RelA P65 Research Articles

Epigallocatechin-3-Gallate, a Histone Acetyltransferase Inhibitor, Inhibits EBV-Induced B Lymphocyte Transformation via Suppression of RelA Acetylation

Because the p300/CBP-mediated hyperacetylation of RelA (p65) is critical for nuclear factor-kappaB (NF-kappaB) activation, the attenuation of p65 acetylation is a potential molecular target for the prevention of chronic inflammation. During our ongoing screening study to identify natural compounds with histone acetyltransferase inhibitor (HATi) activity, we identified epigallocatechin-3-gallate (EGCG) as a novel HATi with global specificity for the majority of HAT enzymes but with no activity toward epigenetic enzymes including HDAC, SIRT1, and HMTase. At a dose of 100 micromol/L, EGCG abrogates p300-induced p65 acetylation in vitro and in vivo, increases the level of cytosolic IkappaBalpha, and suppresses tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. We also showed that EGCG prevents TNFalpha-induced p65 translocation to the nucleus, confirming that hyperacetylation is critical for NF-kappaB translocation as well as activity. Furthermore, EGCG treatment inhibited the acetylation of p65 and the expression of NF-kappaB target genes in response to diverse stimuli. Finally, EGCG reduced the binding of p300 to the promoter region of interleukin-6 gene with an increased recruitment of HDAC3, which highlights the importance of the balance between HATs and histone deacetylases in the NF-kappaB-mediated inflammatory signaling pathway. Importantly, EGCG at 50 micromol/L dose completely blocks EBV infection-induced cytokine expression and subsequently the EBV-induced B lymphocyte transformation. These results show the crucial role of acetylation in the development of inflammatory-related diseases.

Decrease in RelA phosphorylation by inhibiting protein kinase A induces cell death in NF-κB-expressing and drug-resistant tumor cells

The RelA (p65) is a subunit of nuclear transcription factor kappa B (NF-κB) and actively participates in expression of NF-κB-dependent genes involved in inflammation and tumorigenesis. Hence, the regulation of p65 is an important strategy to regulate those responses. In this study, we provide data that the dichlorophenyl derivative of 1,2,4-thiadiazolidine (known as P3-25) induced cell death in NF-κB-expressing and doxorubicin-resistant cells. P3-25 inhibited NF-κB DNA binding activity partially, but inhibited NF-κB-dependent genes expression completely. It inhibited phosphorylation of Rel A (p65) by inhibiting activity of protein kinase A (PKA). The PKA inhibition was independent of adenylate cyclase activity or cAMP level. The PKA activity decreased due to inhibition of catalytic subunit of PKA. P3-25 inhibited almost 80% PKA activity at 100nM concentration, having an IC50 at 10.5nM. P3-25 potentiated different chemotherapeutic agents-mediated cell death. Our results suggest that P3-25 inhibits PKA activity followed by decreased phosphorylation of p65 and transcriptional activity of NF-κB thereby decreasing antiapoptotic proteins resulting in induction of apoptosis in NF-κB-expressing and doxorubicin-resistant cells. The study might help to understand the mechanism of P3-25-mediated apoptosis and to design it as new chemotherapeutic drug for tumor therapy.

The DEAD‐box RNA helicase DDX1 interacts with RelA and enhances nuclear factor kappaB‐mediated transcription

DEAD-box RNA helicases constitute the largest family of RNA helicases and are involved in many aspects of RNA metabolism. In this study, we identified RelA (p65), a subunit of nuclear factor-kappaB (NF-kappaB), as a cellular co-factor of DEAD-box RNA helicase DDX1, through mammalian two hybrid system and co-immunoprecipitation assay. Additionally, confocal microscopy and chromatin immunoprecipitation assays confirmed this interaction. In NF-kappaB dependent reporter gene assay, DDX1 acted as a co-activator to enhance NF-kappaB-mediated transcription activation. The functional domains involved were mapped to the carboxy terminal transactivation domain of RelA and the amino terminal ATPase/helicase domain of DDX1. The DDX1 trans-dominant negative mutant lacking ATP-dependent RNA helicase activity lost it transcriptional inducer activity. Moreover, depletion of endogenous DDX1 by specific small interfering RNAs significantly reduced NF-kappaB-dependent transcription. Taken together, the results suggest that DDX1 may play an important role in NF-kappaB-mediated transactivation, and revelation of this regulatory pathway may help to explore the novel mechanisms for regulating NF-kappaB transcriptional activity.

