Expression of C/EBP delta in rat liver during development and the acute-phase response

Using Western analysis, C/EBP delta was established in the nuclear extract and nuclear matrix throughout rat liver development and in the adult. During the acute-phase response (APR), C/EBP delta increased in the nuclear extract but remained unchanged in the nuclear matrix of fetal and postnatal rats, whereas it increased in both the nuclear extract and nuclear matrix of the adult. The solubility partitioning of gene regulatory proteins in the nucleus is important for their functioning (Uskokovi? et al. 2002). The obtained different solubility partitioning profiles of C/EBP delta suggest that its activity is regulated by different mechanisms during development and in the adult.     

Activity and distribution of protein kinase C in liver during the acute-phase response

Activity and subcellular distribution of protein kinase C were estimated in liver cytosol and membrane fractions of rats carrying a turpentine-induced inflammation. Protein kinase C activity increases significantly 8 h after treatment in the membrane fraction, with concurrent reduction in the cytosol; 10 h after treatment the membrane-associated activity returns to normal, without concomitant recovery of that detected in the cytosol. The specific binding of phorbol dibutyrate to the liver membrane fraction increases but overall the effect is less evident and delayed in time. The changes are associated to alterations in the phosphorylation pattern of some liver proteins. Liver protein kinase C activity and intracellular distribution seem to be affected by a treatment which is known to induce an acute-phase response in the liver cells.     

Melanocortin receptors in rat liver cells: change of gene expression and intracellular localization during acute-phase response

MCRs are known to be expressed predominantly in the brain where they mediate metabolic and anti-inflammatory functions. Leptin plays an important role in appetite and energy regulation via signaling through melanocortin receptors (MCRs) in the brain. As serum levels of MCR ligands are elevated in a clinical situation [acute-phase response (APR)] to tissue damage, where the liver is responsible for the metabolic changes, we studied hepatic gene expression of MCRs in a model of muscle tissue damage induced by turpentine oil (TO) injection in rats. A significant increase in gene expression of all five MCRs (MC4R was the highest) in liver at the RNA and protein level was detected after TO injection. A similar pattern of increase was also found in the brain. Immunohistology showed MC4R in the cytoplasm, but also in the nucleus of parenchymal and non-parenchymal liver cells, whereas MC3R-positivity was mainly cytoplasmic. A time-dependent migration of MC4R protein from the cytoplasm into the nucleus was observed during APR, in parallel with an increase in α-MSH and leptin serum levels. An increase of MC4R was detected at the protein level in wild-type mice, while such an increase was not observed in IL-6ko mice during APR. Moreover, treatment of isolated liver cells with melanocortin agonists (α-MSH and THIQ) inhibited the endotoxin-induced upregulation of the acute-phase cytokine (IL-6, IL1β and TNF-α) gene expression in Kupffer cells and of chemokine gene expression in hepatocytes. MCRs are expressed not only in the brain, but also in liver cells and their gene expression in liver and brain tissue is upregulated during APR. Due to the presence of specific ligands in the serum, they may mediate metabolic changes and exert a protective effect on liver cells.     

The association of acute phase protein genes with the nuclear matrix of rat liver during experimental inflammation

The association of acute phase protein genes with the nuclear matrix in livers from healthy rats and rats suffering from inflammation was studied. alpha 1-Acid glycoprotein and transthyretin are synthesized at low levels in normal liver and no matrix association of their genes was observed. Albumin, transferrin and the beta-chain of fibrinogen are synthesized at much higher levels in normal liver and their genes were found to be associated with the nuclear matrix. An inflammation induced increase in synthesis of alpha 1-acid glycoprotein and the beta-chain of fibrinogen resulted in stronger matrix association of their genes. However, inflammation induced decrease in the synthesis of albumin did not influence matrix association of its gene.     

Changes in expression of albumin and alpha-fetoprotein genes during rat liver development and neoplasia.

