RelA P65 Research Articles

Increased synovial tissue NF-kappa B1 expression at sites adjacent to the cartilage-pannus junction in rheumatoid arthritis.

To compare the expression of the Rel/NF-kappa B subunits, NF-kappa B1 (p50) and RelA (p65), in paired synovial tissue samples selected from sites adjacent to and remote from the cartilage-pannus junction (CPJ) in patients with inflammatory arthritis. Synovial tissue was selected at arthroscopy from sites adjacent to the CPJ and from the suprapatellar pouch of patients who were referred to an early arthritis clinic. Tissue samples from patients with osteoarthritis (OA) undergoing knee arthroplasty were also studied. Rel/NF-kappa B subunit activation and expression were measured by electrophoretic mobility shift assay and supershift analyses and by immunohistochemistry. Tissue samples were obtained from 10 patients with rheumatoid arthritis (RA), 7 with a seronegative arthropathy (SnA), and 6 with OA. Rel/NF-kappa B was abundantly expressed in all samples. In both RA and SnA synovial tissue, the absolute number of NF-kappa B1+ cells at the CPJ was significantly higher than at non-CPJ sites (P = 0.006 and P = 0.02, respectively). The proportion of cells expressing NF-kappa B1 was also significantly higher at the CPJ compared with non-CPJ sites (P = 0.003 in RA, P = 0.009 in SnA). The numbers of RelA+ cells were consistently lower throughout. In RA synovial tissue, but not in SnA synovial tissue, both the absolute number and the proportion of RelA+ cells were significantly higher at the CPJ than at non-CPJ sites (P = 0.003 and P = 0.01, respectively). In OA synovial tissue, the numbers of cells expressing NF-kappa B1 and RelA were similar to those observed at the non-CPJ sites in all inflammatory tissues studied. In this study of early inflammatory arthritis, expression of NF-kappa B1 in synovial tissue was highest at sites most likely to be associated with joint erosion. These observations are consistent with a critical role of NF-kappa B1 in joint destruction, and support the rationale for specific therapeutic inhibition of NF-kappa B in RA.

CD86 and β2-Adrenergic Receptor Signaling Pathways, Respectively, Increase Oct-2 and OCA-B Expression and Binding to the 3′-IgH Enhancer in B Cells

Stimulation of CD86 (formerly known as B7-2) and/or the beta2-adrenergic receptor on a CD40 ligand/interleukin-4-activated B cell increased the rate of mature IgG1 transcription. To identify the mechanism responsible for this effect, we determined whether CD86 and/or beta2-adrenergic receptor stimulation regulated transcription factor expression and binding to the 3'-IgH enhancer in vitro and in vivo. We showed that CD86 stimulation increased the nuclear localization of NF-kappaB1 (p50) and phosphorylated RelA (p65) and increased Oct-2 expression and binding to the 3'-IgH enhancer, in a protein kinase C-dependent manner. These effects were lost when CD86-deficient or NF-kappaB1-deficient B cells were used. CD86 stimulation also increased the level of IkappaB-alpha phosphorylation but in a protein kinase C-independent manner. Beta2-adrenergic receptor stimulation increased CREB phosphorylation, OCA-B expression, and OCA-B binding to the 3'-IgH enhancer in a protein kinase A-dependent manner, an effect lost when beta2-adrenergic receptor-deficient B cells were used. Also, the beta2-adrenergic receptor-induced increase in the level of mature IgG1 transcript was lost when OCA-B-deficient B cells were used. These data are the first to show that CD86 stimulation up-regulates the expression of the transcription factor Oct-2 in a protein kinase C- and NF-kappaB1-dependent manner, and that beta2-adrenergic receptor stimulation up-regulates the expression of the coactivator OCA-B in a protein kinase A-dependent manner to cooperate with Oct-2 binding to the 3'-IgH enhancer.

