Nuclear factor kappa B activation and regulation of cyclooxygenase type-2 expression in human amnion mesenchymal cells by interleukin-1beta

Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin (PG) output by up-regulating the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG synthesis. In this study, we investigated the possible role of the nuclear factor kappa B (NFkappaB) in IL-1beta signaling, leading to the expression of COX-2 in human amnion cell culture. Fetal amnion was obtained following vaginal delivery and digested with collagenase, and the subepithelial (mesenchymal) cells were isolated. Cultures were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Confluent cells were stimulated with human recombinant IL-1beta, and activation of NFkappaB was assessed by measuring changes in the inhibitory protein IkappaB (total IkappaB and phosphorylated IkappaB) using Western blot analysis as well as by nuclear binding of NFkappaB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1beta, phosphorylated IkappaB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IkappaB and increased nuclear NFkappaB DNA-binding activity. In the IL-1beta-treated group, COX-2 protein began to increase after 6 h; this response was time-dependent, with a significant increase until 24 h after IL-1beta stimulation. When NFkappaB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFkappaB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFkappaB is involved in the IL-1beta-induced COX-2 expression in the mesenchymal cells of human amnion.     

Aurintricarboxylic acid structure modifications lead to reduction of inhibitory properties against virulence factor YopH and higher cytotoxicity

Yersinia sp. bacteria owe their viability and pathogenic virulence to the YopH factor, which is a highly active bacterial protein tyrosine phosphatase. Inhibition of YopH phosphatase results in the lack of Yersinia sp. pathogenicity. We have previously described that aurintricarboxylic acid inhibits the activity of YopH at nanomolar concentrations and represents a unique mechanism of YopH inactivation due to a redox process. This work is a continuation of our previous studies. Here we show that modifications of the structure of aurintricarboxylic acid reduce the ability to inactivate YopH and lead to higher cytotoxicity. In the present paper we examine the inhibitory properties of aurintricarboxylic acid analogues, such as eriochrome cyanine R (ECR) and pararosaniline. Computational docking studies we report here indicate that ATA analogues are not precluded to bind in the YopH active site and in all obtained binding conformations ECR and pararosaniline bind to YopH active site. The free binding energy calculations show that ECR has a stronger binding affinity to YopH than pararosaniline, which was confirmed by experimental YopH enzymatic activity studies. We found that ATA analogues can reversibly reduce the enzymatic activity of YopH, but possess weaker inhibitory properties than ATA. The ATA analogues induced inactivation of YopH is probably due to oxidative mechanism, as pretreatment with catalase prevents from inhibition. We also found that ATA analogues significantly decrease the viability of macrophage cells, especially pararosaniline, while ATA reveals only slight effect on cell viability.     

Modulation of DNA binding of glucocorticoid receptor by aurintricarboxylic acid

Effects of aurintricarboxylic acid (ATA) were examined on the DNA binding properties of rat liver glucocorticoid-receptor complex. The DNA-cellulose binding capacity of the glucocorticoid-receptor complex was completely abolished by a pretreatment of receptor preparation with 0.1-0.5 mM ATA at 4 degrees C. The half-maximal inhibition (i.d.50) in the DNA binding of [3H]triamcinolone acetonide-receptor complex [( 3H]TARc) was observed at 130- and 40 microM ATA depending upon whether the inhibitor was added prior to or following the receptor activation. The entire DNA-cellulose bound [3H]TARc could be extracted in a concentration-dependent manner by incubation with 2-100 microns ATA. The [3H]TARc remained intact under the above conditions, the receptor in both control and ATA-treated preparations sedimented in the same region in salt-containing 5-20% sucrose gradients. The action of ATA appeared to be on the receptor and not on DNA-cellulose. The DNA-binding capacity of ATA-treated receptor preparations could be recovered upon exhaustive dialysis. The treatment with ATA did not appear to change the ionic behavior of heat activated GRc; the receptor in both control and the ATA-treated preparations showed similar elution profiles. Therefore, ATA appears to alter the binding to and dissociation of glucocorticoid-receptor complex from DNA. The use of ATA should offer a good chemical probe for analysis of the DNA binding domain(s) of the glucocorticoid receptor.     