Biological Significance of Constitutive NF-κB Activity Levels in Human Myeloma Cell Lines

To elucidate the mechanism of constitutive NF-κB activity in human myeloma cell lines, constitutive expression of all the NF-κB family, RelA (p65), c-Rel, RelB, NF-κB1 (p105/p50) and NF-κB2 (p100/p52), genes in different human myeloma cell lines, such as IL-6-independent (AMO1, NOP2), IL-6-dependent (ILKM2, ILKM8) and IL-6-responsive cell line (U266), were first checked by RT-PCR and western blot analysis. In the electrophoretic mobility shift assay (EMSA) for κB main consensus, total DNA binding activity levels for NF-κB were higher in IL-6-dependent, IL-6-responsive and IL-6-independent human myeloma cell lines respectively. Supershift analysis with anti-NF-κB family antibodies and immunoprecipitation followed by western blotting, confirmed that p50/p50, p50/RelB and p50/p52 as the principal player responsible for constitutive activation of NF-κB in IL-6-independent cell lines. However, beside these major dimers, p50/p65 dimer was also involved in the constitutive NF-κB activation in IL-6-dependent and IL-6-responsive cell lines. Moreover, on the basis of microarray data in human myeloma cell lines, we confirmed that the expression levels of NF-κB target genes, such as NFKB2, RELB, NFKBIA, MALT1 and CD54, showed correlation with the level of DNA binding activity for NF-κB. Therefore, these data suggest that the difference of total DNA-binding activities for NF-κB partly depended on the different p50/p65 activity among the IL-6-independent, IL-6-dependent and IL-6-responsive human myeloma cell lines, and further studies in the different NF-κB-dimers activity levels might contribute to the clarification of the regulation of NF-κB target genes expression in human myeloma cell lines.

A global transcriptional view of apoptosis in human T-cell activation

BackgroundT-cell activation is an essential step of immune response. The process of proper T-cell activation is strictly monitored and regulated by apoptosis signaling. Yet, regulation of apoptosis, an integral and crucial facet during the process of T-cell activation, is not well understood.MethodsIn this study, a Gene-Ontology driven global gene expression analysis coupled with protein abundance and activity assays identified genes and pathways associated with regulation of apoptosis in primary human CD3+ T cells and separately CD4+ and CD8+ T cells.ResultsWe identified significantly regulated apoptotic genes in several protein families, such as BCL2 proteins, CASPASE proteins, and TNF receptors, and detailed their transcriptional kinetics during the T-cell activation process. Transcriptional patterns of a few select genes (BCL2A1, BBC3 and CASP3) were validated at the protein level. Many of these apoptotic genes are involved in NF-κB signaling pathway, including TNFRSF10A, TNFRSF10B, TRAF4, TRAF1, TRAF3, and TRAF6. Upregulation of NF-κB and IκB family genes (REL, RELA, and RELB, NFKBIA, NFKBIE and NFKB1) at 48 to 96 hours, supported by the increase of phosphorylated RELA (p65), suggests that the involvement of the NF-κB complex in the process of T-cell proliferation is not only regulated at the protein level but also at the transcriptional level. Examination of genes involved in MAP kinase signalling pathway, important in apoptosis, suggests an induction of p38 and ERK1 cascades in T-cell proliferation (at 48 to 96 hours), which was explored using phosphorylation assays for p38 (MAPK14) and ERK1 (MAPK3). An immediate and short-lived increase of AP-1 activity measured by DNA-binding activity suggests a rapid and transient activation of p38 and/or JNK cascades upon T-cell activation.ConclusionThis comparative genome-scale, transcriptional analysis of T-cell activation in the CD4+ and CD8+ subsets and the mixed CD3+ population identified many apoptosis genes not previously identified in the context of T-cell activation. Furthermore, it provided a comprehensive temporal analysis of the transcriptional program of apoptosis associated with T-cell activation.