Context Matters

Signaling pathways initiated by inflammatory cytokines enable nuclear factor κB (NF-κB) to translocate to the nucleus so as to activate target genes transcriptionally. NF-κB--specifically the RelA subunit--has been associated with prosurvival responses mediated through the activation of antiapoptotic genes and NF-κB inhibition has been scrutinized for potential use in cancer therapy (see Preview by Miyamoto). NF-κB is also activated by cytotoxic stimuli; however, the effects of cytotoxic activators on NF-κB are incompletely understood. Noting reports of proapoptotic effects of NF-κB, Campbell et al. investigated the effects of NF-κB activation by ultraviolet light (UV-C) and DNA-damaging drugs used in chemotherapy. Treatment of U-2 OS human osteosarcoma cells with UV-C or with daunorubicin and doxorubicin (which inhibit topoisomerase II) stimulated DNA binding of a RelA-containing NF-κB heterodimer but inhibited activity of NF-κB-dependent gene reporters. In contrast, etoposide, which also inhibits topoisomerase II, stimulated NF-κB-dependent transcriptional activity. UV-C and daunorubicin decreased mRNA and protein levels from endogenous antiapoptotic genes regulated by NF-κB. Moreover, UV-C and daunorubicin inhibited NF-κB-dependent transcriptional activity elicited by tumor necrosis factor α. Experiments comparing wild-type mouse embryo fibroblasts with those from mice lacking RelA indicated that RelA was required for repression of Bcl-xL by UV-C and daunorubicin, as did experiments using RNA interference in U-2 OS cells. UV-C and daunorubicin increased RelA association with histone deacetylases and decreased histone H3 acetylation at the Bcl-xL promoter. Thus, the pro- or antisurvival effects of NF-κB appear to depend on how it is activated and (based on comparisons with previous research) may be critically dependent on cellular context. K. J. Campbell, S. Rocha, N. D. Perkins, Active repression of antiapoptotic gene expression by RelA(p65) NF-κB. Mol. Cell 13 , 853-865 (2004). [Online Journal] S. Miyamoto, RelA life and death decisions. Mol. Cell 13 , 763-764 (2004). [Online Journal]

Degradation of NF-kappa B in T cells by gangliosides expressed on renal cell carcinomas.

T cells from cancer patients are often functionally impaired, which imposes a barrier to effective immunotherapy. Most pronounced are the alterations characterizing tumor-infiltrating T cells, which in renal cell carcinomas includes defective NF-kappaB activation and a heightened sensitivity to apoptosis. Coculture experiments revealed that renal tumor cell lines induced a time-dependent decrease in RelA(p65) and p50 protein levels within both Jurkat T cells and peripheral blood T lymphocytes that coincided with the onset of apoptosis. The degradation of RelA/p50 is critical for SK-RC-45-induced apoptosis because overexpression of RelA in Jurkat cells protects against cell death. The loss of RelA/p50 coincided with a decrease in expression of the NF-kappaB regulated antiapoptotic protein Bcl-xL at both the protein and mRNA level. The disappearance of RelA/p50 protein was mediated by a caspase-dependent pathway because pretreatment of T lymphocytes with a pan caspase inhibitor before coculture with SK-RC-45 blocked RelA and p50 degradation. SK-RC-45 gangliosides appear to mediate this degradative pathway, as blocking ganglioside synthesis in SK-RC-45 cells with the glucosylceramide synthase inhibitor, PPPP, protected T cells from tumor cell-induced RelA degradation and apoptosis. The ability of the Bcl-2 transgene to protect Jurkat cells from RelA degradation, caspase activation, and apoptosis implicates the mitochondria in these SK-RC-45 ganglioside-mediated effects.

Active Repression of Antiapoptotic Gene Expression by RelA(p65) NF-κB

With the emerging role of NF-κB in cancer it is important that its responses to stimuli relevant to tumor progression and therapy are understood. Here, we demonstrate that NF-κB induced by cytotoxic stimuli, such as ultraviolet light (UV-C) and the chemotherapeutic drugs daunorubicin/doxorubicin, is functionally distinct to that seen with the inflammatory cytokine TNF and is an active repressor of antiapoptotic gene expression. Surprisingly, these effects are mediated by the RelA(p65) NF-κB subunit. Furthermore, UV-C and daunorubicin inhibit TNF-induced NF-κB transactivation, indicating that this is a dominant effect. Consistent with this, mechanistic studies reveal that UV-C and daunorubicin induce the association of RelA with histone deacetylases. RelA can therefore be both an activator and repressor of its target genes, dependent upon the manner in which it is induced. This has important implications for the role of NF-κB in tumorigenesis and the use of NF-κB inhibitors in cancer therapy.

12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits osteoclastogenesis by suppressing RANKL-induced NF-kappaB activation.