Albumin mRNA was isolated and purified from rat liver polysomes by a combination of immunoprecipitation of specific polysomes, poly(U)-Sepharose 4B chromatography, and fractionation of the resulting poly(A)-containing RNA on a sucrose gradient. alpha-Fetoprotein (AFP) mRNA was isolated from Morris hepatoma 7777 by a similar procedure. The purity of the mRNA preparations was determined by analytical gel electrophoresis under denaturing conditions, analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides synthesized in a wheat germ cell-free system, and the kinetics of hybridization to cDNA transcribed from albumin mRNA and AFP mRNA. The albumin mRNA possessed a chain length of approximately 2265 nucleotides and the AFP mRNA possesed a length of approximately 2235 nucleotides when examined under stringent denaturing conditions on agarose gels containing 10 mM methylmercury hydroxide. Analysis of poly(A) content by a hybridization assay with [3H]poly(U) revealed the presence in albumin mRNA of a poly(A) region containing approximately 100 adenosine residues. The AFP mRNA preparation was found to contain an average poly(A) tract of approximately 190 bases. Thus, albumin mRNA appears to contain approximately 330 untranslated nucleotides, and AFP mRNA appears to contain a similar number (approximately 285) of noncoding, nonpoly(A) bases. The purified albumin and AFP mRNA's were used as templates for synthesis of full-length cDNA hybridization probes. Both of the probes selectively hybridized to their templates with kinetics expected for single RNA species the sizes of albumin and AFP mRNA. ROt analysis was used to quantitate albumin and AFP mRNA sequences during normal liver postnatal development and liver oncogenesis. The number of polysomal AFP mRNA molecules per liver was found to drastically decrease during the first weeks of postnatal life, concomitant with a decline in the AFP synthetic capacity of the livers and in the serum concentrations of AFP. During this period, the concentration of albumin mRNA molecules per cell in the liver remained at high, approximately constant levels. In Morris hepatoma 7777, the concentration of AFP-specifying sequences was at least 10(3)-fold higher than that found in normal adult liver, whereas the content of albumin nRNA was four- to five-fold lower. These changes in concentration of albumin and AFP mRNA sequences closely correlated with a parallel variation in the specific protein synthetic capacity of the tissues.     

Regulation of protein disulfide isomerase gene expression in brain and liver during rat development

A 4-fold increase in protein disulfide isomerase (PDI) mRNA is observed in brain of 10 days-old rats and in liver of 20 days-old foetuses when compared with 20 days-old (brain) and 18 days-old (liver) foetuses respectively. During further postnatal development, the mRNA for PDI decreases in both organs to the initial values present in foetuses and remains practically unchanged in brain till the adult. By contrast in liver by 35-40 days after birth, and coincident with sexual maturation, there is a 2.5-fold increase in PDI mRNA that is maintained by 55 days (adult). These results clearly show that protein disulfide isomerase gene expression is differentially regulated in liver and brain during rat development.     

Comparison of changes in gene expression of transferrin receptor-1 and other iron-regulatory proteins in rat liver and brain during acute-phase response

The “acute phase” is clinically characterized by homeostatic alterations such as somnolence, adinamia, fever, muscular weakness, and leukocytosis. Dramatic changes in iron metabolism are observed under acute-phase conditions. Rats were administered turpentine oil (TO) intramuscularly to induce a sterile abscess and killed at various time points. Tissue iron content in the liver and brain increased progressively after TO administration. Immunohistology revealed an abundant expression of transferrin receptor-1 (TfR1) in the membrane and cytoplasm of the liver cells, in contrast to almost only nuclear expression of TfR1 in brain tissue. The expression of TfR1 increased at the protein and RNA levels in both organs. Gene expression of hepcidin, ferritin-H, iron-regulatory protein-1, and heme oxygenase-1 was also upregulated, whereas that of hemojuvelin, ferroportin-1, and the hemochromatosis gene was significantly downregulated at the same time points in both the brain and the liver at the RNA level. However, in contrast to observations in the liver, gene expression of the main acute-phase cytokine (interleukin-6) in the brain was significantly upregulated. In vitro experiments revealed TfR1 membranous protein expression in the liver cells, whereas nuclear and cytoplasmic TfR1 protein was detectable in brain cells. During the non-bacterial acute phase, iron content in the liver and brain increased together with the expression of TfR1. The iron metabolism proteins were regulated in a way similar to that observed in the liver, possibly by locally produced acute-phase cytokines. The significance of the presence of TfR1 in the nucleus of the brain cells has to be clarified.     