Dynein Light Chain LC8 Negatively Regulates NF-κB through the Redox-dependent Interaction with IκBα

Redox regulation of nuclear factor kappaB (NF-kappaB) has been described, but the molecular mechanism underlying such regulation has remained unclear. We recently showed that a novel disulfide reductase, TRP14, inhibits tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation, and we identified the dynein light chain LC8, which interacts with the NF-kappaB inhibitor IkappaBalpha, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-kappaB activation is redox-dependently regulated through LC8. LC8 inhibited TNFalpha-induced NF-kappaB activation in HeLa cells by interacting with IkappaBalpha and thereby preventing its phosphorylation by IkappaB kinase (IKK), without affecting the activity of IKK itself. TNFalpha induced the production of reactive oxygen species, which oxidized LC8 to a homodimer linked by the reversible formation of a disulfide bond between the Cys(2) residues of each subunit and thereby resulted in its dissociation from IkappaBalpha. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of IkappaBalpha by TNFalpha stimulation. In addition LC8 inhibited NF-kappaB activation by other stimuli including interleukin-1beta and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds IkappaBalpha in a redox-dependent manner and thereby prevents its phosphorylation by IKK. TRP14 contributes to this inhibitory activity by maintaining LC8 in a reduced state.

Regulation of the RelA (p65) transactivation domain

The RelA (p65) NF-kappaB (nuclear factor kappaB) subunit contains an extremely active C-terminal transcriptional activation domain, required for its cellular function. In the present article, we review our knowledge of this domain, its modifications and its known interacting proteins. Moreover, we discuss how analysis of its evolutionary conservation reveals distinct subdomains and conserved residues that might give insights into its regulation and function.

Constitutive Production of NF-κB2 p52 Is Not Tumorigenic but Predisposes Mice to Inflammatory Autoimmune Disease by Repressing Bim Expression

Normal development of the immune system requires regulated processing of NF-kappaB2 p100 to p52, which activates NF-kappaB2 signaling. Constitutive production of p52 has been suggested as a major mechanism underlying lymphomagenesis induced by NF-kappaB2 mutations, which occur recurrently in a variety of human lymphoid malignancies. To test the hypothesis, we generated transgenic mice with targeted expression of p52 in lymphocytes. In contrast to their counterparts expressing the tumor-derived NF-kappaB2 mutant p80HT, which develop predominantly B cell tumors, p52 transgenic mice are not prone to lymphomagenesis. However, they are predisposed to inflammatory autoimmune disease characterized by multiorgan infiltration of activated lymphocytes, high levels of autoantibodies in the serum, and immune complex glomerulonephritis. p52, but not p80HT, represses Bim expression, leading to defects in apoptotic processes critical for elimination of autoreactive lymphocytes and control of immune response. These findings reveal distinct signaling pathways for actions of NF-kappaB2 mutants and p52 and suggest a causal role for sustained NF-kappaB2 activation in the pathogenesis of autoimmunity.

A novel form of NF‐κB is induced by Leishmania infection: Involvement in macrophage gene expression

Leishmania spp. are obligate intracellular parasites that inhabit the phagolysosomes of macrophages. Manipulation of host cell signaling pathways and gene expression by Leishmania is critical for Leishmania's survival and resultant pathology. Here, we show that infection of macrophages with Leishmania promastigotes in vitro causes specific cleavage of the NF-kappaB p65 RelA subunit. Cleavage occurs in the cytoplasm and is dependent on the Leishmania protease gp63. The resulting fragment, p35 RelA, migrates to the nucleus, where it binds DNA as a heterodimer with NF-kappaB p50. Importantly, induction of chemokine gene expression (MIP-2/CXCL2, MCP-1/CCL2, MIP-1alpha/CCL3, MIP-1beta/CCL4) by Leishmania is NF-kappaB dependent, which implies that p35 RelA/p50 dimers are able to activate transcription, despite the absence of a recognized transcriptional transactivation domain. NF-kappaB cleavage was observed following infection with a range of pathogenic species, including L. donovani, L. major, L. mexicana, and L. (Viannia) braziliensis, but not the non-pathogenic L. tarentolae or treatment with IFN-gamma. These results indicate a novel mechanism by which a pathogen can subvert a macrophage's regulatory pathways to alter NF-kappaB activity.

