STAT3/NF-κB interactions determine the level of haptoglobin expression in male rats exposed to dietary restriction and/or acute phase stimuli.

Haptoglobin is a constitutively expressed protein which is predominantly synthesized in the liver. During the acute-phase (AP) response haptoglobin is upregulated along with other AP proteins. Its upregulation during the AP response is mediated by cis-trans interactions between the hormone-responsive element (HRE) residing in the haptoglobin gene and inducible transcription factors STAT3 and C/EBP ß. In male rats that have been subjected to chronic 50% dietary restriction (DR), the basal haptoglobin serum level is decreased. The aim of this study was to characterize the trans-acting factor(s) responsible for the reduction of haptoglobin expression in male rats subjected to 50% DR for 6 weeks. Protein-DNA interactions between C/EBP and STAT families of transcription factors and the HRE region of the haptoglobin gene were examined in livers of male rats subjected to DR, as well as during the AP response that was induced by turpentine administration. In DR rats, we observed associations between the HRE and C/EBPα/ß, STAT5b and NF-κB p50, and the absence of interactions between STAT3 and NF-kB p65. Subsequent induction of the AP response in DR rats by turpentine administration elicited a normal, almost 2-fold increase in the serum haptoglobin level that was accompanied by HRE-binding of C/EBPß, STAT3/5b and NF-kB p65/p50, and the establishment of interaction between STAT3 and NF-κB p65. These results suggest that STAT3 and NF-κB p65 crosstalk plays a central role while C/EBPß acquires an accessory role in establishing the level of haptoglobin gene expression in male rats exposed to DR and AP stimuli.

The acute-phase protein alpha2-macroglobulin plays an important role in radioprotection in the rat.

The importance of alpha2-macroglobulin (alpha2M) in natural radioprotection was studied by examining its radioprotective effectiveness in rat models of exogenously and endogenously, preexposure-increased alpha2M. Radioprotective efficacy was ascertained by the postirradiation survival rate, the restoration of body weight, and the leukocyte count, which were monitored during a 4-week follow-up period. The results were compared with the effects of a pretreatment with the synthetic radioprotective agent amifostine (Ami), which provides 100% protection in rats whole-body-irradiated by x-rays given in a dose of 6.7 Gy (LD50/30). Raising the plasma concentration of alpha2M 15-fold in male rats by a single intraperitoneal injection of purified protein provided 100% survival of irradiated animals. Female rats on the 19th day of pregnancy with endogenously elevated levels of alpha2M displayed improved survival (80%) compared with untreated rats (50% survival). After alpha2M administration, the pregnant, irradiated rats exhibited 100% survival. In both males and pregnant females, alpha2M administration promoted body weight and leukocyte postirradiation recovery as in Ami-pretreated rats. These findings, together with our observation that Ami administration induced a 45-fold increase in alpha2M in the circulation, led us to conclude that alpha2M has an essential role in both natural and amifostine-mediated radioprotection in the rat.

The organophosphate-induced acute-phase response is characterized by synthesis of alpha 1-acid glycoprotein that exhibits an immunomodulatory effect.

The organophosphorus compounds soman and paraoxon induce the acute-phase (AP) response. All phases of the AP response, from macrophage activation and stimulation of glucocorticoid secretion to AP protein expression appear to be under the control of similar molecular mechanisms to those during the turpentine-induced AP response. The AP protein content in the circulation 24 h after either soman, paraoxon or turpentine administration was injury-specific. Both soman and paraoxon poisoning were characterized by significantly increased synthesis of alpha(1)-acid glycoprotein (AGP) that displayed an immunomodulatory effect in vitro. This result suggests that after organophosphate poisoning AGP participates in vivo in a negative feedback mechanism that prevents over-activity of the immune system.

Nuclear localization and binding affinity of STAT5b for the alpha(2)-macroglobulin gene promoter during rat liver development and the acute-phase response.

Expression of the rat alpha(2)-macroglobulin (MG) gene undergoes dynamic changes throughout an individual's life and during the acute-phase (AP) response. Details of the participation of the STAT family of transcription factors in its control remain incompletely understood. Here we examined the involvement of STAT5b in MG gene expression during development and the AP response. Immuno-blot analysis revealed the highest nuclear level of STAT5b in the fetus and during postnatal development, whereas in the adult it decreased. Stimulation of MG expression during the AP response was accompanied by a decrease in STAT5b. Examination of STAT5b localization revealed that the relative concentrations of STAT5b were higher in the nuclear matrix than in the nuclear extract. Affinity chromatography with the extended promoter region of the MG gene (-825/+12), followed by immuno-blot analysis, revealed dynamic changes in STAT5b binding. The highest concentration of the promoter-binding form of STAT5b was observed in the fetus. As postnatal development progressed, the level of promoter-bound STAT5b decreased and in the adult liver it was the lowest. Stimulation of MG gene expression during the AP response in both the fetus and adult was accompanied by significantly decreased STAT5b binding to the MG promoter. The AP response was accompanied by lower levels of STAT5b serine and tyrosine phosphorylation in both fetus and adult. In the nuclear matrix derived from adult tissues, tyrosine phosphorylated species were completely absent. We conclude that developmental-stage differences in the mechanisms that determine STAT5b nuclear localization contribute to its activity in vivo.

