The responses of rat liver glucocorticoid receptors and genes for tyrosine aminotransferase, alpha-2-macroglobulin and gamma-fibrinogen to adrenalectomy-, dexamethasone- and inflammation-induced changes in the levels of glucocorticoids and proinflammatory cytokines.

The responses of liver glucocorticoid receptor (GR) and genes coding for a glucocorticoid-inducible tyrosine aminotransferase (TAT) and two acute-phase proteins (APP) [alpha2-macroglobulin (alpha2-M) and gamma-fibrinogen (Fb)] to changes in glucocorticoid (GC) and proinflammatory (AP) cytokine contents have been examined in rats after single or combined treatments with turpentine oil, dexamethasone (Dex) and adrenalectomy. Activation of two APP genes in turpentine-induced inflammation was accompanied by an increase in the level of GR mRNA and a preferential translocation of GR-GC complexes to the nucleoplasm, while the expression of TAT remained unaltered. Dex alone caused a decrease in the levels of GR and Fb mRNAs, activation of TAT and alpha2-M genes, a decrease in the affinity of hormone binding sites and redistribution of translocated GR-Dex complexes within the nuclei. Inflammation potentiated the effect which Dex alone exerted on the GR content and the number of GR binding sites but counteracted its influence on the affinity of GR binding sites and nuclear distribution of GR-Dex complexes. Adrenalectomy promoted a fall in TAT mRNA, no changes in the GR and Fb mRNA, a decrease in the affinity of GR hormone binding sites and redistribution of GR-hormone complexes within the nuclei. The AP cytokines released in response to inflammation exerted a counteracting effect on the adrenalectomy-induced changes in the affinity of hormone binding sites and nuclear distribution of GR-hormone complexes. They potentiated a fall of TAT mRNA but promoted full expression of the Fb gene. These results argue strongly for the influence of AP cytokines on the functional state of the GR and GC signaling pathways.

Adrenalectomy and dexamethasone treatment alter the patterns of basal and acute phase response-induced expression of acute phase protein genes in rat liver.

Hormonal requirements for full hepatic expression of alpha2-macroglobulin (alpha2M), alpha1-acid glycoprotein (AGP), haptoglobin (Hp) and gamma-fibrinogen (Fb) were assessed at the level of mRNA. Prior to exposure to turpentine-induced inflammation, rats were either depleted of glucocorticoids by adrenalectomy or supplemented with an excess of dexamethasone. Adrenalectomy alone did not affect the basal level of acute phase protein (APP) expression except for alpha2M mRNA, the level of which was enhanced. In contrast, dexamethasone treatment alone promoted full induction of alpha2M, significant, but not maximal increase of AGP and Hp mRNAs and suppression of Fb. In adrenalectomized rats, acute phase (AP)-cytokines, released in response to inflammation, promoted full expression of Fb and Hp and increased the level of AGP mRNA whereas alpha2M mRNA remained at the basal level. Inflammation in dexamethasone pretreated rats elicited changes which, in comparison to mRNA values for dexamethasone unpretreated inflamed rats, were seen as overexpression of alpha2M, full expression of AGP and incomplete expression of Hp, whereas Fb mRNA remained at the basal level. These data suggest that glucocorticoids are the principal inducers of alpha2M and AP-cytokines of Fb. For full induction of AGP, additive actions of glucocorticoids and AP-cytokines are required whereas expression of Hp is predominantly controlled by AP-cytokines.

Structural and functional homology between the 29 kD rat liver nucleoprotein and the high mobility group 1 protein.

A 29 kD soluble rat liver nucleoprotein (p29) has increased binding affinity for the hormone responsive element (RE) of the rat haptoglobin (Hp) gene during the acute-phase reaction. In this work the possibility of its structural and functional homology to the high mobility group 1 (HMG1) nonhistone protein constituent of chromatin was examined. The results of two-dimensional gel electrophoresis, Southwestern and Western immunoblot analyses, showed that p29 and HMG1 are homologous protein species. On the basis of in vitro and in vivo phosphorylation/dephosphorylation experiments, we discuss the modulatory role of phosphate groups in view of the structure and function of p29.

The DNA binding affinity of rat liver nucleoproteins to the regulatory elements of the haptoglobin and alpha 2-macroglobulin genes.

