Miodrag Petrović

STAT3/NFκB Interplay in the Regulation of α2‐Macroglobulin Gene Expression During Rat Liver Development and the Acute Phase Response

The synthesis of alpha‐2‐macroglobulin (α2M) is low in adult rat liver and elevated in fetal liver. During the acute‐phase (AP) response it becomes significantly increased in both adult and fetal liver. In this work, the cross talk of STAT3 and NF‐κB transcription factors during α2M gene expression was analysed. Using immunoblotting, their cellular compartmentalization was examined by comparing the cytoplasmic levels of STAT3 and NF‐κB with their active equivalents, the 86 and 91 kDa isoforms and p65‐subunit, respectively, in the nuclear extract and nuclear matrix. Different partitioning dynamics of the transcription factors were observed. At the level of protein‐DNA interactions, studied by α2M promoter affinity chromatography, it was established that different ratios of promoter‐binding STAT3 isoforms participated in elevated hepatic transcription in the basal state fetus and the AP‐adult, but only the 91 kDa isoform in the AP‐fetus. Unchanged levels of DNA‐bound p65 in the control and AP‐fetus suggest that it participated in constitutive transcription. The promoter‐binding of p65 observed in the AP‐adult suggests that it was involved in transcriptional stimulation of α2M expression. The selective enrichment of the AP‐adult nuclear matrix with promoter‐binding STAT3 disclosed the importance of this association in the induction of transcription. Protein‐protein interactions were examined by co‐immunoprecipitation. Interactions between the 86 kDa STAT3 isoform and p65 that were observed in the control and AP‐fetus and of both the 86 and 91 kDa STAT3 isoforms with p65 in the AP‐adult, suggest that protein‐protein interactions were functionally connected to increased transcription. We concluded that α2M gene expression is driven by developmental‐ and AP‐related mechanisms that rely on STAT3/NF‐κB interplay. IUBMB Life, 59: 170‐178, 2007

C/EBPα and C/EBPβ Regulate Haptoglobin Gene Expression during Rat Liver Development and the Acute-phase Response

The participation of C/EBPα and C/EBPβ in the transcriptional regulation of the haptoglobin (Hp) gene throughout liver development and the acute-phase (AP) response was examined. Western immunoblot analysis revealed that the relative concentrations of C/EBPα and C/EBPβ increased during differentiation in two nuclear protein fractions – the nuclear extract and nuclear matrix. The AP reaction was accompanied by a decrease of the relative concentration of C/EBPα and an increase of C/EBPβ during development in both protein fractions. Using Western analysis after DNA-affinity chromatography it was observed that a 45 kDa C/EBPα isoform displayed a binding affinity towards the Hp gene hormone responsive element (HRE) in both pre- and postnatal livers. In the course of the AP response DNA binding of the 45 kDa isoform was detected only in the adult, when its binding affinity decreased. The 35 kDa C/EBPβ isoform exhibited a binding affinity towards the Hp HRE after the second week from birth, whereas the AP response promoted an enhanced binding of 35 kDa isoform after the first postnatal week. These results indicate that Hp gene transcription is regulated by C/EBPα during normal liver development, whereas C/EBPβ is involved in the AP regulation during the later phase of differentiation and in the adult.

PARTICIPATION OF TWO ISOFORMS OF C/EBPβ TRANSCRIPTION FACTOR IN THE ACUTE‐PHASE REGULATION OF THE RAT HAPTOGLOBIN GENE

Previous analyses of the mechanism of the transcriptional induction of the rat haptoglobin (Hp) gene during acute‐phase (AP)‐reaction have revealed the involvement of several trans ‐acting nucleoproteins (NPs) in controlling this process. In this study, by using antibodies against C/EBPβ factor in Western immunoblot assay, we found that rat liver trans ‐acting NPs p35 and p20 are two characteristic C/EBPβ isoforms whose expression is induced under AP‐conditions. DNA‐binding assays identified the binding sites for these two C/EBPβ proteins in the functionally defined elements A and C of the rat Hp gene and also revealed that they have specific binding affinity towards these elements. Under non‐induced conditions, p35 was the only C/EBPβ binding factor; however, upon AP‐conditions both, 35kDa‐ and 20kDa‐C/EBPβ binding activities were significantly induced suggesting that these interactions are necessary for the activation of the Hp gene. By in vitro phosphorylation assay and selective proteolysis, we also present evidence that p35 requires phosphorylation for its DNA binding ability. Thus, we conclude that increase in binding of C/EBPβ isoforms during AP‐reaction occurs through their upregulation and structural modification.