The expression of alpha-fetoprotein and albumin genes in rat liver during chemical carcinogenesis

The effect of the hepatocarcinogen 3′-methyl-4-dimethylaminoazobenzene on α-fetoprotein (AFP) and albumin gene expression in rat liver was studied. Serum concentrations of AFP and albumin were measured. Amounts of AFP mRNA and albumin mRNA in rat livers were determined by hybridization of total cytoplasmic RNAs to their cDNAs. Dramatic increases in serum AFP concentrations coincided with increases in AFP biosynthesis and amount of AFP mRNA in livers of carcinogen-treated rats. In contrast, no or little change in albumin mRNA concentration was found in livers of rats treated with 3′-methyl-4-dimethylaminoazobenzene. Concomitantly, there was little change in liver albumin biosynthesis or serum albumin concentrations during hepatocarcinogenesis.     

The systemic reaction during inflammation: the acute-phase proteins

The acute-phase response consists in a large number of behavioural, physiologic, biochemical, and nutritional changes involving many organ systems distant from the site, or sites, of inflammation. One of the most investigated, but still not well understood, characteristic of the acute phase is the up-regulation, or down- regulation, of many plasma proteins, known as the acute-phase proteins. The changes in the concentrations of these positive acute-phase proteins and negative acute-phase proteins are due to changes in their liver production. Their increase may vary from 25 percent to 1000 fold, as in the case of C-reactive protein and serum amyloid A. This review summarises the recent advances that have been acquired on the acute-phase proteins, in particular their function in pathologies such as infections or inflammatory lesions.     

Co-culture of primary rat hepatocytes with rat liver epithelial cells enhances interleukin-6-induced acute-phase protein response

Three different primary rat hepatocyte culture methods were compared for their ability to allow the secretion of fibrinogen and albumin under basal and IL-6-stimulated conditions. These culture methods comprised the co-culture of hepatocytes with rat liver epithelial cells (CC-RLEC), a collagen type I sandwich culture (SW) and a conventional primary hepatocyte monolayer culture (ML). Basal albumin secretion was most stable over time in SW. Fibrinogen secretion was induced by IL-6 in all cell culture models. Compared with ML, CC-RLEC showed an almost three-fold higher fibrinogen secretion under both control and IL-6-stimulated conditions. Induction of fibrinogen release by IL-6 was lowest in SW. Albumin secretion was decreased after IL-6 stimulation in both ML and CC-RLEC. Thus, cells growing under the various primary hepatocyte cell culture techniques react differently to IL-6 stimulation with regard to acute-phase protein secretion. CC-RLEC is the preferred method for studying cytokine-mediated induction of acute-phase proteins, because of the pronounced stimulation of fibrinogen secretion upon IL-6 exposure under these conditions.     