The mechanism by which TPA-induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW(264.7) cell culture system and assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL-induced NF-kappaB activation. The protein kinase C (PKC) pathway has been suggested to be an important regulator of osteoclastic bone resorption. The role of PKC in RANKL-induced osteoclastogenesis, however, is not clear. In this study, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, on osteoclastogenesis and studied its role in RANKL-induced signaling. RANKL-induced RAW(264.7) cell differentiation into osteoclast-like cells was used to assess the effect of TPA on osteoclastogenesis. Assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, protein kinase activity, and Western blotting were used to examine the effects of TPA on RANKL-induced NF-kappaB, c-Jun N-terminal kinase (JNK), and MEK/ERK and p38 signal transduction pathways. We found that TPA inhibited RANKL-induced RAW(264.7) cell differentiation into osteoclasts in a dose-dependent manner. Time course analysis showed that the inhibitory effect of TPA on RANKL-induced osteoclastogenesis occurs predominantly at an early stage of osteoclast differentiation. TPA alone had little effect on NF-kappaB activation in RAW(264.7) cells, but it suppresses the RANKL-induced NF-kappaB activation in a dose-dependent fashion. Interestingly, the suppressive effect of TPA on RANKL-induced NF-kappaB activation was prevented by a conventional PKC inhibitor, Go6976. Supershift studies revealed that the RANKL-induced DNA binding of NF-kappaB complexes consisted of C-Rel, NF-kappaB1 (p50), and RelA (p65). In addition, TPA induced the activation of JNK in RAW(264.7) cells but had little effect on RANKL-induced activation of JNK. TPA also inhibited RANKL-induced activation of ERK but had little effect on p38 activation. Given that NF-kappaB activation is obligatory for osteoclast differentiation, our studies imply that inhibition of osteoclastogenesis by TPA is, at least in part, caused by the suppression of RANKL-induced activation of NF-kappaB during an early stage of osteoclastogenesis. Selective modulation of RANKL signaling pathways by PKC activators may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption.

Regulation of Developing B Cell Survival by RelA-Containing NF-κB Complexes

Mice deficient in the RelA (p65) subunit of NF-kappaB die during embryonic development. Fetal liver (FL) hemopoietic precursors from these mice were used to generate RelA-deficient lymphocytes by adoptive transfer into lethally irradiated mature lymphocyte-deficient recombination-activating gene-1(-/-) mice. Strikingly, RelA(-/-) lymphocyte generation was greatly diminished compared with that of RelA(+/+) lymphocytes. The most dramatic reduction was noticed in the numbers of developing B cells, which were considerably increased when RelA(-/-) FL cells that were also TNFR1 deficient were used. The role of RelA was further investigated in FL-derived developing B cells in vitro. Our results show that RelA is a major component of constitutive and TNF-alpha-induced kappaB site-binding activity in developing B cells, and provide evidence for a direct role of TNF-alpha in killing RelA(-/-) B cells. The absence of RelA significantly reduced mRNA expression of the antiapoptotic genes cellular FLICE-inhibitory protein and Bcl-2. Retroviral transduction of RelA(-/-) B cells with either cFLIP or Bcl-2 significantly reduced TNF-alpha killing. Together, these results indicate that RelA plays a crucial role in regulating developing B cell survival by inhibiting TNF-alpha cytotoxicity.

Essential role of RelA Ser311 phosphorylation by zetaPKC in NF-kappaB transcriptional activation.

The activation of the transcription factor NF-kappaB is central to the control of the cellular response triggered by many stimuli. Once released from the inhibitory molecule IkappaB, NF-kappaB is translocated to the nucleus, and it has to be phosphorylated to activate transcription. In zeta protein kinase C (PKC)-deficient cells, NF-kappaB is transcriptionally inactive and the phosphorylation of the RelA subunit in response to tumor necrosis factor (TNF-alpha) is severely impaired. In vitro assays showed that zetaPKC directly phosphorylates RelA. Here we demonstrate that Ser311 accounts for zetaPKC phosphorylation of RelA and that this site is phosphorylated in vivo in response to TNF-alpha. Also, an inactivating mutation of that residue severely impairs RelA transcriptional activity, blocks its anti-apoptotic function and abrogates the interaction of RelA with the co-activator CBP as well as its recruitment, and that of RNA polymerase II (Pol II) with the interleukin-6 (IL-6) promoter. The interaction of endogenous CBP with endogenous RelA is inhibited in zetaPKC-/- cells, as well as the binding of Pol II to the IL-6 promoter. These results demonstrate the mechanism whereby zetaPKC regulates NF-kappaB activation in vivo.

