further specific features of inflammation-associated gene transcription in liver from acute phase onset to resolution are provided.

Up-regulated expression of HGF in rat liver cells after experimental endotoxemia: a potential pathway for enhancement of liver regeneration.

A lipopolysaccharide (LPS)-induced inflammation prior to an hepatic resection has been shown to enhance liver regeneration in rat. The aim of the present study was to investigate the expression of hepatocyte growth factor (HGF) and its c-Met receptor under such experimental conditions. Animals were submitted to a two-third hepatectomy or a LPS challenge carried out 12 h prior to resection. Non parenchymal and parenchymal cells were isolated from livers obtained at various times post-hepatectomy. Quantitative RT-PCR for HGF and c-Met mRNAs were performed from total liver or purified cell fractions and HGF mRNA was also analyzed by in situ RT-PCR on liver sections. A LPS challenge alone induced a marked up-regulation of HGF mRNA level in whole liver and isolated hepatocytes. Furthermore, when partial hepatectomy (PH) was preceded by a LPS challenge, an increase of HGF mRNA level was seen in whole liver and contrasted with a decreased level in non parenchymal cells. These results were confirmed by in situ RT-PCR. In isolated hepatocytes from endotoxemic rats, the mRNA level for the LPS-specific membranous receptor mCD14 was markedly up-regulated and even more so when LPS was followed by PH. Moreover, a TNFalpha challenge alone induced an up-regulation of HGF mRNA in hepatocytes and a down-regulation in non parenchymal cells (NPCs). Overall, when a LPS challenge is given prior to PH the major source of hepatic HGF appears to be the hepatocyte itself rather than NPCs. An autocrine HGF/c-Met loop which promotes the proliferative potential of the hepatic parenchymal cell and participates in liver regeneration is postulated.

Long-range identification of hepatocyte nuclear factor-3 (FoxA) high and low-affinity binding sites with a chimeric nuclease.

Identifying the complete set of transcription factors that bind the promoter and other regulatory regions of a gene of interest is an essential step in functional genomics. We have developed an original assay for the systematic detection of hepatocyte nuclear factor-3 (HNF-3) binding sites within cloned promoters. This assay is based on expression of a recombinant enzyme, HNF-3beta/FN, that is comprised of the rat HNF-3beta DNA-binding domain and the non-specific nuclease domain of the FokI restriction enzyme. Southern analysis of target plasmids with proven HNF-3 binding sites showed that HNF-3beta/FN was able to specifically cut both DNA strands in the vicinity of these binding sites, whereas mutagenized binding sites were no longer cleaved. Likewise, as yet undescribed HNF-3 binding sites were detected easily over a distance spanning several thousand bases. The functionality of such binding sites was confirmed by electromobility shift assay. Furthermore, the extent of cleavage by HNF-3beta/FN at a given binding site was tightly correlated with the affinity of a natural HNF-3beta molecule for this site. This novel approach can be extended to other transcription factors for long-range identification of functional transcription factor binding sites in genes.

Unmasking a hyaluronan-binding site of the BX(7)B type in the H3 heavy chain of the inter-alpha-inhibitor family.

The inter-alpha-inhibitor (I alpha I) family gathers together several plasma protease inhibitors such as I alpha I and pre-alpha-inhibitor (P alpha I) that are variously assembled from a set of polypeptide chain precursors designated H1P to H3P. In addition to their protease inhibitory activity, a major physiological function of I alpha I family members is hyaluronan (HA) binding and HA-dependent stabilization of the extracellular matrix surrounding various cell types. Also, binding of HA to these molecules has been shown to be an important event in tumor cell proliferation and rheumatoid arthritis. However, how HA and I alpha I family members first recognize each other has so far remained elusive. The so-called BX7B domain found in some HA-binding proteins is an HA-binding site in which B represents a basic amino-acid residue and X represents any nonacidic residue. This domain has now been identified in the N-terminal end of H3P that is a precursor of P alpha I. A series of wild-type or mutant recombinant H3P chains produced with a mouse cDNA expressed in Escherichia coli allowed us to demonstrate that this domain binds HA in a noncovalent fashion. Furthermore, unmasking this HA-binding activity required most of H3P to be trimmed off at its C-terminal end. The latter observation was confirmed with a natural, mature H3 chain purified from human plasma. Indeed, a thermolysin-generated, N-terminal fragment of this H3 chain strongly bound HA whereas the intact H3 chain did not. Therefore, in vivo, the HA-binding activity of the mature H3 chain within P alpha I may vary with the folding and/or fragmentation of this protein.

