Acute-phase related binding ability of p53 for the hormone response element of the haptoglobin gene in adult rats.

Interaction between transcription factor p53 and the hormone response element (HRE) of the haptoglobin (Hp) gene in adult rat liver was studied. We detected a sequence homologous to the p53 consensus DNA-binding site in the regulatory promoter element of the Hp gene. DNA-affinity chromatography, followed by Western immunoblot analysis with an antibody to p53 indicated that components of the nuclear extract possessed the same antigen determinants as p53. While p53 was identified in both control and acute-phase (AP) samples, DNA-binding affinity for the Hp gene HRE was detected only in the nuclear extract prepared from rats undergoing the AP response. Whether either as an inducible or as a constitutive transcription factor, p53 could be involved in the transcriptional regulation of the Hp gene in adult rat liver.

Binding of a 23 kD endonuclease to the rat liver nuclear matrix.

In a previous paper we have described a 23 kD nuclear endonuclease (p23) that was mostly found to exist in a state of association with the isolated rat hepatocyte nuclear matrix. To investigate the nature of this interaction, the nuclear matrix was prepared using different procedures and examined for the presence/absence of the enzyme by activity gel analysis. Treatment of isolated nuclei with sodium tetrathionate (NaTT), a sulfhydryl-cross-linking agent, led to the complete recovery of p23 in the nuclear matrix, whereas incubation of nuclei with dithiothreitol (DTT), a sulfhydryl-reducing agent, led to its complete solubilization and resulting absence from the nuclear matrix. Exposure of the isolated nuclear matrix to DTT in high-ionic strength buffer, a procedure that promotes the solubilization of the internal nuclear matrix, caused the nearly complete solubilization of p23. It was concluded that disulfide bonds play an essential role in the association of p23 with the nuclear matrix and that p23 is mostly localized in the nuclear matrix interior.

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 (Uskokovic 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.

The effect of O-glcnac glycosylation of rat liver nucleoproteins on their acute phase-dependent binding ability to the hormone responsive element of the haptoglobin gene.

We examined whether the transcriptional activation of the rat haptoglobin (Hp) gene during the acute phase (AP) response reflects the O-linked N-acetylglucosamine (O-GlcNAc) status of liver nucleoproteins (NPs) and their binding for the hormone responsive element (HRE). After deglycosylation with N-acetylglucosaminidase of the O-GlcNAc glycoproteins obtained by WGA, affinity chromatography and South-Western analysis, it was observed that only increased HRE binding ability of p64/p70 in control and p51 obtained from turpentine-treated rats can be directly attributed to the presence of O-GlcNAc residues. Therefore, expression of the rat Hp gene could be controlled by this modification of certain trans-acting NPs.

Acute-phase-dependent binding affinity of C/EBPbeta from the nuclear extract and nuclear matrix towards the hormone response element of the alpha2-macroglobulin gene in rat hepatocytes.

Interactions of nuclear extract and nuclear matrix proteins from rat hepatocytes with the hormone response element of the alpha2-macroglobulin gene were studied. By Western and South-Western blot analysis we have shown the presence of C/EBPbeta in the examined nuclear fractions as well as its increased binding affinity to the examined gene sequence during the acute-phase response. The results suggest that both nuclear protein fractions could participate in the transcriptional regulation of the alpha2-macroglobulin gene in the rat hepatocytes.

The protein composition of the hepatocyte nuclear matrix is differentiation-stage specific.

The protein composition of hepatocyte nuclear matrices was examined in rats from the 16th day of gestation to 75 days after birth (adult). An overall increase in size of the nuclear matrix was accompanied by quantitative and qualitative changes in its protein content. Quantitative changes of the major proteins of the peripheral lamina surrounding the isolated nuclear matrix were detected. By Western analysis we established that in pre- and postnatal nuclear matrices the relative concentrations of lamin C were greater than lamin A. After birth, the relative concentrations of both lamins progressively increased. In the adult nuclear matrix, the concentration of lamin A was greater than lamin C. In contrast, the relative concentrations of lamin B remained unchanged throughout development and growth. The relative concentrations of two nuclear matrix-associated regulatory proteins studied changed with development and growth: transcription factor C/EBPalpha isoforms, which were detected during the gestation period, increased notably after the first postnatal day, attaining a maximum at the adult stage; the high concentrations of the proliferating cell nuclear antigen (PCNA) perceptibly decreased after the 21st prenatal day. Changes in the composition of the nuclear matrix protein suggest that this structure coordinates nuclear functioning during cell differentiation.