Gastrin-Mediated Interleukin-8 and Cyclooxygenase-2 Gene Expression: Differential Transcriptional and Posttranscriptional Mechanisms

Gastrin induces the expression of cyclooxygenase (COX)-2 and interleukin (IL)-8; however, the mechanism(s), especially in gastric epithelial cells, is not well understood. Here, we have determined the intracellular mechanisms mediating gastrin-dependent gene expression. AGS-E human gastric cancer cell line stably expressing cholecystokinin-2 receptor was treated with amidated gastrin-17. Real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were performed to determine COX-2 and IL-8 expression and Akt, Erk, and p38 phosphorylation. Gene promoter activity was determined by luciferase assay. Electrophoretic mobility shift assay analysis was performed for nuclear factor kappaB (NF-kappaB) and activator protein-1 activity. RNA stability was determined after actinomycin D treatment. HuR localization was determined by immunocytochemistry. Gastrin induced COX-2 and IL-8 expression in AGS-E cells, which was inhibited by phosphatidylinositol 3' kinase (PI3K) and p38 inhibitors. Gastrin-mediated Akt activation was observed to be downstream of p38. IL-8 expression was dependent on COX-2-mediated prostaglandin E(2) synthesis. In the presence of an NF-kappaB inhibitor MG132, IL-8 transcription was inhibited, but not that of COX-2. This was confirmed after knockdown of the p65 RelA subunit of NF-kappaB. Further studies showed that COX-2 gene transcription is regulated by activator protein-1. Gastrin increased the stability of both COX-2 and IL-8 messenger RNA (mRNA) in a p38-dependent manner, the half-life increasing from 31 minutes to 8 hours and approximately 4 hours, respectively. Gastrin, through p38 activity, also enhanced HuR expression, nucleocytoplasmic translocation, and enhanced COX-2 mRNA binding. Gastrin differentially induces COX-2 and IL-8 expression at the transcriptional and posttranscriptional levels by PI3K and p38 mitogen-activated protein kinase pathways, respectively.

Interactions between Bortezomib and Romidepsin and Belinostat in Chronic Lymphocytic Leukemia Cells

The goal of this study was to characterize interactions between the proteasome inhibitor bortezomib and the histone deacetylase (HDAC) inhibitors (HDACI) romidepsin or belinostat in chronic lymphocytic leukemia (CLL) cells. Primary and cultured (JVM-3 and MEC-2) CLL cells were exposed to agents alone or in combination, after which cell death was determined by 7-aminoactinomycin D staining/flow cytometry. Acetylation of target proteins, activation of caspase cascades, and expression of apoptosis-regulatory proteins were monitored by Western blot analysis. Nuclear factor-kappaB (NF-kappaB) activity was determined by luciferase reporter assay. Cells were transiently transfected with wild-type and acetylation site-mutated (inactive) RelA(p65) (e.g., K221R, K310R, or K281/221/310R) and assessed for HDACI sensitivity. Combined exposure to very low concentrations of romidepsin or belinostat (i.e., low nanomolar and submicromolar, respectively) in combination with low nanomolar concentrations of bortezomib synergistically induced cell death in primary and cultured CLL cells. These events were likely associated with prevention of HDACI-mediated RelA acetylation and NF-kappaB activation by bortezomib, down-regulation of antiapoptotic proteins (i.e., Bcl-xL, Mcl-1, and XIAP), as well as up-regulation of the proapoptotic protein Bim, resulting in activation of caspase cascade. Finally, CLL cells transfected with inactive RelA displayed a significant increase in HDACI lethality. Coadministration of the clinically relevant HDACIs romidepsin or belinostat with bortezomib synergistically induces cell death in CLL cells, likely through mechanisms involving, among other factors, NF-kappaB inactivation and perturbation in the expression of proapoptotic and antiapoptotic proteins. A strategy combining HDAC with proteasome inhibition warrants further attention in CLL.