STAT3/NFkappaB interplay in the regulation of alpha2-macroglobulin gene expression during rat liver development and the acute phase response.

The synthesis of alpha-2-macroglobulin (alpha(2)M) 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-kappaB transcription factors during alpha(2)M gene expression was analysed. Using immunoblotting, their cellular compartmentalization was examined by comparing the cytoplasmic levels of STAT3 and NF-kappaB 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 alpha(2)M 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 alpha(2)M 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 alpha(2)M gene expression is driven by developmental- and AP-related mechanisms that rely on STAT3/NF-kappaB interplay.

Establishment of association of an Mg2+-dependent endonuclease with the rat liver nuclear matrix in cryonecrosis.

Previously, we characterized the endonucleolytic activity of the nuclear matrix prepared from rat liver cryopreserved in liquid nitrogen. The enzymic activity was attributed to a 23 kDa, Mg(2+)-dependent and sequence non-specific endonuclease (p23) stably associated with the nuclear matrix. Here we show that p23 was absent from the nuclear matrix prepared from fresh liver. Instead, both ex vivo (cryopreservation), as well as in vivo-induced necrosis by repeated freezing/thawing of liver tissue in an anaesthetized rat, promoted the activation and translocation of p23 to the nuclear matrix. Considering that ex vivo and in vivo freezing/thawing of the liver were accompanied by morphological (nuclear compaction) and biochemical events (increased LDH activity, disorderly genomic DNA degradation, absence of lamin proteolysis, appearance of 62 and 50 kDa necrotic cleavage products of PARP-1) commonly observed during necrosis, and because the association of p23 with the nuclear matrix was saturable, reflecting the existence of a limited number of distinct high affinity sites on the nuclear matrix for p23, we concluded that the activation of the nuclear matrix-associated endonuclease p23 is a feature of liver cryonecrosis. Although cryonecrosis represents a typical example of acute cell damage, our results suggest that it is realized by ordered molecular events.

C/EBP alpha and C/EBP beta regulate haptoglobin gene expression during rat liver development and the acute-phase response.

The participation of C/EBP alpha and C/EBP beta 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/EBP alpha and C/EBP beta 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/EBP alpha and an increase of C/EBP beta during development in both protein fractions. Using Western analysis after DNA-affinity chromatography it was observed that a 45 kDa C/EBP alpha 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/EBP beta 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/EBP alpha during normal liver development, whereas C/EBP beta is involved in the AP regulation during the later phase of differentiation and in the adult.

The radioprotective activities of turpentine-induced inflammation and alpha2-macroglobulin: the effect of dexamethasone on the radioprotective efficacy of the inflammation.

This work was aimed at the radioprotective efficacy of turpentine oil (TO), alpha2-Macroglobulin (alpha2-M), Amifostine (Ami) and/or dexamethasone (Dex). These agents were administrated, alone or in combination, prior to irradiation of rats with 6.7 Gy (LD(50/30)). The survival was recorded daily for 4 weeks after irradiation and body weight, peripheral leukocytes and thrombocytes were measured. The plasma concentration of alpha2-M and other acute phase proteins were determined by crossed immunoelectrophoresis. All rats receiving alpha2-M and Ami alone or in combination survived the radiation injury, whereas the rate of survival of TO-treated rats was 90%. Radiation and therapy-induced changes in the expression of acute phase protein genes were atypical for the acute phase reaction. Dex alone was lethal for 45% and 55% of control and irradiated rats, respectively. Pretreatment with 1mg Dex reduced radioprotective efficacy of TO and Ami to 30% and 40%, respectively. Given together TO and Ami provided 70% protection to rats receiving Dex. The TO and alpha2-M enhanced the rate of survival from 50% to 90% and 100%, respectively. In the presence of 1mg Dex the TO-induced radioprotectors and Ami exhibited radiosensitizing rather than radioprotecting activities.

p53-like protein binding affinity to the hormone responsive element of the haptoglobin gene in fetal rat livers.

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 53 kDa, 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 53 kDa-nucleoprotein could be a homologue of transcription factor p53, participating in the transcriptional modulation of the Hp gene throughout prenatal hepatic development.