The regulation of the UCH-L1 gene by transcription factor NF-κB in podocytes

In kidney, the ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) is involved in podocyte injury and proteinuria but details of the mechanism underlying its regulation are not known. Activation of NF-κB is thought to be the predominant risk factor for kidney disease; therefore, it is postulated that UCH-L1 may be one of the NF-κB target genes. In this study, we investigated the involvement of NF-κB activation in the regulation of UCH-L1 expression and the function of murine podocytes. Stimulation of podocytes with the cytokines TNF-α and IL-1β up-regulated UCH-L1 expression rapidly at the mRNA and protein levels and the NF-κB-specific inhibitor pyrrolidine dithiocarbamate resulted in down-regulation. NF-κB up-regulates UCH-L1 via binding the ? 300 bp and ? 109 bp sites of its promoter, which was confirmed by the electrophoretic mobility shift assay of DNA–nuclear protein binding. In the renal biopsy from lupus nephritis patients, the expressions of NF-κB and UCH-L1 increased in immunohistochestry staining and were positively correlated. Activation of NF-κB up-regulates UCH-L1 expression following changing of other podocytes molecules, such as nephrin and snail. These results suggest that activation of the NF-κB signaling pathway could be the major pathogenesis to up-regulate UCH-L1 in podocyte injury, followed by the turnover of other molecules, which might result in morphological changes and dysfunction of podocytes. This work help us to understand the effect of NF-κB on specific target molecules of podocytes, and suggest that targeting the NF-κB–UCH-L1 interaction could be a novel therapeutic strategy for the treatment of podocyte lesions and proteinuria.     

NF-κB in the regulation of epithelial homeostasis and inflammation

The IκB kinase/NF-κB signaling pathway has been implicated in the pathogenesis of several inflammatory diseases. Increased activation of NF-κB is often detected in both immune and non-immune cells in tissues affected by chronic inflammation, where it is believed to exert detrimental functions by inducing the expression of proinflammatory mediators that orchestrate and sustain the inflammatory response and cause tissue damage. Thus, increased NF-κB activation is considered an important pathogenic factor in many acute and chronic inflammatory disorders, raising hopes that NF-κB inhibitors could be effective for the treatment of inflammatory diseases. However, ample evidence has accumulated that NF-κB inhibition can also be harmful for the organism, and in some cases trigger the development of inflammation and disease. These findings suggested that NF-κB signaling has important functions for the maintenance of physiological immune homeostasis and for the prevention of inflammatory diseases in many tissues. This beneficial function of NF-κB has been predominantly observed in epithelial cells, indicating that NF-κB signaling has a particularly important role for the maintenance of immune homeostasis in epithelial tissues. It seems therefore that NF-κB displays two faces in chronic inflammation: on the one hand increased and sustained NF-κB activation induces inflammation and tissue damage, but on the other hand inhibition of NF-κB signaling can also disturb immune homeostasis, triggering inflammation and disease. Here, we discuss the mechanisms that control these apparently opposing functions of NF-κB signaling, focusing particularly on the role of NF-κB in the regulation of immune homeostasis and inflammation in the intestine and the skin.     

The role of calcium,NF-κB and NFAT in the regulation of CXCL8 and IL-6 expression in Jurkat T-cells

T-cells play an important role in host immunity against invading pathogens. Determining the underlying regulatory mechanisms will provide a better understanding of T-cell-derived immune responses. In this study, we have shown the differential regulation of IL-6 and CXCL8 by NF-κB and NFAT in Jurkat T-cells, in response to PMA, heat killed Escherichia coli and calcium. CXCL8 was closely associated with the activation pattern of NFAT, while IL-6 expression was associated with NF-κB. Furthermore, increasing the intracellular Ca²⁺ concentration by calcium ionophore treatment of the cells resulted in NFAT induction without affecting the NF-κB activity. Interestingly, NF-κB activation by heat killed E. coli, as well as CXCL8 and IL-6 expression was significantly suppressed following addition of the calcium ionophore. This indicates that calcium plays an important role in regulating protein trafficking and T-cell signalling, and the subsequent inflammatory gene expression infers an involvement of NFAT in CXCL8 regulation.Understanding these regulatory patterns provide clarification of conditions that involve altered intracellular signalling leading to T-cell-derived cytokine expression.     