The prostacyclin analogue treprostinil blocks NFkappaB nuclear translocation in human alveolar macrophages

Primary pulmonary hypertension (PPH) is characterized by increased pulmonary arterial pressure and vascular resistance. We and others have observed that inflammatory cytokines and infiltrates are present in the lung tissue, but the significance is uncertain. Treprostinil (TRE), a prostacyclin analogue with extended half-life and chemical stability, has shown promise in the treatment of PPH. We hypothesize that TRE might exert beneficial effects in PPH by antagonizing inflammatory cytokine production in the lung. Here we show that TRE dose-dependently inhibits inflammatory cytokine (tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and granulocyte macrophage colony-stimulating factor) secretion and gene expression by human alveolar macrophages. TRE blocks NFkappaB activation, but IkappaB-alpha phosphorylation and degradation are unaffected. Moreover, TRE does not affect the formation of the NFkappaB.DNA complex but blocks nuclear translocation of p65. These results are the first to illustrate the anti-cytokine actions of TRE in down-regulating NFkappaB, not through its inhibitory component or by direct binding but by blocking nuclear translocation. These data indicate that inflammatory mechanisms may be important in the pathogenesis of PPH and cytokine antagonism by blocking NFkappaB may contribute to the efficacy of TRE therapy in PPH.     

Glutathione supplementation potentiates hypoxic apoptosis by S-glutathionylation of p65-NFkappaB

In murine embryonic fibroblasts, N-acetyl-L-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFkappaB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFkappaB that are responsible for NFkappaB inactivation. In MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFkappaB DNA binding and NFkappaB transactivation by 2.6- and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFkappaB protein levels and p65-NFkappaB nuclear translocation during hypoxia. Pharmacological inhibition of the NFkappaB pathway also induced hypoxic apoptosis, indicating that the NFkappaB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after hypoxia and treatment with N-ethylmaleimide (thiol alkylating agent), dithiothreitol (disulfide reducing agent) was not able to increase binding of p65-NFkappaB to DNA, suggesting that most sulfhydryls in p65-NFkappaB protein were in reduced and activated forms after hypoxia, thereby being blocked by N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC, dithiothreitol increased p65-NFkappaB DNA binding. Glutaredoxin (GRx), which specifically catalyzes reduction of protein-SSG mixed disulfides, reversed inhibition of p65-NFkappaB DNA binding in extracts from cells treated with hypoxia plus NAC and restored NFkappaB activity. This finding indicated that p65-NFkappaB-SSG was formed in situ under hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFkappaB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFkappaB is most likely responsible for NAC-mediated NFkappaB inactivation and enhanced hypoxic apoptosis.     

Construction, expression, purification and functional analysis of recombinant NFkappaB p50/p65 heterodimer.

NFkappaB plays an important role in mediating the gene expression of numerous cellular processes such as growth, development, the inflammatory response and virus proliferation. The p50/p65 heterodimer is the most abundant form of the NFkappaB dimers and plays a more elaborate role in gene regulation. Biochemical research on p50/p65 NFkappaB has not benefited however from the availability of easily purified recombinant protein. We report two methods for the large scale expression and purification of recombinant NFkappaB p50/p65 heterodimer. The first utilizes a bacterial double expression vector which contains two ribosomal binding sites to facilitate the coexpression of the polypeptides in the p50/p65 NFkappaB heterodimer. The second method uses a mixed protein refolding strategy. Both methods yield crystallizable protein. Electrophoretic mobility shift assays confirm that the DNA binding affinity is independent of the method used to purify the protein. These methods will facilitate the numerous studies on various NFkappaB/Rel family members.     