The proapoptotic effects of sulindac, sulindac sulfone and indomethacin are mediated by nucleolar translocation of the RelA(p65) subunit of NF-κB

Understanding the mechanisms that underlie the antitumour activity of non-steroidal anti-inflammatory drugs (NSAIDs) against colorectal cancer will allow the development of more effective and specific chemopreventative agents. Modulation of the NF-kappaB pathway has been implicated as a key effector of the antitumour effect of aspirin, but the effects of non-aspirin NSAIDs on this pathway have yet to be fully defined. Here, we demonstrate that sulindac, sulindac sulfone and indomethacin activate the NF-kappaB pathway in colorectal cancer cells, as determined by western blot analysis of cytoplasmic levels of IkappaBalpha and immunocytochemical analysis of nuclear NF-kappaB/RelA. Furthermore, we show that all of these NSAIDs induce nucleolar translocation of the RelA subunit of NF-kappaB. Using RelA deleted for the previously described nucleolar localization signal, we demonstrate that this response is causally involved in the apoptotic effects of these agents. Finally, we demonstrate that NSAID-mediated nucleolar translocation of RelA is associated with downregulation of NF-kappaB-driven transcription and of the NF-kappaB target gene, ICAM-1. These data identify nucleolar translocation of RelA and the associated repression of the NF-kappaB-driven transcription as a central molecular mechanism of NSAID-mediated growth inhibition and apoptosis. As well as providing new understanding of the molecular determinants of RelA function, these findings also have relevance to the development of novel chemotherapeutic and chemopreventative agents.

Hypo-phosphorylation leads to nuclear retention of NF-κB p65 due to impaired IκBα gene synthesis

Subcellular localization guided by IκBα is crucial for regulation of nuclear factor-κB function. Here, we show that p65 Rel homology domain phosphorylation mutants are transported into the nucleus after IκBα degradation, but as a consequence of lower IκBα levels their relocation to the cytosol is blocked. We demonstrate that phosphorylation of residues S205, S276, and S281 of p65 is not required for interaction between p65 and IκBα, but is pivotal for regulating cellular IκBα levels by positively affecting gene synthesis. Our findings indicate that reduction of phosphorylation leads to nuclear retention of p65, which might be partly responsible for altered transcriptional behavior of p65 serine mutants.

A cell cycle regulatory network controlling NF-kappaB subunit activity and function.

Aberrantly active NF-kappaB complexes can contribute to tumorigenesis by regulating genes that promote the growth and survival of cancer cells. We have investigated NF-kappaB during the cell cycle and find that its ability to regulate the G1-phase expression of key proto-oncogenes is subject to regulation by the integrated activity of IkappaB kinase (IKK)alpha, IKKbeta, Akt and Chk1. The coordinated binding of NF-kappaB subunits to the Cyclin D1, c-Myc and Skp2 promoters is dynamic with distinct changes in promoter occupancy and RelA(p65) phosphorylation occurring through G1, S and G2 phases, concomitant with a switch from coactivator to corepressor recruitment. Akt activity is required for IKK-dependent phosphorylation of NF-kappaB subunits in G1 and G2 phases, where Chk1 is inactive. However, in S-phase, Akt is inactivated, while Chk1 phosphorylates RelA and associates with IKKalpha, inhibiting the processing of the p100 (NF-kappaB2) subunit, which also plays a critical role in the regulation of these genes. These data reveal a complex regulatory network integrating NF-kappaB with the DNA-replication checkpoint and the expression of critical regulators of cell proliferation.

Hsp72 induces inflammation and regulates cytokine production in airway epithelium through a TLR4- and NF-kappaB-dependent mechanism.

Heat shock proteins are generally regarded as intracellular proteins acting as molecular chaperones; however, Hsp72 is also detected in the extracellular compartment. Hsp72 has been identified in the bronchoalveolar lavage fluid (BALF) of patients with acute lung injury. To address whether Hsp72 directly activated airway epithelium, human bronchial epithelial cells (16HBE14o-) were treated with recombinant Hsp72. Hsp72 induced a dose-dependent increase in IL-8 expression, which was inhibited by the NF-kappaB inhibitor parthenolide. Hsp72 induced activation of NF-kappaB, as evidenced by NF-kappaB trans-activation and by p65 RelA and p50 NF-kappaB1 binding to DNA. Endotoxin contamination of the Hsp72 preparation was not responsible for these effects. Next, BALB/c mice were challenged with a single intratracheal inhalation of Hsp72 and killed 4 h later. Hsp72 induced significant up-regulation of KC, TNF-alpha, neutrophil recruitment, and myeloperoxidase in the BALF. A similar challenge with Hsp72 in TLR4 mutant mice did not stimulate the inflammatory response, stressing the importance of TLR4 in Hsp72-mediated lung inflammation. Last, cultured mouse tracheal epithelial cells (MTEC) from BALB/c and TLR4 mutant and wild-type mice were treated ex vivo with Hsp72. Hsp72 induced a significant increase in KC expression from BALB/c and wild-type MTEC in an NF-kappaB-dependent manner; however, TLR4 mutant MTEC had minimal cytokine release. Taken together, these data suggest that Hsp72 is released and biologically active in the BALF and can regulate airway epithelial cell cytokine expression in a TLR4 and NF-kappaB-dependent mechanism.