Deubiquitinases in the regulation of NF-κB signaling

Nuclear factor-kappa B (NF-κB) is a critical regulator of multiple biological functions including innate and adaptive immunity and cell survival. Activation of NF-κB is tightly regulated to preclude chronic signaling that may lead to persistent inflammation and cancer. Ubiquitination of key signaling molecules by E3 ubiquitin ligases has emerged as an important regulatory mechanism for NF-κB signaling. Deubiquitinases (DUBs) counteract E3 ligases and therefore play a prominent role in the downregulation of NF-κB signaling and homeostasis. Understanding the mechanisms of NF-κB downregulation by specific DUBs such as A20 and CYLD may provide therapeutic opportunities for the treatment of chronic inflammatory diseases and cancer.     

Essential role of NF-κB and AP-1 transcription factors in TNF-α-induced TSLP expression in human airway smooth muscle cells

Human airway smooth muscle (HASM) cells are a rich source of inflammatory mediators that may propagate the airway inflammatory responses. Recent studies from our laboratory and others demonstrate that HASM cells express the proallergic cytokine thymic stromal lymphopoietin (TSLP) in vitro and in vivo. Compelling evidence from in vitro studies and animal models suggest that the TSLP is a critical factor sufficient and necessary to induce or maintain the allergic airway inflammation. Despite of an immense interest in pathophysiology of TSLP in allergic inflammation, the triggers and mechanisms of TSLP expression remain inadequately understood. In this study, we found that TNF-α upregulates the TSLP mRNA and induces high levels of TSLP protein release in primary human ASM cells. Interestingly, TNF-α induced the TSLP promoter activity (P < 0.05; n = 4) in HASM that was mediated by upstream NF-κB and activator protein-1 (AP-1) binding sites. Mutation in NF-κB and AP-1 binding sites completely abrogated the effect of TNF-α-mediated TSLP promoter activity and so did the expression of a dominant-negative mutant construct of IκB kinase. Furthermore, the peptide inhibitors of IκB kinase or NF-κB inhibited the TNF-α-induced TSLP protein release (P < 0.05; n = 3) in HASM. Collectively, our data suggest a novel important biological role for NF-κB pathway in TNF-α-induced TSLP expression in HASM and recommend this as a prime target for anti-inflammatory drugs.     

Enteroaggregative Escherichia coli infection induces IL-8 production via activation of mitogen-activated protein kinases and the transcription factors NF-κB and AP-1 in INT-407 cells

Multiple mucosal immune factors, such as TNF-α and IL-1β, are thought to be key mediators involved in inflammatory bowel disease. We evaluated the role of the pro-inflammatory cytokine TNF-α on nitric oxide synthase (NOS) expression in indomethacin-induced jejunoileitis in rats. Jejunoileitis was induced in rats with subcutaneous injections of indomethacin (7.5 mg/kg) 24 h apart for two consecutive days, and animals were randomized into four groups. Group 1 received only indomethacin. Group 2 was treated with a daily dose of phosphodiesterase (PDE) inhibitor (theophylline or pentoxifylline) by oral gavage for 2 days before and 4 days after indomethacin. Group 3 received a single dose of anti-TNF-α monoclonal antibody (TNF-Ab, IP) 30 min before indomethacin. Group 4 was treated with 1 h hyperbaric oxygenation (HBO₂) for 5 days after indomethacin. Rats were sacrificed at 12 h or 4 days after final indomethacin injection. PDE inhibitor, TNF-Ab, or HBO₂ treatment significantly decreased indomethacin-induced ulceration, myeloperoxidase activity, and disease activity index. Although indomethacin significantly increased serum TNF-α and nitrate/nitrite (NOx) concentrations above control values at 12 h, inducible NOS (iNOS) expression was detected only at day 4. Serum IL-1β levels did not change at 12 h but increased 4-fold after 4 days. Indomethacin had no effect on constitutive NOS. Treatment with PDE inhibitor, TNF-Ab, or HBO₂ significantly reduced serum/tissue TNF-α, IL-1β, NOx, and iNOS expression. Our data show TNF-α plays an early pro-inflammatory role in indomethacin-induced jejunoileitis. Additionally, down-regulation of NOx by PDE inhibitors, TNF-Ab, or HBO₂ suggests that TNF-α modulates iNOS expression.     