Transcriptional regulation and binding interactions between soluble nucleoproteins and the hormone response elements (REs) of the rat haptoglobin (Hp) and alpha 2-macroglobulin (alpha 2MG) genes was examined in the livers of rats during the acute-phase reaction. Our results demonstrate that the elevation of the Hp and alpha 2MG genes' transcription rates in acute-phase liver relies essentially on an increase in the binding-affinity of pre-existing soluble nucleoproteins, enhancing their capability to bind the examined cis-regulatory elements. The 35kD nucleoprotein that displayed an acute-phase inducible affinity to bind hormone REs of rat Hp and alpha 2MG genes, was identified as a C/EBP beta isoform.

Acute phase-dependent changes in the binding of rat liver nucleoproteins to the cytokine response element of the rat haptoglobin gene.

Hormones released during the acute phase reaction promote the transcriptional activation of the haptoglobin (Hp) gene and a consequent increase of Hp protein synthesis in the liver. The mechanisms underlying the alterations of basal transcription rates of eukaryotic genes are assumed to result from modulations of the binding affinities between nucleoproteins and specific DNA sequences in the enhancer and promoter elements. In order to characterize the changes in the interaction of nucleoproteins with the promoter that accompany the induction of the Hp gene, nuclear extracts from normal and inflamed livers were probed with hormone responsive element (HRE) of the rat Hp gene by gel mobility shift and Southwestern assays. Each of the three cis-acting sequences of the HRE, elements A, B, and C, recognized a distinct set of proteins. Together they conferred an additional level of specificity to the protein binding sites of the entire ABC-element. These sites were recognized by proteins in liver nuclear extracts isolated from both control and treated rats. The differences in the gel shift and Southwestern patterns of the corresponding DNA-protein complexes suggested that transcriptional activation of the Hp gene relied on changes in the concentrations and/or functional modifications of preexisting proteins rather than on the induction of new trans-acting factors.

Inflammation of the dam has different effects on the binding of maternal and fetal rat liver nucleoproteins to the rat haptoglobin gene promoter.

Transcriptional regulation of binding interactions between nucleoproteins and the hormone response element (RE) of the rat haptoglobin (Hp) gene was investigated in adult and fetal livers of rats exposed to inflammation on day 19 of pregnancy. Nuclear extracts from the embryonal liver displayed a barely detectable binding-affinity for hormone RE, but in extracts from the adult liver it was noticeable. The acute phase reaction of the mother promoted an increase of Hp gene expression in both adult and fetal livers, relying on stage-specific changes in hormone RE binding activities of nucleoplasmic proteins. The results indicated that the elevation of Hp gene expression in fetal liver to the steady basal level found in adults required the induction of new trans-acting proteins, whereas an overexpression of this gene in adult acute phase liver relied essentially on an increase in the binding-affinity of the preexisting hormone RE binding proteins.

Pretreatment with alpha 2-macroglobulin leads to recovery of rats exposed to a lethal scald.

The effect of alpha 2-macroglobulin (alpha 2M) administration on the survival rate of lethally injured rats and molecular mechanisms regulating its hepatic expression after sublethal and lethal scalding were examined. Transcriptional activity of nuclei for the alpha 2M gene increased after a sublethal 20 per cent TBSA scald reaching a maximal three-fold increase by 12 h, whereas concentrations of the corresponding mRNA and protein attained the maximal nine- and 18-fold enhancements by 24 h, respectively. After the second, lethal scald, the plasma alpha 2M level increased during the first few hours, then dropped rapidly below the control value although the abundance of its mRNA was several fold enhanced. This anomaly was ascribed to inhibition of the alpha 2M mRNA translation caused by the second scald-induced disturbance of the haemodynamic equilibria. Eighty per cent of rats receiving alpha 2M prior to rescalding survived the second injury. Their recovery proceeded in parallel with normalization of the plasma volume and reactivation of the process of acute phase protein synthesis in the liver. A functional link between these events is discussed.

Regions of the haptoglobin 5' flanking gene sequence show different binding affinities to nuclear matrix proteins during the acute phase response.