Chromosomal assignments of mammalian genes with an acute inflammation-regulated expression in liver.

A set of acute inflammation-regulated genes expressed in liver has been assigned to rat, mouse, and human chromosomes by detecting species-specific PCR amplicons in rat(x)mouse or mouse(x)hamster somatic cell hybrids or radiation hybrids or by in silico matches of corresponding rat cDNAs to various libraries of previously assigned rat, mouse, or human genes or expressed-sequence tags. This allowed us to assign 24, 22, and 21 inflammation-regulated genes to rat, mouse, and human chromosomes, respectively. From these assignments as well as those previously determined for a larger set of genes with an acute inflammation-regulated transcription in liver, we further investigated whether such genes are clustered onto given chromosomes. A cluster was found on rat Chromosome (Chr) 6q with a conserved synteny on mouse Chr 12 and human Chr 14q13-q32, and another cluster previously reported on human Chr 1q has been extended with five further genes. Our data suggest that during an acute inflammation, a higher-order regulation may control some liver-expressed genes that share a given chromosome area.

Chromosomal location, exon/intron organization and evolution of lipocalin genes.

Lipocalins exhibit low sequence similarity that contrasts with a tightly conserved folding shared by all members of this superfamily. This conserved folding can be, at least partly, accounted for by a highly conserved gene structure. The array of lipocalin genes that have so far been studied mostly in mammals indicate a large conservation of a typical seven exon/six intron arrangement. Other conserved features include a partly coding exon 1 of variable size, fixed sizes of exons 2-5 that code for an array of lipocalin-specific beta-strands and a tendency of the last exons to either fuse or expand into further exons without major changes in the length of the resulting open reading frame. The conserved exon/intron arrangement as well as a clustering of most lipocalin genes in given chromosomes of human and mouse indicate that the lipocalin genes diverged from a shared ancestor by successive rounds of duplications followed by late changes in exon arrangements.

Fetuin-B, a second member of the fetuin family in mammals.

A set of orthologous plasma proteins found in human, sheep, pig, cow and rodents, now collectively designated fetuin-A, constitutes the fetuin family. Fetuin-A has been identified as a major protein during fetal life and is also involved in important functions such as inhibition of the insulin receptor tyrosine kinase activity, protease inhibitory activities and development-associated regulation of calcium metabolism and osteogenesis. Furthermore, fetuin-A is a key partner in the recovery phase of an acute inflammatory response. We now describe a second protein of the fetuin family, called fetuin-B, which is found at least in human and rodents. On grounds of domain homology, overall conservation of cysteine residues and chromosomal assignments of the corresponding genes in these species, fetuin-B is unambiguously a paralogue of fetuin-A. Yet, fetuin-A and fetuin-B exhibit significant differences at the amino acid sequence level, notably including variations with respect to the archetypal fetuin-specific signature. Differences and similarities in terms of gene regulation were also observed. Indeed, studies performed during development in rat and mouse showed for the first time high expression of a member of the fetuin family in adulthood, as shown with the fetuin-B mRNA in rat. However, like its fetuin-A counterpart, the fetuin-B mRNA level is down-regulated during the acute phase of experimentally induced inflammation in rat.

Positive and negative elements modulate the promoter of the human liver-specific alpha2-HS-glycoprotein gene.

The human alpha2-HS-glycoprotein (AHSG) and the 63-kDa rat phosphoprotein (pp63) are homologous plasma proteins that belong to the fetuin family. AHSG and pp63 are involved in important functions such as inhibition of insulin receptor tyrosine kinase activity, inhibition of protease activities, and regulation of calcium metabolism and osteogenesis. Studies of the AHSG proximal promoter performed in vitro in rat and human cells indicate that several NF-1 and C/EBP binding sites exert a positive effect on its transcriptional activity. However, until now, no distal elements have been examined in this gene, in either species. We report that the human AHSG gene promoter acts in a liver-specific manner and is further controlled by three distal, 5'-flanking elements. The negative elements III and I are, respectively, located 5' and 3' of the positive element II. All three elements require the natural context of the human AHSG gene to fully exert their negative or positive effect. Element I harbours a single binding site for NF-1. This nuclear factor thus appears to be able to up- or downregulate the AHSG gene depending on the site it binds to. Elements I, II and possibly III are absent in the rodent Ahsg gene encoding pp63.

Differential expression of apoptosis-associated genes post-hepatectomy in cirrhotic vs. normal rats.