Regulation of airway epithelial cell NF-kappa B-dependent gene expression by protein kinase C delta.

Airway epithelial cells synthesize proinflammatory molecules such as IL-8, GM-CSF, RANTES, and ICAM-1, the expression of which is increased in the airways of patients with asthma. We investigated the regulation of these NF-kappa B-dependent genes by the novel protein kinase C (PKC) isoform PKC delta in 16HBE14o- human airway epithelial cells, focusing on IL-8 expression. Transient transfection with the constitutively active catalytic subunit of PKC delta (PKC delta-CAT), and treatment with bryostatin 1, an activator of PKC delta, each increased transcription from the IL-8 promoter, whereas overexpression of PKC epsilon had minor effects. Expression of a dominant negative PKC delta mutant (PKC delta-KR) or pretreatment of cells with rottlerin, a chemical PKC delta inhibitor, attenuated TNF-alpha- and phorbol ester-induced transcription from the IL-8 promoter. Bryostatin 1 treatment increased IL-8 protein abundance in primary airway epithelial cells. Selective activation of PKC delta by bryostatin also activated NF-kappa B, as evidenced by p65 RelA and p50 NF-kappa B1 binding to DNA, NF-kappa B trans-activation, and I kappa B degradation. The sufficiency of PKC delta to induce NF-kappa B nuclear translocation and binding to DNA was confirmed in a 16HBE14o- cell line inducibly expressing PKC delta-CAT under the tet-off system. Deletion of the NF-kappa B response element severely attenuated PKC delta-induced IL-8 promoter activity. Finally, PKC delta-CAT induced transcription from the GM-CSF, RANTES, and ICAM-1 promoters. Together these data suggest that PKC delta plays a key role in the regulation of airway epithelial cell NF-kappa B-dependent gene expression.

Mitogenic and antiapoptotic role of constitutive NF-kappaB/Rel activity in pancreatic cancer.

The transcription factor NF-kappaB/Rel was found to be constitutively activated in human pancreatic cancer. RelA is present in the nucleus in primary human pancreatic cancer samples as well as in pancreatic cancer cell lines. NF-kappaB/Rel-binding activity consists of NF-kappaB1(p50) and RelA(p65). Constitutive NF-kappaB/Rel activity correlates with IkappaB kinase (IKK) activity and can be blocked by dominant negative mutants of IKKbeta and to a lesser extent by IKKalpha. Constitutive NF-kappaB/Rel activity and the transactivation potential of RelA(p65) can be inhibited by dominant negative mutant Ras, the PI3 kinase inhibitor LY294002, or dominant negative mutant Akt kinase. Transfection of a dominant negative mutant epidermal growth factor receptor (EGF-R), EGF-R kinase inhibitor Tyrphostin and LY 294002 blocked IKK activity and NF-kappaB-dependent transcription. Inhibition of constitutive IKK or NF-kappaB/Rel activity increased the number of apoptotic cells. Stably expressing a nondegradable form of IkappaBalpha inhibited anchorage-dependent and -independent proliferation in MiaPaCa2 and Panc1 cells. Our data demonstrate that an EGF-R/Ras/PI3 kinase/Akt/IKK-dependent pathway contributes to constitutive NF-kappaB/Rel activity in pancreatic cancer. Inhibition of NF-kappaB/Rel activity reveals a mitogenic and antiapoptotic role for NF-kappaB/Rel in pancreatic cancer.

The p38 mitogen‐activated protein kinase regulates interleukin‐1β‐induced IL‐8 expression via an effect on the IL‐8 promoter in intestinal epithelial cells

Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB.

Differential induction of nuclear factor-kappaB and activator protein-1 activity after CD40 ligation is associated with primary human hepatocyte apoptosis or intrahepatic endothelial cell proliferation.