Glutathione Depletion Down-regulates Tumor Necrosis Factor α-induced NF-κB Activity via IκB Kinase-dependent and -independent Mechanisms

Reduced glutathione (GSH) plays a crucial role in hepatocyte function, and GSH depletion by diethyl maleate was shown previously to inhibit expression of NF-kappaB target genes induced by tumor necrosis factor alpha (TNFalpha) and sensitize primary cultured mouse hepatocytes to TNF-mediated apoptotic killing. Here we demonstrate in the same system that GSH depletion down-regulates TNF-induced NF-kappaB transactivation via two mechanisms, depending on the extent of the depletion. With moderate GSH depletion (approximately 50%), the down-regulation is IkappaB kinase (IKK)-independent and likely acts on NF-kappaB transcriptional activity because TNF-induced IKK activation, IkappaBalpha phosphorylation and degradation, NF-kappaB nuclear translocation, NF-kappaB DNA binding in vitro, and NF-kappaB subunit RelA(p65) recruitment to kappaB sites of target gene promoters all appear unaltered. On the other hand, with profound GSH depletion (approximately 80%), the down-regulation also is IKK-dependent, and a timeline is established linking the inhibition of polyubiquitination of receptor-interacting protein 1 in TNF receptor 1 complex to partial blockage of IKK activation, IkappaBalpha phosphorylation and degradation, and NF-kappaB nuclear translocation. Of note, pretreatment with antioxidant trolox protects against the inhibitory effect of profound GSH depletion on IKK activation and NF-kappaB nuclear translocation but fails to restore expression of NF-kappaB target genes, revealing both IKK-dependent and -independent inhibition. These findings provide new insights into the complex effects of oxidative stress and redox perturbations on the NF-kappaB pathway.

Genome-wide Mapping of RELA(p65) Binding Identifies E2F1 as a Transcriptional Activator Recruited by NF-κB upon TLR4 Activation

NF-kappaB is a key mediator of inflammation. Here, we mapped the genome-wide loci bound by the RELA subunit of NF-kappaB in lipopolysaccharide (LPS)-stimulated human monocytic cells, and together with global gene expression profiling, found an overrepresentation of the E2F1-binding motif among RELA-bound loci associated with NF-kappaB target genes. Knockdown of endogenous E2F1 impaired the LPS inducibility of the proinflammatory cytokines CCL3(MIP-1alpha), IL23A(p19), TNF-alpha, and IL1-beta. Upon LPS stimulation, E2F1 is rapidly recruited to the promoters of these genes along with p50/RELA heterodimer via a mechanism that is dependent on NF-kappaB activation. Together with the observation that E2F1 physically interacts with p50/RELA in LPS-stimulated cells, our findings suggest that NF-kappaB recruits E2F1 to fully activate the transcription of NF-kappaB target genes. Global gene expression profiling subsequently revealed a spectrum of NF-kappaB target genes that are positively regulated by E2F1, further demonstrating the critical role of E2F1 in the Toll-like receptor 4 pathway.