Liver regeneration after partial hepatectomy or liver injury is controlled by a wide variety of growth factors that are proven activators or inhibitors of hepatocyte proliferation. Liver regeneration post-hepatectomy has been proven to be decreased and delayed in cirrhotic vs. normal liver. Apoptosis seems to play an important role in cellular proliferation and in liver regeneration. Therefore, this study has analyzed the expression of apoptosis-associated genes following 2/3 hepatectomy in cirrhotic vs. normal rats. Cirrhosis was induced by a weekly intragastric administration of CCl4 for 16 weeks followed by hepatectomy and histological examination of the resected liver. Rats were sacrificed at 6 h, 12 h, 24 h, or 72 h after liver resection. The expression of proapoptotic (Bad, Bak, Bax) and antiapoptotic (Bcl-2, Bcl-XL) genes was analyzed by quantitative RT-PCR. We have observed an early increase in antiapoptotic mRNA levels and a delayed increase in proapoptotic mRNA levels in normal liver following hepatectomy. Before resection, proapoptotic mRNA levels were significantly higher in cirrhotic vs. normal liver. After hepatectomy, apoptotic mRNA levels were decreased and delayed as compared with that observed following hepatectomy in normal liver. These results indicate that apoptosis takes place in liver during CCl4-induced cirrhosis and could participate in the impaired regenerative response observed in cirrhotic liver.

Changes in growth factor and cytokine mRNA levels after hepatectomy in rat with CCl(4)-induced cirrhosis.

Cirrhotic liver is considered to regenerate less actively than normal liver after hepatic resection. However, the mechanisms responsible for this impaired regeneration and the cross talk of implicated factors still remain unclear. In the present study, mRNA levels for cyclins, growth factors, and cytokines were quantitatively assessed by a RT-PCR method at different times after hepatectomy in order to determine the relationships between these factors and the impaired regenerative process observed in cirrhotic liver. In our model of CCl(4)-induced cirrhosis, mRNA levels for cyclins and thymidine kinase provide evidence for the impaired and delayed hepatic regeneration. Moreover, we observed a significant decrease in interleukin (IL)-6 and tumor necrosis factor-alpha mRNA and a significant increase for IL-1beta mRNA. No significant change of hepatocyte growth factor (HGF) mRNA level was detected, contrasting with the decrease both at mRNA and protein levels in the expression of the c-Met/HGF receptor. Therefore, the impaired regeneration of the cirrhotic liver is associated not only with a lowered level of signals that normally promote liver growth but also with a strong decrease in c-Met receptor despite a normal expression of its specific ligand.

The alpha1-microglobulin/bikunin gene: characterization in mouse and evolution.

The 129Sv mouse gene coding for the alpha1-microglobulin/bikunin precursor has been isolated and characterized. The 11kb long gene contains ten exons, including six 5'-exons coding for alpha1-microglobulin and four 3'-exons encoding bikunin. Exon 7 also codes for the tribasic tetrapeptide RARR which connects the alpha1-microglobulin and bikunin parts. The sixth intron, which separates the alpha1-microglobulin and bikunin encoding parts, was compared in the human, mouse and a fish (plaice) gene. The size of this intron varies considerably, 6.5, 3.3 and 0.1kb in man, mouse and plaice, respectively. In all three genes, this intron contains A/T-rich regions, and retroposon elements are found in the first two genes. This indicates that this sixth intron is an unstable region and a hotspot for recombinational events, supporting the concept that the alpha1-microglobulin and bikunin parts of this gene are assembled from two ancestral genes. Finally, the nonsynonymous nucleotide substitution rate of the gene was determined by comparing coding sequences from ten vertebrate species. The results indicate that the alpha1-microglobulin part of the gene has evolved faster than the bikunin part.

A novel set of hepatic mRNAs preferentially expressed during an acute inflammation in rat represents mostly intracellular proteins.

A cloning of hepatic cDNAs associated with the early phase of an acute, systemic inflammation was carried out by differential screening of arrayed cDNA clones from rat livers obtained at 4-8 h postchallenge with Freund's complete adjuvant. End sequencing of 174 selected clones provided three cDNA groups that coded for: (i) 23 known acute-phase proteins, (ii) 31 known proteins whose change in hepatic synthesis during an acute phase was so far unsuspected, and (iii) 36 novel proteins whose cDNAs were completely sequenced. For 16 proteins in the third group the hepatic mRNA could be detected and quantitated by Northern blot hybridization in Freund's adjuvant-challenged animals, and an extrahepatic expression in healthy animals was further investigated. Matching the open reading frames of the 36 novel proteins with general and specialized data libraries indicated the potential relationships of 16 of these proteins with known protein families/superfamilies and/or the presence of functional domains previously described in other proteins. Overall, our search for novel inflammation-associated proteins selected mostly known or as yet undescribed proteins with an intracellular or membrane location, which extends our knowledge of the proteins involved in the intracellular metabolism of hepatic cells during a systemic, acute-phase response. Finally, some of the cDNAs above allowed us to successfully identify hepatic mRNAs that are differentially expressed in acute vs chronic (polyarthritis) inflammatory conditions in rat.

