Regulation Of Nuclear factor-κB Research Articles

Inhibitory effects of chelidonic acid on IL-6 production by blocking NF-κB and caspase-1 in HMC-1 cells

Chelidonic acid (CA) is a γ-pyrone which is contained in the rhizome of Chelidonium majus L. It has multiple pharmacological effects including those of a mild analgesic, an antimicrobial, an oncostatic and a central nervous system sedative, but the anti-inflammatory effect of CA and its molecular mechanisms are poorly understood. In this study, we investigated the regulatory mechanism of CA in mast cell-mediated inflammatory response by phorbol 12-myristate 13-acetate and calcium ionophore A23187. The results indicate that CA inhibits the production of interleukin-6 (IL-6) and the expression of IL-6 mRNA through the regulation of nuclear factor-κB. In addition, CA suppresses the activation and expression of caspase-1. These results provide new insights into the pharmacological actions of CA as a potential molecule for use in therapy in mast cell-mediated inflammatory diseases.

Sigma-1 Receptors Regulate Bcl-2 Expression by Reactive Oxygen Species-Dependent Transcriptional Regulation of Nuclear Factor κB

The expression of Bcl-2, the major antiapoptotic member of the Bcl-2 family, is under complex controls of several factors, including reactive oxygen species (ROS). The sigma-1 receptor (Sig-1R), which was recently identified as a novel molecular chaperone at the mitochondria-associated endoplasmic reticulum membrane (MAM), has been shown to exert robust cellular protective actions. However, mechanisms underlying the antiapoptotic action of the Sig-1R remain to be clarified. Here, we found that the Sig-1R promotes cellular survival by regulating the Bcl-2 expression in Chinese hamster ovary cells. Although both Sig-1Rs and Bcl-2 are highly enriched at the MAM, Sig-1Rs neither associate physically with Bcl-2 nor regulate stability of Bcl-2 proteins. However, Sig-1Rs tonically regulate the expression of Bcl-2 proteins. Knockdown of Sig-1Rs down-regulates whereas overexpression of Sig-1Rs up-regulates bcl-2 mRNA, indicating that the Sig-1R transcriptionally regulates the expression of Bcl-2. The effect of Sig-1R small interfering RNA down-regulating Bcl-2 was blocked by ROS scavengers and by the inhibitor of the ROS-inducible transcription factor nuclear factor kappaB (NF-kappaB). Knockdown of Sig-1Rs up-regulates p105, the precursor of NF-kappaB, while concomitantly decreasing inhibitor of nuclear factor-kappaBalpha. Sig-1R knockdown also accelerates the conversion of p105 to the active form p50. Lastly, we showed that knockdown of Sig-1Rs potentiates H(2)O(2)-induced apoptosis; the action is blocked by either the NF-kappaB inhibitor oridonin or overexpression of Bcl-2. Thus, these findings suggest that Sig-1Rs promote cell survival, at least in part, by transcriptionally regulating Bcl-2 expression via the ROS/NF-kappaB pathway.

Truncation of the Catalytic Domain of the Cylindromatosis Tumor Suppressor Impairs Lung Maturation

Cyld encodes a 956-amino acid deubiquitinating enzyme (CYLD), which is a negative regulator of nuclear factor κB and mitogen-activated protein kinase pathways. Mutations that truncate and inactivate the carboxyl-terminal deubiquitinating domain of CYLD underlie the development of skin appendage tumors in humans, whereas down-regulation of Cyld expression has been associated with the development of various types of human malignancies including lung cancer. To establish an animal model of human CYLD inactivation and characterize the biological role of CYLD in vivo, we generated mice carrying a homozygous deletion of Cyld exon 9 (CyldΔ9/Δ9 mice) using a conditional approach. Deletion of exon 9 would cause a carboxyl-terminal truncation of CYLD and inactivation of its deubiquitinating activity. In accordance with previous studies, fibroblasts from CyldΔ9/Δ9 embryos had hyperactive nuclear factor κB and c-Jun kinase pathways compared with control fibroblasts. CyldΔ9/Δ9 newborn mice were smaller than wild-type littermates with a short and kinky tail and nomajor developmental defects. However, CyldΔ9/Δ9 mice died shortly after birth from apparent respiratory dysfunction. Histological examination of E18.5 CyldΔ9/Δ9 lungs demonstrated an immature phenotype characterized by hyperplasic mesenchyme but apparently normal epithelial, smooth muscle. and endothelial structures. Our study identifies an important role of CYLD in lung maturation, which may underlie the development of many cases of lung cancer.