Comparative analysis of host-cell signalling mechanisms activated in response to infection with Rickettsia conorii and Rickettsia typhi

The Gram-negative intracellular bacteria Rickettsia conorii and Rickettsia typhi are the aetiological agents of Mediterranean spotted fever and endemic typhus, respectively, in humans. Infection of endothelial cells (ECs) lining vessel walls, and the resultant vascular inflammation and haemostatic alterations are salient pathogenetic features of both of these rickettsial diseases. An important consideration, however, is that dramatic differences in the intracellular motility and accumulation patterns for spotted fever versus typhus group rickettsiae have been documented, suggesting the possibility of unique and potentially different interactions with host cells. This study characterized and compared R. conorii- and R. typhi-mediated effects on cultured human ECs. The DNA-binding activity of nuclear transcription factor-kappaB (NF-kappaB) and the phosphorylation status of stress-activated p38 kinase were determined as indicators of NF-kappaB and p38 activation. R. conorii infection resulted in a biphasic activation of NF-kappaB, with an early increase in DNA-binding activity at 3 h, followed by a later peak at 24 h. The activated NF-kappaB species were composed mainly of RelA p65-p50 heterodimers and p50 homodimers. R. typhi infection of ECs resulted in only early activation of NF-kappaB at 3 h, composed primarily of p65-p50 heterodimers. Whilst R. conorii infection induced increased phosphorylation of p38 kinase (threefold mean induction) with the maximal response at 3 h, a considerably less-intense response peaking at about 6 h post-infection was found with R. typhi. Furthermore, mRNA expression of the chemokines interleukin (IL)-8 and monocyte chemoattractant protein-1 in ECs infected with either Rickettsia species was higher than the corresponding controls, but there were distinct differences in the secretion patterns for IL-8, suggesting the possibility of involvement of post-transcriptional control mechanisms or differences in the release from intracellular storage sites. Thus, the intensity and kinetics of host-cell responses triggered by spotted fever and typhus species exhibit distinct variations that could subsequently lead to differences in the extent of endothelial activation and inflammation and serve as important determinants of pathogenesis.

Proenkephalin assists stress-activated apoptosis through transcriptional repression of NF-κB- and p53-regulated gene targets

The respective pro- and antiapoptotic functions of the transcription factors p53 and nuclear factor kappaB (NF-kappaB), and their potential impact on tumorigenesis and response to tumor therapy are well recognized. The capacity of the RelA(p65) subunit of NF-kappaB to specify a pro-apoptotic outcome in response to some stimuli is less well recognized, but needs to be understood if rational manipulation of the NF-kappaB pathway is to be deployed in cancer therapy. In this report, we provide evidence that the growth-responsive nuclear protein, proenkephalin (Penk), is required, in part, for apoptosis induction, in response to activation or overexpression of p53 and RelA(p65). We describe UV-C-inducible physical associations between endogenous Penk and p53 and RelA(p65) in mammalian cell lines. Depletion of Penk by RNA interference (RNAi) substantially preserves viable cell number following exposure to UV-C irradiation or hydrogen peroxide and confers transient protection in cells exposed to the genotoxin etoposide. In virally transformed and human tumor cell lines, overexpression of nuclear Penk with overabundant or activated p53, or RelA(p65) even in the absence of p53, enhances apoptosis to the point of synergy. We have further shown that Penk depletion by RNAi substantially derepresses transcription of a range of antiapoptotic gene targets previously implicated in repression-mediated apoptosis induction by NF-kappaB and p53. Physical association of endogenous Penk with the transcriptional co-repressor histone deacetylase suggests that it may be a component of a transcriptional repression complex that contributes to a pro-apoptotic outcome, following activation of the NF-kappaB and p53 pathways, and could therefore help to facilitate an antitumor response to a broad range of agents.

Nuclear factor-κB1: Regulation and function

The transcription factor NF-κB is a critical regulator of many cellular processes including cell survival and inflammation. NF-κB functions as a hetero- or homo-dimer which can be formed from five NF-κB subunits, NF-κB1 (p50 and its precursor p105), NF-κB2 (p52 and its precursor p100), RelA (p65), RelB and c-Rel. The most studied dimer is p50:p65, which is activated by the classical pathway and usually promotes gene expression. Activation of p50:p65 is linked with cell survival and promoting inflammation. This review provides a detailed overview of the structure, synthesis and function of the lesser characterised NF-κB subunit; NF-κB1 (p105 and p50). The diverse interactions of NF-κB1 with co-activators, co-repressors and other signaling networks that influence NF-κB1 gene expression are discussed. Finally the anti-inflammatory actions of NF-κB1 signaling will be assessed and the crucial need to design novel therapeutic drugs which exploit and amplify the anti-inflammatory actions of p50 will be explored.