The nuclear protein PH5P of the inter-alpha-inhibitor superfamily: a missing link between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and a novel actor of DNA repair?

Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-alpha-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1-H4. The latter originate from precursor polypeptides H1P-H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P-H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and is likely to be involved in DNA repair.

An array of binding sites for hepatocyte nuclear factor 4 of high and low affinities modulates the liver-specific enhancer for the human alpha1-microglobulin/bikunin precursor.

Alpha1-Microglobulin and bikunin are two plasma glycoproteins encoded by a gene for alpha1-microglobulin/bikunin precursor (AMBP). The strict liver-specific transcription of the AMBP gene is controlled by an elaborate and remote enhancer made of six clustered boxes numbered 1 to 6 (core enhancer) that are binding sites for the hepatocyte-enriched nuclear factors HNF-1, HNF-4, HNF-3, HNF-1, HNF-3 and HNF-4 respectively. Three further boxes, 7 to 9, have now been found in the enhancer area in a position 5' of box 2, 5' of box 1 and 3' of box 6, respectively. Electrophoretic mobility-shift assays with nuclear extracts from the HepG2 hepatoma cell line demonstrated that boxes 7 and 8 are both functional HNF-4-binding sites of high and low affinity respectively, whereas no binding capacity of box 9 was detected by this method. Transfection of HepG2 and Chinese hamster ovary cells with chloramphenicol acetyltransferase constructs harbouring the core or extended AMBP enhancer with wild-type or mutated boxes and co-transfection with expression plasmids for a wild-type or defective HNF-4 identified box 7 as an essential element for the basal activity of this enhancer. The response of boxes 7 and 8 varies with the level of HNF-4 in cells. Box 9 exhibits a repressor activity that can be detected when box 8 is ablated. In vivo this corresponds to conditions of low box 8 occupancy when the intracellular level of HNF-4 is limited. These results reinforce the view that the AMBP enhancer is a quite elaborate and unusual example of a modular enhancer whose activity is fine-tuned by the level of cognate nuclear factors in the cell.

Down-regulation of negative acute-phase response genes by hypotonic stress in HepG2 hepatoma cells.

An increased hepatocellular hydration state (HS) that can be induced by hypotonic stress or a high glutamine uptake modulates the transcription of given genes in liver. This could be important in the acute phase (AP) of a systemic inflammation where both HS and glutamine uptake transiently increase in liver. In HepG2 hepatoma cells cultured in conditions of hypotonic stress or a high extracellular glutamine availability, a specifically decreased expression of two human mRNAs, namely those of alphal-microglobulin/bikunin precursor (AMBP) and alpha2-HS-glycoprotein, that are also down-regulated in liver by AP, could be seen. A functional analysis of the AMBP promoter indicated that this hypotonic stress-induced down-regulation takes place at a transcriptional level. In these experiments, the mRNA level and transcription of the glyceraldehyde-3-phosphate dehydrogenase gene that are known to be unmodified in AP did not exhibit any change. Given that hypotonic stress also upregulates the transcription of a liver gene that is also upregulated in AP [Meisse et al. (1998) FEBS Lett. 422, 3463481, the AP-associated increase in hepatocellular HS now appears to participate in the transcriptional control of both sets of genes that are up- or down-regulated in AP.

Structural and functional analysis of the 5'-transcription control region for the human alpha2-HS glycoprotein gene.

The human alpha2-HS glycoprotein (A2HS) and rat phosphoprotein of Mr 63000 (pp63) are homologous plasma proteins and members of the fetuin superfamily. A2HS is involved in important functions such as inhibition of the tyrosine kinase activity of the insulin receptor, regulation of calcium metabolism and osteogenesis as well as protease inhibitory activity. We report an analysis of the 5' transcription control region (4 kb) of the A2HS gene. Its most proximal 300 nt display a very potent transcriptional activity. The latter is likely accounted for by C/EBP and NF1 binding sites that are conserved from the human A2HS gene to the rat pp63 gene. In contrast, these human and rat genes appear to largely diverge beyond their proximal promoter.