Poly(ADP-Ribose) Polymerase Inhibition Down-Regulates Expression of Metastasis-Related Genes in CT26 Colon Carcinoma Cells

Objectives: The current study was designed to test the hypothesis that inhibition of poly(ADP-ribose) polymerase in colorectal cancer mediates down-regulation of metastasis-related gene expression through the regulation of nuclear factor-κB (NF-κB) activity. Methods: Mouse colon carcinoma cells (CT26) were treated with and without the PARP inhibitor 5-aminoisoquinolin-1(2H)-one hydrochloride (5-AIQ). We investigated adhesion, migration and invasion of differently treated CT26 cells. In addition, the expression levels of PARP, NF-κB, integrin β₁, MMP-9 and MMP-2 as well as the activities of NF-κB, MMP-9 and MMP-2 were determined by Western blot, electrophoretic mobility gel shift assay and zymography, respectively. Results: Inhibition ofPARP attenuated the adhesion of CT26 cells to the extracellular matrix and their migration and invasion through Matrigel. In addition, the results of Western blot showed that the expression levels of PARP, NF-κB, integrin β₁, MMP-9 and MMP-2 were reduced in 5-AIQ-treated CT26 cells; the activities of NF-κB, MMP-9 and MMP-2 were also suppressed. Conclusions: Inhibition of PARP down-regulates the expression of metastasis-related genes in mouse colon carcinoma cells. This could be, at least in part, through the regulation of NF-κB activity, but the precise mechanisms of action remain to be elucidated.

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.

Menatetrenone, a Vitamin K2 Analogue, Inhibits Hepatocellular Carcinoma Cell Growth by Suppressing Cyclin D1 Expression through Inhibition of Nuclear Factor κB Activation

Menatetrenone, a vitamin K2 analogue, plays an important role in the production of blood coagulation factors. Menatetrenone has also bee shown to have antineoplastic effects against several cancer cell lines including hepatocellular carcinoma (HCC) cells. However, the mechanisms by which vitamin K2 inhibits HCC cell growth have not bee fully clarified, and we therefore investigated the molecular basis of vitamin K2-induced growth inhibition of HCC cells. HCC cells were treated with vitamin K2 and the expression of several growth-related genes including cyclin-dependent kinase inhibitors and cyclin D1 was examined at the mRNA and protein levels. A reporter gene assay of the cyclin D1 promoter was done under vitamin K2 treatment. The regulation of nuclear factor kappaB (NF-kappaB) activation was investigated by a NF-kappaB reporter gene assay, an electrophoretic mobility shift assay, a Western blot for phosphorylated IkappaB, and an in vitro kinase assay for IkappaB kinase (IKK). We also examined the effect of vitamin K2 on the growth of HCC cells transfected with p65 or cyclin D1. Vitamin K2 inhibited cyclin D1 mRNA and protein expression in a dose-dependent manner in the HCC cells. Vitamin K2 also suppressed the NF-kappaB binding site-dependent cyclin D1 promoter activity and suppressed the basal, 12-O-tetradecanoylphorbol-13-acetate (TPA)-, TNF-alpha-, and interleukin (IL)-1-induced activation of NF-kappaB binding and transactivation. Concomitant with the suppression of NF-kappaB activation, vitamin K2 also inhibited the phosphorylation and degradation of IkappaBalpha and suppressed IKK kinase activity. Moreover, HCC cells overexpressing cyclin D1 and p65 became resistant to vitamin K2 treatment. Vitamin K2 inhibits the growth of HCC cells via suppression of cyclin D1 expression through the IKK/IkappaB/NF-kappaB pathway and might therefore be useful for treatment of HCC.

Disease-associated mutations in CIAS1 induce cathepsin B–dependent rapid cell death of human THP-1 monocytic cells

AbstractMutations in the cold-induced autoinflammatory syndrome 1 (CIAS1) gene are associated with a spectrum of autoinflammatory diseases, including familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and chronic infantile neurologic, cutaneous, articular syndrome, also known as neonatal-onset multisystem inflammatory disease. CIAS1 encodes cryopyrin, a protein that localizes to the cytosol and functions as pattern recognition receptor. Cryopyrin also participates in nuclear factor-κB regulation and caspase-1–mediated maturation of interleukin 1β. In this study, we showed that disease-associated mutations in CIAS1 induced rapid cell death of THP-1 monocytic cells. The features of cell death, including 7-AAD staining, the presence of cellular edema, and early membrane damage resulting in lactate dehydrogenase (LDH) release, indicated that it was more likely to be necrosis than apoptosis, and was effectively blocked with the cathepsin B–specific inhibitor CA-074-Me. CA-074-Me also suppressed induced by disease-associated mutation lysosomal leakage and mitochondrial damage. In addition, R837, a recently identified activator of cryopyrin-associated inflammasomes, induced cell death in wild type CIAS1-transfected THP-1 cells. These results indicated that monocytes undergo rapid cell death in a cathepsin B–dependent manner upon activation of cryopyrin, which is also a specific phenomenon induced by disease-associated mutation of CIAS1.