Antagonistic crosstalk between NF-κB and SIRT1 in the regulation of inflammation and metabolic disorders

Recent studies have indicated that the regulation of innate immunity and energy metabolism are connected together through an antagonistic crosstalk between NF-κB and SIRT1 signaling pathways. NF-κB signaling has a major role in innate immunity defense while SIRT1 regulates the oxidative respiration and cellular survival. However, NF-κB signaling can stimulate glycolytic energy flux during acute inflammation, whereas SIRT1 activation inhibits NF-κB signaling and enhances oxidative metabolism and the resolution of inflammation. SIRT1 inhibits NF-κB signaling directly by deacetylating the p65 subunit of NF-κB complex. SIRT1 stimulates oxidative energy production via the activation of AMPK, PPARα and PGC-1α and simultaneously, these factors inhibit NF-κB signaling and suppress inflammation. On the other hand, NF-κB signaling down-regulates SIRT1 activity through the expression of miR-34a, IFNγ, and reactive oxygen species. The inhibition of SIRT1 disrupts oxidative energy metabolism and stimulates the NF-κB-induced inflammatory responses present in many chronic metabolic and age-related diseases. We will examine the molecular mechanisms of the antagonistic signaling between NF-κB and SIRT1 and describe how this crosstalk controls inflammatory process and energy metabolism. In addition, we will discuss how disturbances in this signaling crosstalk induce the appearance of chronic inflammation in metabolic diseases.     

Mechanism of NF-κB signaling pathway and autophagy in the regulation of osteoblast differentiation

Objective: The objective of the present work was to investigate a possible mechanism of NF-κB signaling pathway and autophagy in the regulation of osteoblast differentiation, and provide experimental basis for the study of tooth eruption disorder.

Methods: Mouse osteoblast-like (MC3T3-E1) cells were inoculated with a cell density of 70%. According to the grouping experimental design, Western blot and monodansylcadaverine (MDC) detection were conducted after dosing for 24?h. The cells were divided into the following five groups: blank control group; 6.25?µg/mL SN50 group; 12.5?µg/mL SN50 group; 25?µg/mL SN50 group and 50?µg/mL SN50 group.

Results: Western blot analysis revealed that the expression of LC3 protein was present in the blank control group; 6.25?µg/mL SN50 group; 12.5?µg/mL SN50 group and 50?µg/mL SN50 group, with no significant differences among these groups. However, the expression of LC3 protein was significantly lower in the 25?µg/mL SN50 group. MDC detection showed that, in the blank control group; 6.25?µg/mL SN50 group; 12.5?µg/mL SN50 group and 50?µg/mL SN50 group, there was obvious green fluorescence in the cytoplasm of the osteoblasts. However, in the 25?µg/mL SN50 group, it was found that there were significantly fewer green fluorescent particles.

Conclusion: The osteoblast itself had a strong function of autophagy. The appropriate concentration of SN50 in blocking the NF-κB pathway of the osteoblast was associated with the obvious inhibition of autophagy. However, the relationship between NF-κB signaling pathway and autophagy in the process of tooth eruption requires further study.     