C/EBPalpha and C/EBPbeta are persistently associated with the rat liver nuclear matrix throughout development and the acute phase response.

The partitioning of C/EBPalpha and C/EBPbeta on the nuclear matrix structure was examined during the different transcriptional activities accompanying liver development and the acute phase (AP) response. The presence of C/EBPalpha and C/EBPbeta was established on the nuclear matrix. Their relative concentrations on the matrix always reflected the developmental stage- and AP-related fluctuations observed in the nuclear extract. Thus, they progressively increased as development proceeded, whereas during the AP response, C/EBPalpha decreased and C/EBPbeta increased. In addition, the levels of both transcription factors were always notably higher in the nuclear matrix than in the extracts. We conclude that the observed changes and overall enrichment of the nuclear matrix with regulatory proteins is a reflection of the importance of such interactions for the in vivo functioning of C/EBP proteins.

Identification of nuclear matrix and associated proteins that bind the haptoglobin gene cis-element.

To identify the major nuclear matrix proteins that bind to the rat haptoglobin gene cis-element, we isolated a soluble nuclear matrix protein fraction and analysed it by gel retardation. Two major DNA-binding proteins exhibiting different types of protein-DNA interactions were detected: a DNA sequence-specific 32-kDa isoform of transcription factor C/EBP beta, and a nuclear matrix protein p55 that bound to the DNA nonspecifically. During increased transcription of the haptoglobin gene in the course of the acute-phase reaction, the DNA-binding affinities and concentrations of these proteins in the soluble nuclear matrix fraction were increased. These data lend further evidence that the nuclear matrix is an active support structure that localizes gene regulatory proteins and participates in transcriptional regulation.

Re-establishment of homeostasis and the acute-phase proteins.

The stimulation of transcription of acute-phase protein (APP) genes in the liver is incorporated in the complex interchange of cytokines, growth factors and glucocorticoid hormones that are released during the systemic defence reaction in response to trauma. Through the broad spectrum of their activities, this heterogeneous group of circulating proteins assists the injured organism in restoring homeostasis by assuming a protective role. APPs accomplish this by inactivating vasoactive, proteolytic and cytotoxic molecules liberated from damaged tissues and accumulating phagocytic cells, and by participating in a feedback control mechanism that prevents an overload by the organisms' immune response. APP synthesis represents a non-specific response of the liver, in so much as different types of trauma elicit the production of the same proteins. However, data obtained from different laboratory models and clinical observations revealed a certain relationship between the severity and type of trauma and the magnitude of activation of APP gene expression. The observed variations of the overall pattern of APP synthesis point to the existence of different interplays between humoral and cellular mediators capable of adjusting the production of individual proteins to suit different traumas. Hence, changes in the serum concentrations of some APPs have been shown to be useful in monitoring complications such as infection or sepsis after surgery or trauma, and predicting the clinical course of malignant and other diseases. Of the APPs studied in humans, information obtained on CRP and SAA has in particular proved to be a useful indicator of the progression of different pathological states.

Properties of nuclear matrix proteins that bind the 5' flanking region of the haptoglobin gene are changed during the acute-phase response.

It was previously shown that during the acute-phase response-induced elevated transcription of the rat haptoglobin gene, protein p55 and the lamins mediate the increased binding of restriction fragment II (-541/-146) via a 38 bp adenine tract lying 147 bp upstream from the haptoglobin gene cis element (-165/-56), to scaffold type II-like nuclear matrices. Here we show that the fragment II binding pattern to > or = 40 kDa proteins of nuclear matrices analogous to type I scaffolds is more complex. Fragment II bound conspicuously to a 40 and less so to p55, a 60 kDa protein and the lamins of control matrices. During increased gene transcription, it bound prominently to p55, the lamins, and less so to 45 and 52 kDa proteins. This was accompanied by the tenacious binding of the DNA to isolated nuclear matrices in vitro and post-translational modifications of certain matrix proteins. The lamins and p55 demonstrated a greater N-acetylglucosamine/sialic acid content and p55 an increased in vitro phosphorylation by a nuclear matrix-associated cyclic-nucleotide-independent kinase. The acute-phase response also caused an increased partitioning of p55 with the nuclear matrix. It was concluded that, as a result of a molecular remodelling of the nuclear matrix at the point of contact with chromatin, the nature of its association with region II DNA changed during elevated haptoglobin gene expression.