Regulation of Nuclear Factor κB in the Hippocampus by Group I Metabotropic Glutamate Receptors

An increasing amount of evidence suggests that the family of nuclear factor kappaB (NF-kappaB) transcription factors plays an important role in synaptic plasticity and long-term memory formation. The present study investigated the regulation of NF-kappaB family members p50, p65/RelA, and c-Rel in the hippocampus in response to metabotropic glutamate receptor (mGluR) signaling. Activation of group I metabotropic glutamate receptors (GpI-mGluRs) with the agonist (S)-3,5-dihydroxyphenylglycine (DHPG) resulted in a time-dependent increase in DNA binding activity of p50, p65, and c-Rel in area CA1 of the hippocampus. An antagonist of mGluR5, 2-Methyl-6-(phenylethynyl)pyridine, inhibited the DHPG-induced activation of NF-kappaB, whereas an antagonist of mGluR1, (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid, did not. Using a series of inhibitors, we investigated the signaling pathways necessary for DHPG-induced activation of NF-kappaB and found that they included the phosphatidyl inositol 3-kinase, protein kinase C, mitogen-activated protein kinase kinase, and p38-mitogen-activated protein kinase pathways. To determine the functional significance of mGluR-induced regulation of NF-kappaB, we measured long-term depression (LTD) of Schaffer-collateral synapses in the hippocampus of c-Rel knock-out mice. Early phase LTD was normal in c-rel(-/-) mice. However, late-phase LTD (>90 min) was impaired in c-rel(-/-) mice. The observations of this deficit in hippocampal synaptic plasticity prompted us to further investigate long-term memory formation in c-rel(-/-) mice. c-rel(-/-) mice exhibited impaired performance in a long-term passive avoidance task, providing additional evidence for c-Rel in long-term memory formation. These results demonstrate that the NF-kappaB transcription factor family is regulated by GpI-mGluRs in the hippocampus and that the c-Rel transcription factor is necessary for long-term maintenance of LTD and formation of long-term memory.

Regulation of nuclear factor-κB (NF-κB) activity and apoptosis by estradiol in bovine granulosa cells

Although atresia of bovine follicles is associated with apoptosis of granulosa cells, the signals initiating this cell death have not been resolved. NF-κB has been implicated as an important regulator of genes controlling apoptosis, and previous studies indicate that estradiol may modulate NF-κB activation. We hypothesized that estradiol activates NF-κB, and thus, inhibits apoptosis in granulosa cells of dominant follicles. Dominant follicles were collected on Days 4, 6, and 8 of the first follicular wave following ovulation and assayed for NF-κB activity. Concentrations of estradiol in follicular fluid decreased on Day 6 compared to Day 4 of the wave. NF-κB activity in granulosa cells was lower on Day 8 compared to Days 4 and 6. In vitro, NF-κB activity and minimal incidence of apoptosis (<4%), as measured by Annexin V and TUNEL assays, were associated with production of estradiol. However, inhibition of NF-κB in vitro led to a suppression of apoptosis. These results indicate that follicular NF-κB activation is associated with estradiol production. However, whether NF-κB is playing a pro- or anti-apoptotic role in granulosa cells of the dominant bovine follicle remains to be elucidated.

Poly(ADP-Ribose) Polymerase Is Involved in the Development of Diabetic Retinopathy via Regulation of Nuclear Factor-κB