Opposite nuclear level and binding activity of STAT5B and STAT3 proteins with rat haptoglobin gene under normal and turpentine induced acute phase conditions.

Transcription of the rat gene encoding haptoglobin (Hp) is highly induced during acute phase (AP) response which has been previously shown to be mediated by inducible STAT3 member of the Signal Transducer and Activators of Transcription (STATs) family proteins. In this study, we observed that under normal but not in the turpentine induced AP conditions, another member of the STAT family proteins, STAT5b is expressed and binds to the hormone regulatory element (HRE) of the rat Hp gene. We found that the nuclear amounts of constitutively active STAT5b in rat liver decreased significantly with time of turpentine treatment as opposed to that of cytosol STAT5b, suggesting possible export of constitutive STAT5b from the nucleus. Nuclear accumulation and binding of inducible STAT3 proteins to the rat Hp gene HRE following turpentine treatment implicated that STAT5b negatively regulates Hp gene expression during normal conditions.

Pretreatment with α2-macroglobulin leads to recovery of rats exposed to a lethal scald

The effect of α 2 -macroglobulin (α2M) 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 α2M 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 α2M 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 α2M mRNA translation caused by the second scald-induced disturbance of the haemodynamic equilibria. Eighty per cent of rats receiving α2M 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.

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.

STAT3 involvement in the acute phase-related expression of the rat haptoglobin gene

Turpentine-induced acute-phase (AP) response in rats is followed by transcriptional activation of the haptoglobin (Hp) gene in liver. Analysing the promoter sequence of the rat Hp gene we postulated an involvement of Signal Transducer and Activator of Transcription 3 (STAT3) in the regulation of this process. Results obtained by using a combination of Western immunoblot and DNA-binding assays revealed AP-induced binding of constitutive 86kD- and inducible 91kD-STAT3 isoforms to the rat Hp gene inducible promoter element. On the basis of these data we assumed that AP-related interactions of these two STAT3 isoforms correlates with an activated transcriptional status of the rat Hp gene.

p53-LIKE PROTEIN BINDING AFFINITY TO THE HORMONE RESPONSIVE ELEMENT OF THE HAPTOGLOBIN GENE IN FETAL RAT LIVER

In order to identify nucleoproteins involved in transcriptional regulation of the haptoglobin (Hp) gene, fetal rat livers of dams exposed to inflammation on day 19 of pregnancy were used. Previously observed acute phase‐dependent elevation of Hp gene transcriptional activity in prenatal liver was accompanied by increased binding affinities of several fetal soluble nucleoproteins and the hormone response element (RE) of the Hp gene (‐170/‐56). One of these proteins, a hepatic nucleoprotein of 53kDa, was identified by Western blotting analysis as a protein within the same molecular mass and epitopes as transcription factor p53. Also, in vitro phosphorylation experiments revealed that the examined fetal nucleoprotein could be liable to the same phosphorylative post‐translational modification as p53. The obtained results suggest that the fetal 53kDa‐nucleoprotein could be a homologue of transcription factor p53, participating in the transcriptional modulation of the Hp gene throughout prenatal hepatic development.

The effects of repeated thermal injury on rat liver and serum protein synthesis rates

Abstract 1. 1. The effects of single and repeated thermal injury on liver cells and blood serum proteins metabolism were investigated in experiments performed on rats. 2. 2. During the first three postburn days amino acid incorporation into nuclear and mitochondrial proteins proceeded at an increased rats, whereas the effects of the repeated injury dependent on time interval elapsed between two injuries.

The DNA binding affinity of rat liver nucleoproteins to the α1-acid glycoprotein gene

Transcriptional regulation and binding interactions between soluble nucleoproteins and the distal regulatory element (DRE) of the rat α1‐acid glycoprotein (α1‐AGP) gene were examined in the liver of rats during the acute‐phase response. Our results show that the elevation of the α1‐AGP gene transcription activity in acute phase liver relies basically on an increase in the binding‐affinity of the constitutive soluble nucleoproteins with molecular masses 35 and 45 kD, enhancing their capability to bind to the distal regulatory element (DRE) of α1‐AGP gene. On the basis of in vitro phosphorylation/dephosphorylation experiments we discuss that the role of these proteins during α1‐AGP transcription may be dependent on their behavior as phosphoproteins. The 35kD nucleoprotein that displayed an acute‐phase inducible affinity to bind DRE was indentified as a C/EBPβ isoform.