HI-6 therapy and the acute phase response in the rat.

The propensity of therapeutic doses of HI-6 (50 mg/kg) in combination with atropine sulphate (17 mg/kg), antidotes used to treat organophosphate poisoning, to induce the acute phase response (APR) in the laboratory rat was examined. A single intraperitoneal injection of HI-6, either alone or with atropine, caused a rapid doubling of the plasma corticosterone concentration. However, this increase was short-lived in comparison with corticosterone kinetics during the typical, turpentine-induced APR. The elevated glucocorticosteroid concentration did not affect acute phase protein (APP) gene transcription or mRNA and protein synthesis in the livers of oxime/atropine-treated rats. On the basis of these findings, we concluded that the administered doses of HI-6 and atropine did not induce the generalised, non-specific APR.

Ionizing radiation-induced expression of the genes associated with the acute response to injury in the rat.

Total-body irradiation of rats with doses ranging from an LD10/30 to an LD100/30 induced a dose-dependent increase in the concentration of serum protein associated with the acute response to the irradiation. However, this increase was reached at a later time and was not as pronounced as described previously during the typical acute phase of the response found experimentally (A. Koj, in Structure and Function of Plasma Proteins, Vol. 1, pp. 73-131, Plenum Press, London, 1974). The greatest increase in the serum concentrations of acute-phase proteins was found from the third to the seventh days postirradiation. At these times, the serum concentrations of alpha 2-macroglobulin, haptoglobin, fibrinogen and cysteine protease inhibitor were raised from two- to fivefold, whereas alpha 1-acid glycoprotein was increased sixfold. Incorporation of [35S]methionine into total serum and acute-phase proteins indicated that the increase in the concentration of the acute-phase proteins was preceded by their de novo synthesis in the liver. The results that were obtained by dot-blot analysis showed that the basic course of change in the relative mRNA concentrations in the liver for the acute-phase proteins examined correlated with the changes in their protein concentrations in the serum; only the relative increase in the concentration of alpha 1-acid glycoprotein mRNA was significantly lower than the increase in proteins in the serum, suggesting that a fraction of the serum alpha 1-acid glycoprotein had an extrahepatic origin. On the basis of these results we concluded that total-body irradiation increased the expression of acute-phase protein genes.

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 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.

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.

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.

Thermal injury-induced expression of acute-phase proteins in rat liver.

Relative changes in acute-phase protein and albumin mRNAs from the liver of rats exposed to sublethal and lethal scaldings were examined by hybridization and cell-free translation. Infliction of a sublethal scalding comprising 20 per cent of the total skin area resulted in a seven-fold increase of alpha 1-acid glycoprotein (AGP), alpha 1-cysteine protease inhibitor (CPI) and the alpha- and gamma-fibrinogens' (Fb) mRNAs, whereas the level of haptoglobin (Hp) mRNA increased three times. The simultaneous infliction of two such sublethal scaldings were lethal and accompanied by a significant decrease in the concentration of AGP while the levels of CPI. Hp and Fb mRNAs remained similar to those observed after a single 20 per cent injury. A 4 h time delay between the two scaldings was also fatal and followed by an additional increase in Hp mRNA concentration whereas the levels of the other mRNAs were close to those observed after a single 20 per cent scalding. These results demonstrated that the fatal effect of the second scalding was not related to any inhibition of acute-phase reactants mRNA synthesis in the liver.

Soman intoxication-induced increase in the levels of mRNAs coding for acute phase reactants.

The effect of lethal and repetitive sublethal soman intoxication on the composition of rat liver mRNA was examined by cell-free translation and hybridization. It was found that lethal as well as sustained sublethal soman poisoning of rats elicited a typical acute phase response as judged by a several-fold increase in levels of mRNAs coding for the major acute phase proteins and the vast number of systemic and metabolic changes creating the acute phase response should be taken into account when the metabolic events during the recovery from organophosphate intoxication are under consideration.