The current study investigated the role of poly(ADP-ribose) polymerase (PARP) in the development of diabetic retinopathy. Activity of PARP was increased in whole retina and in endothelial cells and pericytes of diabetic rats. Administration of PJ-34 (a potent PARP inhibitor) for 9 months to diabetic rats significantly inhibited the diabetes-induced death of retinal microvascular cells and the development of early lesions of diabetic retinopathy, including acellular capillaries and pericyte ghosts. To further investigate how PARP activation leads to cell death in diabetes, we investigated the possibility that PARP acts as a coactivator of nuclear factor-kappaB (NF-kappaB) in the retinal cells. In bovine retinal endothelial cells (BRECs), PARP interacted directly with both subunits of NF-kappaB (p50 and p65). More PARP was complexed to the p50 subunit in elevated glucose concentration (25 mmol/l) than at 5 mmol/l glucose. PJ-34 blocked the hyperglycemia-induced increase in NF-kappaB activation in BRECs. PJ-34 also inhibited diabetes-induced increase expression of intercellular adhesion molecule-1, a product of NF-kappaB-dependent transcription in retina, and subsequent leukostasis. Inhibition of PARP or NF-kappaB inhibited the hyperglycemia (25 mmol/l glucose)-induced cell death in retinal endothelial cells. Thus, PARP activation plays an important role in the diabetes-induced death of retinal capillary cells, at least in part via its regulation of NF-kappaB.

Viral and host cofactors facilitate HIV-1 replication in macrophages.

Human immunodeficiency virus type 1 (HIV-1) infection of CD4+ T lymphocytes leads to their progressive loss, whereas HIV-1-infected macrophages appear to resist HIV-1-mediated apoptotic death. The differential response of these two host-cell populations may be critical in the development of immunodeficiency and long-term persistence of the virus. Multiple contributing factors may favor the macrophage as a resilient host, not only supporting infection by HIV-1 but also promoting replication and persistence of this member of the lentivirus subfamily of primate retroviruses. An encounter between macrophages and R5 virus engages a signal cascade eventuating in transcriptional regulation of multiple genes including those associated with host defense, cell cycle, nuclear factor-kappaB regulation, and apoptosis. It is important that enhanced gene expression is transient, declining to near control levels, and during this quiescent state, the virus continues its life cycle unimpeded. However, when viral replication becomes prominent, an increase in host genes again occurs under the orchestration of viral gene products. This biphasic host response must fulfill the needs of the parasitic virus as viral replication activity occurs and leads to intracellular and cell surface-associated viral budding. Inroads into understanding how HIV-1 co-opts host factors to generate a permissive environment for viral replication and transmission to new viral hosts may provide opportunities for targeted interruption of this lethal process.

Regulation of intestinal nuclear factor-κB activity and E-selectin expression during sepsis: A role for peroxynitrite

Background & Aims: During sepsis, the production of both nitric oxide and superoxide are increased. Furthermore, NO and O2− may interact to produce peroxynitrite. The major aim of the present study was to assess the relative roles of NO, O2−, and ONOO− in the regulation of nuclear factor κB (NF-κB) activity and subsequent E-selectin expression during the early stages of sepsis. Methods: Mice were administered 5 μg lipopolysaccharide (LPS) intraperitoneally, and NF-κB activity and E-selectin expression in the small intestine were assessed 3 hours later. Results: In wild-type mice, increased levels of NF-κB in nuclear extracts were noted. By contrast, in both inducible nitric oxide synthase–deficient and transgenic Cu/Zn superoxide dismutase–overexpressing mice, NF-κB mobilization to the nucleus was diminished. Pretreatment with a ONOO− decomposition catalyst (5,10,15,20-tetrakis[4-sulfonatophenyl]porphyrinato iron [III] [FeTPPS]) resulted in a diminished impact of LPS on NF-κB activation. LPS increased vascular E-selectin expression in wild-type mice. E-selectin expression was diminished in inducible nitric oxide synthase–deficient mice after LPS challenge, but E-selectin expression remained elevated in both Cu/Zn superoxide dismutase transgenic mice and wild-type mice pretreated with FeTPPS. Conclusions: Our observations suggest that NO, O2−, and ONOO− production are all important mediators in the induction of NF-κB activity during endotoxemia and that, in vivo, E-selectin expression on endothelium may not always be associated with whole-organ NF-κB activation.GASTROENTEROLOGY 2003;124:118-128

Regulation of Nuclear Factor-κB by Corticotropin-Releasing Hormone in Mouse Thymocytes

CRH, a major mediator of the stress response, has been shown to exert potent immunomodulatory effects in vivo, through mechanisms that have not been elucidated yet. To determine the molecular pathways mediating the proinflammatory effects of peripheral CRH, we studied its role in the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor crucial for the regulation of a variety of inflammatory mediator genes. Our studies demonstrate that, in mouse thymocytes, CRH induces the NF-kappaB DNA-binding activity in a time- and dose-dependent manner, with parallel degradation of its inhibitor protein inhibitor of NF-kappaB. The effect of CRH is not inhibited by dexamethasone and is mediated by the protein kinase A and protein kinase C signaling pathways. In vivo, we show that CRH-deficient mice respond to lipopolysaccharide administration by reduced activation of thymus NF-kappaB, despite their significantly elevated proinflammatory cytokine and their low corticosterone levels. These findings suggest a putative molecular pathway mediating the proinflammatory effects of peripheral CRH through induction of the NF-kappaB DNA binding activity.