CD40, a tumor necrosis factor receptor superfamily member, is up-regulated on intraheptatic endothelial cells (IHEC) and epithelial cells during inflammatory liver disease, and there is evidence that the functional outcome of CD40 ligation differs between cell types. Ligation of CD40 on cholangiocytes or hepatocytes results in induction of Fas-mediated apoptosis, whereas ligation of IHEC CD40 leads to enhanced chemokine secretion and adhesion molecule expression. We now report that differential activation of two transcription factors, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), in primary human hepatocytes or IHEC, is associated with and may explain, in part, the different responses of these cell types to CD40 ligation. CD40 ligation induced a rise in NF-kappaB activity in hepatocytes,which peaked at 2 h and returned to baseline by 24 h; however, IHEC CD40 ligation resulted in a sustained up-regulation of NF-kappaB (>24 h). In hepatocytes, CD40 ligation led to sustained up-regulation of AP-1 activity >24 h associated with increased protein levels of RelA (p65), c-Jun, and c-Fos, whereas no induction of AP-1 activity was observed in IHECs. Analysis of mitogen-activated protein kinase phosphorylation (phospho-extracellular signal-regulated kinase 1/2 and phospho-c-Jun NH(2)-terminal kinase 1/2) and expression of inhibitor kappaBalpha were entirely consistent, and thus confirmed the profiles of NF-kappaB and AP-1 signaling and the effects of the selective inhibitors assessed using electrophoretic mobility shift assay or Western immunoblotting. CD40 ligation resulted in induction of apoptosis in hepatocytes after 24 h, but on IHECs, CD40 ligation resulted in proliferation. Inhibition of (CD40-mediated) NF-kappaB activation prevented IHEC proliferation and led to induction of apoptosis. Selective extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase inhibitors reduced levels of apoptosis in (CD40-stimulated) hepatocytes by approximately 50%. We conclude that differential activation of these two transcription factors in response to CD40 ligation is associated with differences in cell fate. Transient activation of NF-kappaB and sustained AP-1 activation is associated with apoptosis in hepatocytes, whereas prolonged NF-kappaB activation and a lack of AP-1 activation in IHECs result in proliferation.

Regulation of RelA (p65) Function by the Large Subunit of Replication Factor C

The RelA (p65) subunit of NF-kappaB is an important regulator of inflammation, proliferation, and apoptosis. We have discovered that the large subunit, p140, of replication factor C (RFC) can function as a regulator of RelA. RFC is a clamp loader, facilitating the addition and removal of proliferating-cell nuclear antigen from DNA during replication and repair but can also interact directly with the retinoblastoma tumor suppressor protein and the transcription factor C/EBPalpha. We find that RFC (p140) interacts with RelA both in vitro and in vivo and stimulates RelA transactivation. In contrast, coexpression of fragments of RFC (p140) that mediate the interaction with RelA results in transcriptional inhibition. The significance of this regulation was confirmed by using short interfering RNA oligonucleotides targeted to RFC (p140). Down regulation of endogenous RFC (p140) inhibits expression from a chromosomally integrated reporter plasmid induced by endogenous, TNF-alpha-activated NF-kappaB. Dominant negative fragments of RFC (p140) also cooperate with overexpressed RelA to induce cell death. Interestingly, RFC (p140) also interacts with the tumor suppressor p53. Taken together, these observations suggest that, in addition to its previously described function in DNA replication and repair, RFC (p140) has an important role as a regulator of transcription and NF-kappaB activity.

The Transcriptional Coactivator CREB-binding Protein Cooperates with STAT1 and NF-κB for Synergistic Transcriptional Activation of the CXC Ligand 9/Monokine Induced by Interferon-γ Gene

Signal transducers and activators of transcription 1 (STAT1) and NF-kappaB cooperatively regulate the expression of many inflammatory genes. In the present study, we demonstrate that the transcriptional coactivator CREB-binding protein (CBP) mediated the STAT1/NF-kappaB synergy for transcription of the gene for CXC ligand 9 (CXCL9), an interferon-gamma (IFN-gamma)-inducible chemokine. Reporter gene analysis showed that expression of CBP potentiated IFN-gamma and tumor necrosis factor (TNFalpha)-induced promoter activity and that the CBP-mediated synergy depended upon STAT1- and NF-kappaB-binding sites in the promoter. Experiments with CBP mutants indicated that the N-terminal and C-terminal regions were necessary for the transcriptional synergy, although the histone acetyltransferase activity of CBP was dispensable. A co-immunoprecipitation assay demonstrated that STAT1 and NF-kappaB RelA (p65) simultaneously associated with CBP in vivo. Furthermore, chromatin immunoprecipitation revealed that, although costimulation with IFN-gamma and TNFalpha did not cooperatively enhance the levels of acetylated histones, it did result in increased recruitment of STAT1, CBP, and RNA polymerase II at the promoter region of the CXCL 9 gene. Together, these results demonstrate that the STAT1/NF-kappaB-dependent transcriptional synergy could result from the enhanced recruitment of RNA polymerase II complex to the promoter via simultaneous interaction of CBP with STAT1 and NF-kappaB.