The effect of the acute phase response on the affinity of binding between nuclear matrix proteins and the rat haptoglobin (Hp) gene region was examined. Nuclear matrices isolated from acute phase livers were enriched with the 5' Hp gene flanking region (-705/+159), but not with the spliced, protein-coding sequence. Reassociation experiments with isolated nuclear protein matrix spheres and end-labeled fragments I (-146/+156), II (-146/-541), and III (-541/-705) revealed that the matrix proteins displayed an increased binding potential during the acute phase response for all of the examined regions, this being most pronounced for fragment II. BAL 31 digestion of fragment II showed that the sequence element that was responsible for the increased association with nuclear matrix proteins during the acute phase response was a tract of 38 adenine bases. The DNA region established stable associations with nuclear lamin B (67 kDa, pI 5.7) in the controls, and with lamins A (69 kDa, pI 7.0), B, isoforms of lamin C (62 kDa, pI 6.55-6.95), and a 55-kDa (pI 5.9) polypeptide during the acute phase response. Sequence ABC (-165/-56), which overlaps fragments I and II and represents the Hp cis-acting element, did not bind to the non-histone nuclear matrix proteins.

The 70 kD rat liver nucleoplasmic protein binding hormone responsive element of rat haptoglobin gene shares size, charge and epitopes with lamin A.

The 70 kD rat liver nucleoplasmic protein was shown previously to display binding affinity for hormone responsive element of rat haptoglobin gene which markedly increased during the acute-phase reaction. In this work the possibility of its structural homology to lamin-type intermediate filament proteins is assessed. The results of two-dimensional Southwestern and Western analyses pinpointed to size, charge and epitope homology between the 70 kD rat liver nucleoplasmic protein binding the hormone responsive element and the lamin A constituent of nuclear matrix structure.

The acute phase response of rats to soman intoxication.

The capacity of an organophosphate to elicit the acute phase response (APR) was assessed by studying the effects of acute soman intoxication on two major processes which characterize inflammation, cytokine production in macrophages and the expression of acute phase protein (APP) genes in the liver. It was established that the concentration of lymphostimulatory substances secreted by the macrophages of soman-intoxicated rats was increased to a level characteristic of the primary inflammatory reaction. Macrophage activation was followed by increased transcription rates of APP genes and the corresponding mRNA and protein synthesis in the liver. The pattern of the DNA-protein complexes obtained with nuclear extracts and the cis-element of the rat haptoglobin gene in the gel-retardation assay suggested that the molecular events which underlie the expression of APP genes of intoxicated rats are similar to those that occur during the APR. From these findings we concluded that soman intoxication was a metabolic injury which elicited the typical APR.

The expression of liver acute-phase protein genes during rat development and in response to inflammation of the dam.

The hepatic expression of albumin (Al) and plasma acute phase protein genes (APP) was examined during the development of rat liver and in response to inflammation of the dam. Throughout the 10- to 20-day gestation period the level of alpha 2-macroglobulin (alpha 2M) mRNA in fetal liver exceeded twice that of the adult liver. The concentrations of the other APP and Al mRNAs were 10-30% of those of the adult liver between days 10 and 13 of gestation, then increased to values which ranged from 40% for haptoglobin (Hp) to 80% for Al and alpha 1-acid glycoprotein (AGP) mRNAs on day 19 of gestation. The transition of fetuses to an extrauterine environment was followed by a temporary overexpression of the Hp gene and an increase of the fibrinogen (Fb), AGP and thiostatin (TST) mRNAs to adult levels. Fetal liver responded to inflammation of the mother by a transcriptional induction of all of the investigated APP genes, except for the Fb gene whose level of expression remained unchanged. The pattern of individual APP genes expression in maternal and fetal livers was similar and characteristic for the acute phase reaction.

The effect of Yoshida sarcoma cell transplantation to rats on the rate of acute-phase protein synthesis in the liver.

The effect of transplantation of Yoshida sarcoma cells into rats on the concentrations of both the acute-phase proteins (APPs) in the serum and their mRNAs in the liver has been investigated. In rats that responded to transplantation by forming solid tumors, the serum APP levels increased with tumor size. Some of the APP was found to be infiltrated in the area of the tumor cells. Concentrations of the APP mRNAs in the liver were enhanced to an extent comparable to that observed for the encoded proteins, indicating that hepatocytes were the major site of their synthesis. In rats in which no formation of solid tumors occurred, the serum APP concentrations expressed a tendency to decrease below the control values. An inverse relationship between the rates of APP synthesis and the capability of rats to prevent proliferation of transplanted ascites tumor cells in the solid tumor form was discussed.