Expression and Regulation of Inducible IκB Kinase (IKK-i) in Human Fibroblast-like Synoviocytes

IκB kinase (IKK) plays a key role in the regulation of nuclear factor κB (NF-κB). We previously demonstrated the expression of two kinases, IKK1 and IKK2, in fibroblast-like synoviocytes (FLS) and determined their functional consequences for inflammatory gene expression in vitro and in vivo. Recently, a novel inducible IκB kinase has been described, namely, IKK-i or IKK-ϵ, which is functionally and structurally distinct from constitutively expressed IKK1 and IKK2. Therefore, we investigated the expression and regulation of this novel kinase in FLS from patients with rheumatoid arthritis and osteoarthritis. Interestingly, constitutive gene expression and protein expression were observed in all cell lines examined. TNFα stimulation for 24 h increased IKK-i expression 7.2 ± 1.8-fold in FLS (P < 0.02). IL-1 also significantly increased IKK-i gene expression. Time course experiments demonstrated that IKK-i gene expression increased within 3 h of TNFα stimulation and persisted for at least 24 h. Dose–response studies showed that as little as 1 ng/ml of TNFα increased IKK-i gene expression. Constitutive IKK-1 gene expression was also noted in rheumatoid arthritis, osteoarthritis, and normal synovium. This is the first report demonstrating constitutive expression and cytokine regulation of this novel kinase in primary human synovial cells.

Regulation of nuclear factor-κB, activator protein-1, and glutathione levels by tumor necrosis factor-α and dexamethasone in alveolar epithelial cells

The development of an oxidant/antioxidant imbalance in lung inflammation may activate redox-sensitive transcription factors such as nuclear factor- kappaB (NF-κB) and activator protein-1 (AP-1), which regulate the genes for proinflammatory mediators and protective antioxidant genes. GSH, a ubiquitous tripeptide thiol, is a vital intra- and extracellular protective antioxidant against oxidative stress, which plays a key role in the control of proinflammatory processes in the lungs. The rate-limiting enzyme in GSH synthesis is γ-glutamylcysteine synthetase (γ-GCS), which consists of a catalytic heavy and a regulatory light subunit. The promoter regions of the human γ-GCS subunits contain AP-1, NF-κB, and antioxidant response elements and are regulated by oxidants, growth factors, inflammatory cytokine tumor necrosis factor-α (TNF-α), and anti-inflammatory agent (dexamethasone) in lung cells. TNF-α depletes intracellular GSH, concomitant with an increase in oxidised glutathione levels in alveolar epithelial cells. TNF-α also induces the activation of NF-κB and AP-1 and the subsequent increase in γ-GCS heavy subunit transcription in these cells. Dexamethasone depleted both basal and TNF-α-stimulated GSH levels by down-regulating the γ-GCS-heavy subunit transcription via a mechanism involving AP-1 (c-Jun). The existence of this fine tuning between the redox GSH levels and the activation of transcription factors may determine the balance of transcription for proinflammatory and antioxidant γ-GCS genes in inflammation. More studies are required to understand the signalling mechanism of the redox regulation of NF-κB and AP-1 and gene transcription in inflammation. This could lead to the development of therapeutic strategies based on the pharmacological manipulation of the production of this important antioxidant in inflammation.

HIV-1 Vpr suppresses immune activation and apoptosis through regulation of nuclear factor kappa B.

The HIV-1 accessory gene product Vpr can influence viral pathogenesis by affecting viral replication as well as host cell transcription and proliferation. We have investigated the effects of Vpr on host cell activation and confirm that it influences cellular proliferation. However, we have also found that Vpr modulates T-cell receptor (TCR)-triggered apoptosis in a manner similar to that of glucocorticoids. In the absence of TCR-mediated activation, Vpr induces apoptosis whereas in its presence, Vpr interrupts the expected induction of apoptosis. This regulation of apoptosis is linked to Vpr suppression of NF-kappa B activity via the induction of I kappa B, an inhibitor of NF-kappa B. Further, Vpr suppresses expression of IL-2, IL-10, IL-12, TNF alpha and IL-4, all of which are NF-kappa B-dependent. The effects of Vpr could be reversed by RU486. Our finding that Vpr can regulate NF-kappa B supports the hypothesis that some aspects of viral pathogenesis are the consequence of cell dysregulation by Vpr.