Hepatic and extra-hepatic transcription of inter-alpha-inhibitor family genes under normal or acute inflammatory conditions in rat.

The expression and level of the mRNAs for the five genes that code for a set of plasma proteins collectively referred to as the inter-alpha-inhibitor family have been studied in rat under a normal condition or in the course of a turpentine-induced, systemic inflammation. In healthy rats, all five mRNAs [H1, H2, H3, H4, and alpha1-microglobulin/bikunin precursor (AMBP)] are expressed primarily in liver and two of them (H2 and H3) are found to a lower extent in brain. By in situ hybridization onto sections of a normal brain, the H3 mRNA has been precisely localized to the hypothalamus, amygdala, pontine area, optic tectum, and cerebellum. By reverse transcriptase-polymerase chain reaction of total RNAs obtained from a panel of organs, low amounts of one or more mRNA(s) could be detected in other locations (e.g., intestine and stomach). Furthermore, the extrahepatic expressions of several of these genes are up- or downregulated at 20 h after the start of a turpentine-induced inflammation. In liver, the contents of H3 and H4 mRNA are upregulated, whereas those of AMBP and H2 are downregulated during the acute phase. This is accounted for by changes in gene transcription, the kinetics of which is gene-specific. This behavior of H1, H2, H3, H4, and AMBP mRNAs in rat liver is in keeping with more limited analyses made at mRNA and/or protein levels in other species (human, pig) suffering from an acute inflammation. Therefore, the inflammation-associated regulation of these five genes that is conserved between species indicates that the inter-alpha-inhibitor family members are likely to be important partners of the acute phase response.

Induction and modulation of acute-phase response by protein malnutrition in rats: comparative effect of systemic and localized inflammation on interleukin-6 and acute-phase protein synthesis.

The acute-phase protein (APP) response is regulated by cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1) and tumor necrosis factor (TNF), but may also be influenced by malnutrition. The aims of this study were as follows: 1) to determine in rats the effect of a protein-deficient diet on IL-6 mRNA expression in intestine, liver and peripheral blood mononuclear cells (PBMC), and on alpha-1 acid glycoprotein (AGP) and alpha-2 macroglobulin (A2M) serum levels and hepatic mRNA expression; 2) to compare, in protein-deficient rats, the IL-6 and APP responses after a turpentine (TO)- or a lipopolysaccharide (LPS)-induced inflammation; and 3) to determine the effect of a protein malnutrition on IL-6 mRNA expression in rat PBMC treated ex vivo with LPS. Interleukin-6 mRNA was present in intestine and PBMC but not in the liver of malnourished rats, and was absent in any tissue or cells of controls. A2M was present in the serum from malnourished rats but not after refeeding. AGP mRNA expression was not influenced by protein malnutrition. In malnourished rats, IL-6 serum level peaked later than in controls after TO and LPS treatment. In malnourished TO-treated rats, A2M mRNA increased earlier than in controls and remained detectable later than in controls. AGP mRNA expression after TO was not influenced by protein malnutrition. In PBMC of malnourished rats, LPS-induced IL-6 mRNA expression occurred earlier and lasted longer than in controls. Our results indicate that protein malnutrition by itself induces IL-6 and A2M expression, and that it modulates the APP response to inflammation.

The H4P heavy chain of inter-alpha-inhibitor family largely differs in the structure and synthesis of its prolin-rich region from rat to human.

The family of plasma proteins collectively referred to as Inter-alpha-Inhibitor (I alpha I) family is comprised of a set of multi-polypeptide molecules and a single-chain molecule designated I alpha IH4P. Although the 4 heavy chain precursors H1P to H4P that lead to these molecules are evolutionarily related, only H4P harbours a Pro-rich region (PRR) in its C-terminal third. A comparison of hepatic H4P cDNAs in human and rat has now unraveled an extensive variability of this PRR. Within the rat PRR, 6 repeats of a Gly-X-Pro motif participate in a collagen-like pattern that is absent in human. Within the human PRR, a domain that is absent in rat can be transcribed or deleted by alternative splicing which results in two variant forms of human H4P. In rat liver, the single mRNA is up-regulated by an acute, systemic inflammation whereas neither mRNA is up-regulated in human liver. Finally the shortest human mRNA is also transcribed in peripheral blood mononuclear cells where it is down-regulated by bacterial lipopolysaccharides. Therefore, in contrast to what is seen for the ITIH1 to -3 genes, the rat and human ITIH4 gene transcriptions and products thereof present marked differences, which suggests species-specific functions for I alpha IH4P.