Effect of lethal scald on the mechanisms of acute-phase protein synthesis in rat liver.

Acute-phase protein (APR) synthesis was studied under conditions in which the acute-phase response to a sublethal scald was interrupted at the onset of APR synthesis by the infliction of a second scald that overwhelmed the defense mechanisms. The rate of APR synthesis increased shortly after the second scald and then declined rapidly to the control level. At this time point, APR messenger RNA (mRNA) concentrations exceeded severalfold the control values, whereas the APR gene transcription rates fell to the control level. These mRNAs were active in APR synthesis in a cell-free system, as well as in hepatocytes grown in a standard culture medium. These results and those demonstrating a drop in the free amino acid pool level in the liver after the second scald suggested that the lethal outcome was preceded by an impaired supply of liver cells with amino acids and resulting inhibition of APR mRNA translation. Changes in amino acid transport were considered to occur secondarily to those causing hypovolemia and circulatory shock.

Acute-phase response to scalding: changes in serum properties and acute-phase protein concentrations.

The time course of changes in the concentrations of individual acute-phase proteins (APRs) in the plasma was examined in the model of rats exposed to a single (sublethal) and repeated (fatal) scalding. These data were interrelated with the rate of survival, plasma volume, corticosterone level, and immunosuppressive potency of the serum. The infliction of a sublethal scalding composing 20% body surface area resulted in an initial 50% reduction of the plasma volume and a severalfold increase of the corticosterone level and immunosuppressive activity of the serum. A subsequent normalisation of these deviations proceeded in parallel with an increased rate of APR synthesis. An early infliction of a second scald was fatal. It led to a 60% reduction of the plasma volume, a lack of plasma APRs, and an additional enhancement of the immunosuppressive activity of serum.

Regulation of plasma acute-phase protein and albumin levels in the liver of scalded rats.

At 12 h after scalding of rats a doubling of the hepatocyte nuclear DNA content, which arose from the presence of additional complete genomes and not from amplification of genes coding for the major acute-phase proteins or albumin, was observed. Examination of relative transcription rates per control DNA mass revealed that alpha 1-acid-glycoprotein and cysteine-proteinase-inhibitor genes remained constitutive, alpha- and gamma-fibrinogen and haptoglobin genes underwent transcriptional activation for 290 and 339% respectively, whereas the relative transcription rate of albumin decreased to 65% of the control level. Along with these changes, the alpha 1-acid glycoprotein, cysteine-proteinase inhibitor and the fibrinogen mRNA concentrations increased about 500%, haptoglobin mRNA 250%, whereas the albumin mRNA concentration fell to 86% of the control. The regulation of the mRNA levels was assessed by comparing the relative change in transcription rates expressed per control DNA content with the relative changes of mRNA concentrations. We arrived at the conclusion that the concentrations of alpha 1-acid-glycoprotein and cysteine-proteinase-inhibitor mRNAs were predominantly regulated by a post-transcriptional mechanism, albumin mRNA by a transcriptional mechanism, and the fibrinogen and haptoglobin mRNAs by a combination of both. The degree of change of the serum levels of the examined proteins was similar to that of their mRNA concentrations and was the result of the complete use of the available RNA templates in protein synthesis.

Soman intoxication-induced changes in serum acute phase protein levels, corticosterone concentration and immunosuppressive potency of the serum.

We have studied the effect of soman intoxication on serum acute phase reactants (APR) levels, and the relationship of the APR and corticosterone concentrations and the immunosuppressive activity of the serum. One day after the injection of 1.8 LD50 soman the concentrations of alpha 2-macroglobulin (alpha 2-MG) and alpha 1-acid glycoprotein (AGP) in the serum of antidote protected rats increased 4- and 7-fold, respectively, whereas those of hemopexin (Hx), haptoglobin (Hp) and cysteine protease inhibitor (CPI) were two to three times higher than in the controls. A similar magnitude of increase of serum acute phase reactants levels was observed when 0.3 LD50 soman was administered at 24-h intervals over the 5-day period. The relationship of changes in the APR concentration, corticosterone level and immunosuppressive activity of the serum was also comparable to that observed in the acute phase response to tissue injury.