Hyperosmotic stress activates p65/RelB NFkappaB in cultured cardiomyocytes with dichotomic actions on caspase activation and cell death

NFkappaB is a participant in the process whereby cells adapt to stress. We have evaluated the activation of NFkappaB pathway by hyperosmotic stress in cultured cardiomyocytes and its role in the activation of caspase and cell death. Exposure of cultured rat cardiomyocytes to hyperosmotic conditions induced phosphorylation of IKKalpha/beta as well as degradation of IkappaBalpha. All five members of the NFkappaB family were identified in cardiomyocytes. Analysis of the subcellular distribution of NFkappaB isoforms in response to hyperosmotic stress showed parallel migration of p65 and RelB from the cytosol to the nucleus. Measurement of the binding of NFkappaB to the consensus DNA kappaB-site binding by EMSA revealed an oscillatory profile with maximum binding 1, 2 and 6h after initiation of the hyperosmotic stress. Supershift analysis revealed that p65 and RelB (but not p50, p52 or cRel) were involved in the binding of NFkappaB to DNA. Hyperosmotic stress also resulted in activation of the NFkappaB-lux reporter gene, transient activation of caspases 9 and 3 and phosphatidylserine externalization. The effect on cell viability was not prevented by ZVAD (a general caspase inhibitor). Blockade of NFkappaB with AdIkappaBalpha, an IkappaBalpha dominant negative overexpressing adenovirus, prevented activation of caspase 9 (more than that caspase 3) but did not affect cell death in hyperosmotically stressed cardiomyocytes. We conclude that hyperosmotic stress activates p65 and RelB NFkappaB isoforms and NFkappaB mediates caspase 9 activation in cardiomyocytes. However cell death triggered by hyperosmotic stress was caspase- and NFkappaB-independent.     

TNFalpha induces NFkappaB/p50 in association with the growth and morphogenesis of normal and transformed rat mammary epithelial cells

In contrast to the cytotoxic or cytostatic effect of TNFalpha on many breast cancer cell lines, TNFalpha stimulates growth and morphogenesis of normal rat mammary epithelial cells (MEC). The present studies were carried out to determine whether there are intrinsic differences between normal and malignant MEC which may explain the differing responsiveness to TNFalpha. Freshly isolated rat MEC organoids from normal mammary gland or 1-methyl-1-nitrosourea-induced mammary tumors were treated with TNFalpha for 21 days. Unexpectedly, TNFalpha stimulated growth and morphogenesis of both normal and transformed MEC in primary culture, although in transformed cells its effects were delayed and the majority of the colonies were histologically abnormal, with multiple cell layers and no lumen. Since NFkappaB is a key mediator of TNFalpha action and has been implicated in carcinogenesis, the expression of the p50, p52, p65, and c-rel NFkappaB proteins in normal and transformed MEC was determined. Expression of p52 was significantly reduced in tumor cells, and p50 was absent, although its putative precursor, p105 was abundant. There were no changes in the levels of p65 or c-rel. TNFalpha induced a pronounced and sustained increase of a p50 homodimeric NFkappaB/DNA complex in both normal and transformed MEC. However, in transformed MEC, NFkappaB binding was initially undetectable but then increased in response to TNFalpha. Thus, NFkappaB expression and DNA binding activity are altered during mammary carcinogenesis. In addition, the significant increase in NFkappaB/p50 DNA-binding was temporally coincident with TNFalpha-induced growth and morphogenesis, suggesting that it may play a significant role in both normal development and carcinogenesis.     

RelA, p50 and inhibitor of kappa B alpha are elevated in human metastatic melanoma cells and respond aberrantly to ultraviolet light B.