Mechanism of action of a dominant negative c-jun mutant in inhibiting activator protein-1 activation.

The dominant negative c-jun TAM-67 has been shown to inhibit tumor promotion induced by 12-O-tetradecanoylphorbol-13-acetate and okadaic acid (OA). To better understand this phenomenon, we investigated the mechanism of action of TAM-67 in response to OA. To identify the mechanism of action, we used a 6xHis-tagged TAM-67 as well as chimeric constructs of TAM-67 that either cannot bind DNA or cannot heterodimerize with wild-type transcription factors. The results of these studies indicated that TAM-67 acts by blocking or squelching. The results of elecrophoretic mobility-shift assays showed that TAM-67 must act by squelching in response to OA, as TAM-67 cannot be found in DNA-binding complexes. We then identified some of the proteins with which TAM-67 interacts. They include all members of the jun and fos families as well as the cAMP response element binding protein, activating transcription factor-1, activating transcription factor-2, and RelA (p65). Thus, we have shown that TAM-67 squelches the induction of activating transcription factor-1 transactivation in response to OA and that TAM-67 is capable of interacting with proteins that control transactivation by binding to the 12-O-tetradecanoylphorbol-13-acetate response element, cAMP response element and nuclear factor-kappaB sites.

Solution structure and design of dithiophosphate backbone aptamers targeting transcription factor NF-κB

A variety of monothio- and dithiosubstituted duplex aptamers targeting NF-κB have been synthesized and designed. The specificity and affinity of the dithioate aptamers of p50 and RelA(p65) NF-κB homodimers was determined by gel shift experiments. The NMR solution structures for several unmodified and dithioate backbone modified 14-base paired duplex aptamers have been determined by a hybrid, complete matrix (MORASS)/restrained molecular dynamics method. Structural perturbations of the dithioate substitutions support our hypothesis that the dithioate binds cations less tightly than phosphoryl groups. This increases the electrostatic repulsion across the B-form narrow minor groove and enlarges the minor groove, similar to that found in A-form duplexes. Structural analysis of modeled aptamer complexes with NF-κB homo- and heterodimers suggests that the dithioate backbone substitution can increase the aptamer’s relative affinity to basic groups in proteins such as NF-κB by helping to “strip” the cations from the aptamer backbone.

A novel form of the RelA nuclear factor kappaB subunit is induced by and forms a complex with the proto-oncogene c-Myc.

Members of both Myc and nuclear factor kappaB (NF-kappaB) families of transcription factors are found overexpressed or inappropriately activated in many forms of human cancer. Furthermore, NF-kappaB can induce c-Myc gene expression, suggesting that the activities of these factors are functionally linked. We have discovered that both c-Myc and v-Myc can induce a previously undescribed, truncated form of the RelA(p65) NF-kappaB subunit, RelA(p37). RelA(p37) encodes the N-terminal DNA binding and dimerization domain of RelA(p65) and would be expected to function as a trans-dominant negative inhibitor of NF-kappaB. Surprisingly, we found that RelA(p37) no longer binds to kappaB elements. This result is explained, however, by the observation that RelA(p37), but not RelA(p65), forms a high-molecular-mass complex with c-Myc. These results demonstrate a previously unknown functional and physical interaction between RelA and c-Myc with many significant implications for our understanding of the role that both proteins play in the molecular events underlying tumourigenesis.

Inhibition of glutathione-related enzymes augments LPS-mediated cytokine biosynthesis: involvement of an IκB/NF-κB-sensitive pathway in the alveolar epithelium