The acute-phase response of the liver in relation to thyroid hormone-induced redox signaling

Recently, we reported that 3,3',5-triiodothyronine (T3) induces the expression of redox-sensitive genes as a nongenomic mechanism of T3 action. In this study, we show that T3 administration to rats (daily doses of 0.1 mg/kg ip for 3 consecutive days) induced a calorigenic response and liver glutathione depletion as an indication of oxidative stress, with higher levels of interleukin (IL)-6 in serum (ELISA) and hepatic STAT3 DNA binding (EMSA), which were maximal at 48-72 h after treatment. Under these conditions, the protein expression of the acute-phase proteins haptoglobin and beta-fibrinogen is significantly augmented, a change that is suppressed by pretreatment with alpha-tocopherol (100 mg/kg ip) or gadolinium chloride (10 mg/kg iv) before T3. It is concluded that T3 administration induces the acute-phase response in rat liver by a redox mechanism triggered at the Kupffer cell level, in association with IL-6 release and activation of the STAT3 cascade, a response that may contribute to reestablishing homeostasis in the liver and extrahepatic tissues exhibiting oxidative stress.     

Apolipoprotein-E-gene expression in rat liver during development in relation to insulin and glucagon

An apolipoprotein-E (apo-E) cDNA probe, cloned by immunoscreening of a lambda GT11 rat liver cDNA library, was used to further characterize the expression of the apo-E gene in rat liver during development, in relation to plasma insulin and glucagon levels. The apo-E mRNA level was low in fetus liver, then abruptly increased at birth and rose further during the suckling period. It returned to the level at birth in 10-week-old adults. These variations were paralleled with dramatic changes in plasma glucagon, which rose at birth and remained high during suckling. At the same time, the insulin/glucagon molar ratio fell. Administration of N6,O2-dibutyryl cAMP to 5-day-old rats resulted in a significant induction of liver apo-E mRNA. Moreover, liver apo-E mRNA rose in 10-h-fasted suckling rats as compared to controls, while plasma glucagon increased and the insulin/glucagon ratio decreased. Conversely, glucose feeding of suckling rats did not induce any increase in liver apo-E mRNA, the insulin/glucagon ratio was 10-fold higher than in fasted animals. Our results are consistent with liver apo-E gene expression being under the control of plasma glucagon and of the glucagon/insulin balance.     

Differential expression of protein S genes during Myxococcus xanthus development

Protein S, the most abundant protein synthesized during development of the fruiting bacterium Myxococcus xanthus, is coded by two highly homologous genes called protein S gene 1 (ops) and protein S gene 2 (tps). The expression of these genes was studied with fusions of the protein S genes to the lacZ gene of Escherichia coli. The gene fusions were constructed so that expression of beta-galactosidase activity was dependent on protein S gene regulatory sequences. Both the gene 1-lacZ fusion and the gene 2-lacZ fusion were expressed exclusively during fruiting body formation (development) in M. xanthus. However, distinct patterns of induction of fusion protein activity were observed for the two genes. Gene 2 fusion activity was detected early during development on an agar surface and could also be observed during nutritional downshift in dispersed liquid culture. Gene 1 fusion activity was not detected until much later in development and was not observed after downshift in liquid culture. The time of induction of gene 1 fusion activity was correlated with the onset of sporulation, and most of the activity was spore associated. This gene fusion was expressed during glycerol-induced sporulation when gene 2 fusion activity could not be detected. The protein S genes appear to be members of distinct regulatory classes of developmental genes in M. xanthus.     

Peroxisome proliferator-activated receptor-alpha expression in rat liver during postnatal development

The expression of the peroxisome proliferator-activated receptor-alpha (PPARalpha) as well as of some related genes was studied in rat liver at different stages of development (from 19-day-old fetuses to 1 month-old rats). The level of PPARalpha mRNA appeared higher in neonates than in fetuses or 1 month-old rats. Whereas the pattern for phosphoenolpyruvate carboxykinase (PEPCK) mRNA level was similar to that of PPARalpha, the mRNA level of both acyl-CoA oxidase (ACO) and apolipoprotein CIII (apo CIII) showed diverse profiles. Western blotting analysis also revealed an increased level of PPARalpha protein in liver of suckling rats. Similarities of mRNA PEPCK and PPARalpha expression indicate a common control mechanism, where both nutritional and hormonal factors may be involved.