Metastatic melanomas are typically resistant to radiation and chemotherapy. The underlying basis for this phenomenon may result in part from defects in apoptotic pathways. Nuclear factor kappa B (NFkappaB) has been shown to control apoptosis in many cell types and normally functions as an immediate stress response mechanism that is rigorously controlled by multiple inhibitory complexes. We have previously shown that NFkappaB binding is elevated in metastatic melanoma cells relative to normal melanocytes. In the current study, Western blot analysis showed that, compared with normal melanocytes, melanoma cell lines have higher nuclear levels of the NFkappaB subunits p50 (7-fold) and RelA (5-10-fold). In response to tumor necrosis factor-alpha (TNFalpha), both melanocytes and melanoma cells showed increased nuclear p50 and RelA levels, but levels in melanoma cells remained higher than in melanocytes. We also found that melanoma cells expressed higher cytoplasmic levels of RelA, p105/p50 and the inhibitory protein, inhibitor of kappa B alpha (IkappaBalpha) than melanocytes. To directly test whether RelA expression has an impact on melanoma cell survival, we used antisense RelA phosphorothioate oligonucleotides and found that melanoma cell viability was significantly decreased compared with untreated or control cultures. The constitutive activation of NFkappaB in metastatic melanoma cell cultures may, therefore, support an inappropriate cell survival pathway that can be therapeutically manipulated.     

Hyperosmotic stress-dependent NFkappaB activation is regulated by reactive oxygen species and IGF-1 in cultured cardiomyocytes

We have recently shown that hyperosmotic stress activates p65/RelB NFkappaB in cultured cardiomyocytes with dichotomic actions on caspase activation and cell death. It remains unexplored how NFkappaB is regulated in cultured rat cardiomyocytes exposed to hyperosmotic stress. We study here: (a) if hyperosmotic stress triggers reactive oxygen species (ROS) generation and in turn whether they regulate NFkappaB and (b) if insulin-like growth factor-1 (IGF-1) modulates ROS production and NFkappaB activation in hyperosmotically-stressed cardiomyocytes. The results showed that hyperosmotic stress generated ROS in cultured cardiac myocytes, in particular the hydroxyl and superoxide species, which were inhibited by N-acetylcysteine (NAC). Hyperosmotic stress-induced NFkappaB activation as determined by IkappaBalpha degradation and NFkappaB DNA binding. NFkappaB activation and procaspase-3 and -9 fragmentation were prevented by NAC and IGF-1. However, this growth factor did not decrease ROS generation induced by hyperosmotic stress, suggesting that its actions over NFkappaB and caspase activation may be due to modulation of events downstream of ROS generation. We conclude that hyperosmotic stress induces ROS, which in turn activates NFkappaB and caspases. IGF-1 prevents NFkappaB activation by a ROS-independent mechanism.     

Aurintricarboxylic Acid Prevents NMDA-Mediated Excitotoxicity: Evidence for Its Action as an NMDA Receptor Antagonist

Abstract: The effect of the endonuclease inhibitor aurintricarboxylic acid (ATA) versus NMDA-mediated delayed cell death was examined in an ex vivo chick retinal preparation. Transient exposure to 100 μM NMDA for 60 min followed by a 24-h recovery period resulted in a sevenfold increase in lactate dehydrogenase (LDH) release into the medium. ATA at 100 μM significantly reduced NMDA-mediated LDH release by 60%. In clarifying the mechanism of protection versus NMDA, ATA was found to inhibit several acute NMDA-mediated effects: ATA attenuated NMDA-mediated GABA release in a dose-dependent manner (IC₅₀= 29.5 μM ), prevented NMDA-stimulated cyclic GMP formation, and blocked NMDA-mediated ²²Na⁺ influx. These acute inhibitory effects of ATA were overcome by increasing the NMDA concentration, which suggested a competitive interaction between NMDA and ATA. In a binding assay using membranes prepared from adult rat forebrain, ATA displaced the competitive NMDA receptor ligand [³H]CGS 19755 with an IC₅₀ of 26.9 μM. Maximal displacement was 88% with 100 μM ATA. These studies demonstrate that ATA protected neurons from NMDA-mediated cell death upstream of endonuclease inhibition, i.e., by antagonizing NMDA receptor activity in a manner consistent with competitive antagonism.