The regulation of lipopolysaccharide (LPS)-mediated pro-inflammatory cytokine biosynthesis by reduction–oxidation (redox)-sensitive enzymes involved in maintaining intracellular glutathione homeostasis was investigated in fetal alveolar type II epithelial cells (fATII). Inhibition of glutathione-oxidized disulfide reductase, which recycles GSSG→2GSH, by the action of 1,3- bis-(2-chloroethyl)-1-nitrosourea (BCNU) augmented LPS-dependent secretion of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α. BCNU increased [GSSG] concentration at the expense of [GSH], thereby favoring oxidation equilibrium. Inhibition of γ-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of GSH, by the action of l-buthionine-( S, R)-sulfoximine (BSO), potentiated LPS-induced IL-1β, IL-6 and TNF-α production. Similar to BCNU, BSO depleted [GSH] and induced the accumulation of [GSSG]. BCNU and BSO reduced LPS-mediated phosphorylation of inhibitory-κB (IκB-α), allowing its cytosolic accumulation. This effect was associated with the inhibition of the nuclear translocation of selective nuclear factor (NF)-κB subunits: NF-κB 1 (p50), RelA (p65), RelB (p68) and c-Rel (p75), but not NF-κB 2 (p52). BCNU and BSO reduced LPS-induced NF-κB activation as determined by the electrophoretic mobility shift DNA-binding assay. Analytical analysis of the effect of modulating the dynamic redox ratio ([GSH]+[GSSG])/[GSSG] revealed a novel role for GSSG as a disulfhydryl compound which mediates an inhibitory effect on NF-κB activation. It is concluded that selective modulation of redox-sensitive enzymes has an immunopharmacological potential in regulating pro-inflammatory cytokines and that the IκB-α/NF-κB pathway is redox-sensitive and differentially involved in mediating redox-dependent regulation of cytokine signaling.

Combinatorial selection and binding of phosphorothioate aptamers targeting human NF-kappa B RelA(p65) and p50.

Previously, we reported the in vitro combinatorial selection of phosphorothioate aptamers or "thioaptamers" targeting the transcription factor NF-IL6. Using the same approach and purified recombinant human NF-kappa B proteins RelA(p65) and p50, duplex thioaptamers have been selected that demonstrate high-affinity, competitive binding with the duplex 22-mer binding site, Ig kappa B. Binding energetics of RelA(p65) and p50 homodimers were studied using a quantitative electrophoretic mobility shift assay or EMSA. As a reference system for competitive aptamer binding, the duplex 22-mer phosphoryl binding site known as Ig kappa was determined to bind each p65 and p50 homodimer with a 1:1 stoichiometry and with affinities, determined by global analysis, K(d) = 4.8 +/- 0.2 nM for p65 and K(d) = 0.8 +/- 0.2 nM for p50. A global analysis tool for competitive NF-kappa B/Ig kappa binding was developed and utilized to measure the affinity of thioaptamers selected by both p65 and p50. The competition results indicate that the thioaptamers bind and compete for the same NF-kappa B site as the known promoter element Ig kappa B (K(d) = 78.9 +/- 1.9 nM for a p65-selected aptamer and 19.6 +/- 1.3 nM for a p50-selected thioaptamer). Qualitative gel shift binding experiments with p50 also demonstrate that the nature of enhanced affinity and specificity can be attributed to the presence of sulfur. Collectively, these results demonstrate the feasibility of the thioaptamer in vitro combinatorial selection technology as a method for producing specific, high-affinity ligands to proteins.

APC0576, a novel inhibitor of NF-κB-dependent gene activation, prevents pro-inflammatory cytokine-induced chemokine production in human endothelial cells

Endothelial cells participate in the inflammatory and immune reactions. Endothelial cell activation is a recurrent phenomenon linked to the pathogenesis of diverse human diseases, such as acute and chronic inflammation and cardiovascular disorders. Pro-inflammatory cytokines (e.g., IL-1, TNF) are well-known activators of endothelial cells, since they strongly induce production of chemokines (e.g., IL-8, MCP-1) and cell adhesion molecules, resulting in an activation of inflammatory transcription factors such as NF-κB. We have established a cell-based reporter assay for the NF-κB-dependent gene activation in HUVEC. Using this assay system, we have identified a novel synthetic small molecule, APC0576, 5-((( S)-2,2-dimethylcyclopropanecarbonyl)amino)-2-(4-((( S)-2,2-dimethylcyclopropanecarbonyl)amino)phenoxy)pyridine, as an inhibitor of IL-1-induced NF-κB-dependent gene activation without any adverse effects on the cell viability. APC0576 represses the IL-1-induced release of chemokines (e.g., IL-8, MCP-1) in HUVEC. This inhibitory effect occurred at the level of mRNA expression. Despite having a strong inhibitory effect on the NF-κB-dependent transcriptional activation, APC0576 does not inhibit the IL-1-induced DNA binding of NF-κB, degradation of I-κB-α, or phosphorylation of RelA (p65). Although its molecular mechanism of action is not yet clear, APC0576 is a promising therapeutic candidate for diverse diseases involved in the pathogenic endothelial activation.