Ilijana Grigorov

Toxicity of oral cadmium intake: Impact on gut immunity

Gastrointestinal tract is one of the main targets of cadmium (Cd), an important food and drinking water contaminant. In the present study, the effect of subchronic (30 days) oral (in water) intake of 5ppm and 50ppm of cadmium on immune responses in the gut was examined in rats. Cadmium consumption resulted in reduction of bacteria corresponding to Lactobacillus strain, tissue damage and intestinal inflammation [increases in high mobility group box 1 (HMGB1 molecules), superoxide dismutase (SOD) and catalase (CAT) activity and proinflammatory cytokine (TNF, IL-1β, IFN-γ, IL-17) content]. Draining (mesenteric) lymph node (MLN) stress response was observed [elevation of MLN glutathione (GSH) and metallothionein (MT) mRNA levels] and stimulation of both adaptive [cellularity, proliferation, proinflammatory (IFN-γ and IL-17) MLN cell cytokine responses] as well as innate immune activity (increases in numbers of NK and CD68(+) cells, oxidative activities, IL-1β). In contrast to proinflammatory milieu in MLN, decreased or unchanged antiinflammatory IL-10 response was observed. Stimulation of immune activities of MLN cells have, most probably, resulted from sensing of cadmium-induced tissue injury, but also from bacterial antigens that breached compromised intestinal barrier. These effects of cadmium should be taken into account when assessing dietary cadmium as health risk factor.

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

Association of the glucocorticoid receptor with STAT3, C/EBPβ, and the hormone-responsive element within the rat haptoglobin gene promoter during the acute phase response

Upregulation of haptoglobin (Hp) expression in the rat during the acute phase (AP) response is the result of synergistic effects of IL‐6–, IL‐1β–, and corticosterone‐activated signaling pathways. IL‐6 signaling terminates in cis–trans interactions of the Hp gene hormone‐responsive element (HRE) with transcription factors STAT3 and C/EBPβ. The aim of this study was to examine the unresolved molecular mechanism of glucocorticoid action. A 3‐fold rise in serum corticosterone at 2 and 4 h of the AP response induced by turpentine administration preceded a 2.3‐fold increase in the rate of Hp gene transcription at 12 h that was accompanied by a 4.8‐fold increase in glucocorticoid receptor (GR), the appearance of an 86‐kDa STAT3 isoform and 3.9‐, 1.9‐, and 1.7‐fold increased amounts of 91‐kDa STAT3, 35‐ and 42‐kDa C/EBPβ isoforms in the nucleus. These events resulted in 4.6‐ and 2.5‐fold increased Hp levels in the liver and serum at 24 h. HRE affinity chromatography and immunoblot analysis revealed that maximal occupancy of the HRE with GR, STAT3, and C/EBPβ at 12 h correlated with increased transcriptional activity of the Hp gene. Coimmunoprecipitation experiments showed that activated GR established de novo interaction with STAT3 isoforms while GR–C/EBPβ interactions observed during basal transcription increased during the AP response. Computer analysis of the HRE disclosed two potential GR‐binding sites: one overlapping STAT3, another adjacent to a C/EBPβ‐binding site. This finding and the experimental results suggest that activated GR through direct interactions with STAT3 and C/EBPβ, participates in Hp gene upregulation as a transcriptional coactivator.

Activation Level of JNK and Akt/ERK Signaling Pathways Determinates Extent of DNA Damage in the Liver of Diabetic Rats

Aims: Diabetes-related oxidative stress conditions lead to progressive tissue damage and disfunctionality. Mechanisms underlying liver pathophysiology during diabetes are not fully understood. The aim of this study was to find relationship between diabetes-related DNA damage in the rat liver and activities of prosurvival signaling pathways. Methods: Effect of diabetes was analyzed two (development stage) and eight weeks (stable diabetes) after single intraperitoneal injection of streptozotocin. Extent of DNA damage, analysed by comet assay, was corelated with oxidative status (plasma level of ROS, liver antioxidant capacity) and activity/abundance of kinases (Akt, p38, ERK1, JNK, JAK) and transcription factors NF-ĸB p65 and STAT3. Results: Significant DNA damage in development stage is accompanied by elevated plasma levels of O2-and H2O2, decreased activities of CAT, MnSOD, and GST in the liver and increased activation of proapoptotic JNK signal pathway. Lower DNA damage in stable diabetes, is accompanied by elevated plasma level of O2-, restored antioxidative liver enzyme activity, decreased activation of JNK and increased activation of prosurvival Akt and ERK signal pathways. Conclusion: These findings indicate that level of DNA damage in diabetic liver depends on the extent of oxidative stress, antioxidant activity and balance between JNK and Akt/ERK signal pathways activation.

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.

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.

Oxidative stress-dependent contribution of HMGB1 to the interplay between apoptosis and autophagy in diabetic rat liver

The progression of oxidative stress, resulting cell damage, and cell death underlies the etiology of liver damage/dysfunction as a complication of diabetes. High-mobility group box 1 (HMGB1) protein, a chromatin-binding nuclear protein and damage-associated molecular pattern molecule, is integral to oxidative stress and signaling pathways regulating cell death and cell survival. We previously found that in streptozotocin (STZ)-induced diabetic rats, reduction of oxidative stress after melatonin administration lowered necrotic cell death and increased expression of HMGB1 and hepatocellular damage. In the present study, we examined whether alleviation of diabetes-attendant oxidative stress and ensuing change in HMGB1 expression influence the dynamic equilibrium between apoptosis/autophagy and liver damage. We observed that elevated HMGB1 protein levels in diabetic rat liver accompanied increased interactions of HMGB1 with TLR4 and RAGE, and activation of the intrinsic apoptotic pathway and Beclin 1-dependent autophagy. The absence of p62 degradation in diabetic rat liver pointed to defective autophagy which was responsible for lower autophagosome/autophagolysosome formation and an increased apoptosis/autophagy ratio. Compared to diabetic rats, in melatonin-treated diabetic rats, the structure of liver cells was preserved, HMGB1/TLR4 interaction and downstream apoptotic signaling were significantly reduced, HMGB1/Beclin 1 colocalization and interactions were augmented and Beclin 1-mediated autophagy, mithophagy in particular, were increased. We concluded that in mild oxidative stress, HMGB1 is cytoprotective, whereas in intense oxidative stress, HMGB1 actions promote cell death and liver damage. Since reduced HMGB1 binds to RAGE but not to TLR4, redox modification of HMGB1 as a mechanism regulating the cross-talk between apoptosis and autophagy in diabetes is discussed.

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.