IKKγ (NEMO) is involved in the coordination of the AP-1 and NF-κB pathways

Tumor necrosis factor alpha (TNFα) activates the nuclear factor-kappaB (NF-κB) pathway in various cell types, leading to expression of cell survival and inflammatory proteins. One mechanism of cell survival brought about by NF-κB is the inhibition of Activator Protein-1 (AP-1), which when activated, could lead to cell death. However, TNFα can also induce the AP-1 pathway, and the mechanisms by which these two pathways are regulated in response to TNFα are poorly understood. We proposed that Inhibitor of κB Kinase gamma (IKKγ) (which is also known as NF-κB essential modulator, NEMO) plays a key role in integrating and coordinating these two pathways. Our results showed that IKKγ activates the AP-1 pathway, via a mechanism that is dependent on the first leucine zipper (LZ) domain of IKKγ, by interacting with two proteins of the AP-1 complex, c-Jun and c-Fos, and changing the phosphorylation status of c-Jun. Even though IKKγ is required for the activation of NF-κB, we found that it reduced the activity of NF-κB when it was overexpressed. In summary, we demonstrated that transfected IKKγ, while inhibiting the NF-κB pathway, directly interacts with the AP-1 proteins and activates the AP-1 pathway independent of its effects on NF-κB. Our results indicate that IKKγ regulates TNFα signaling by coordinating cell responses mediated by the AP-1 and NF-κB pathways. A. S. Shifera and J. M. Friedman contributed equally to this article. Marshall S. Horwitz—Deceased: This article is dedicated to his loving memory.     

The IκB kinase complex in NF-κB regulation and beyond

The IκB kinase (IKK) complex is the signal integration hub for NF-κB activation. Composed of two serine-threonine kinases (IKKα and IKKβ) and the regulatory subunit NEMO (also known as IKKγ), the IKK complex integrates signals from all NF-κB activating stimuli to catalyze the phosphorylation of various IκB and NF-κB proteins, as well as of other substrates. Since the discovery of the IKK complex components about 15 years ago, tremendous progress has been made in the understanding of the IKK architecture and its integration into signaling networks. In addition to the control of NF-κB, IKK subunits mediate the crosstalk with other pathways, thereby extending the complexity of their biological function. This review summarizes recent advances in IKK biology and focuses on emerging aspects of IKK structure, regulation and function.     

Rugulactone derivatives act as inhibitors of NF-κB activation and modulates the transcription of NF-κB dependent genes in MDA-MB-231cells

Rugulactone and its analogues were synthesized following Horners–Wadsworth–Emmons and ring-closing metathesis as the key reactions. A library of new rugulactone analogues were designed, synthesized and evaluated for their anticancer activity in breast cancer cells. All analogues have shown anti-proliferative activity, while some of them exhibited significant cytotoxicity. In assays related to cell-cycle distribution, these conjugates induced G1 cell-cycle arrest in MDA-MB-231 cells. The cell cycle arrest nature was further confirmed by examining the effect on Cyclin E and Cdk2 proteins that acts at G1-S phase transition. Immunocytochemistry assay revealed that these compounds inhibited nuclear translocation of NF-κB protein, thereby activation of NF-κB was inhibited. The expression of NF-κB target genes such as Cyclin D1 and Bcl-xL were severely affected. Apart from acting on NF-κB, these compounds also regulate class I Histone deacetylase proteins such as (HDAC-3 and 8) that have a crucial and regulatory role in cell-proliferation. Simultaneously, the apoptotic inducing nature of these compounds was confirmed by activation of PARP protein, a protein that plays a key role in DNA damage and repair pathways. Among all compounds of this series 3g is the most potent compound and can be used for further studies.     

Differential expression of NF-κB in mycobacteria infected THP-1 affects apoptosis

The present study was conducted to see the role of NF-κB in virulent (Mycobacterium tuberculosis H37Rv) and avirulent (M. tuberculosis H37Ra) mycobacterial infection in THP-1 cells. To inactivate NF-κB, pCMV-IκBαM dn containing THP-1 cell line was generated which showed marked increase in apoptosis with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Infected THP-1-IκBαM dn cells showed decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and enhanced TNF-α production. Increase in apoptosis of infected THP-1-IκBαM dn cells resulted in inhibition of intracellular mycobacterial growth. Differential NF-κB activation potential was observed with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Both the strains activated NF-κB after 4 h in THP-1 cells however after 48 h only M. tuberculosis H37Rv activated NF-κB which lead to up-regulation of bcl-2 family anti-apoptotic member, bfl-1/A1. Our results indicated that NF-κB activation may be a determinant factor for the success of virulent mycobacteria within macrophages.