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.     

Matrix metalloproteinase-2: Mechanism and regulation of NF-κB-mediated activation and its role in cell motility and ECM-invasion

Matrix metalloproteinases belong to a family of enzymes that degrade the extracellular matrix (ECM) components and play an important role in tissue repair, tumor invasion, and metastasis. ECM proteins, cytokines, and certain other factors regulate MMP activity. OPN, an ECM protein, has been found to be overexpressed in various cancers, and it has been shown to correlate with the metastatic potential. Although such reports indicate that OPN plays an important role in the ability of tumor cells to survive and metastasize to secondary sites, the mechanism by which OPN regulates these processes is yet to be understood. In this study we report that native purified human OPN can induce cell migration and ECM invasion. Increased invasiveness and migration correlates with enhanced expression and activation of MMP-2. Our study provides evidence showing that OPN increases gelatinolytic activity by inducing MT1-MMP expression via activation of the NF-B pathway. Suppression of MMP-2 by ASMMP-2 reduces the OPN-induced cell migration and ECM invasion. Curcumin blocks OPN-induced MT1-MMP expression and pro-MMP-2 activation. Curcumin, a known anti-inflammatory and anticarcinogenic compound, suppresses OPN-induced cell migration, invasion and induces apoptotic morphology in OPN-treated cells. The mechanism by which curcumin suppresses the OPN-induced effects has also been delineated. Curcumin inhibits MT1-MMP gene expression by blocking signals leading to IKK activation. This in turn inhibits IB phosphorylation and NF-B activation. Published in 2004.     

The role of PKC isoforms in the inhibition of NF-κB activation by vitamin K2 in human hepatocellular carcinoma cells

Vitamin K (VK) has diverse protective effects against osteoporosis, atherosclerosis and carcinogenesis. We recently reported that menatetrenone, a VK2 analogue, suppressed nuclear factor (NF)-κB activation in human hepatoma cells. Although NF-κB is regulated by isoforms of protein kinase C (PKC), the involvement of PKCs in VK2-mediated NF-κB inhibition remains unknown. Therefore, the effects of VK2 on the activation and the kinase activity of each PKC isoform were investigated. The human hepatoma Huh7 cells were treated with PKC isoform-specific inhibitors and/or siRNAs against each PKC isoform with or without 12-O-tetradecanoylphorbol-13-acetate (TPA). VK2 inhibited the TPA-induced NF-κB activation in Huh7 cells. NF-κB activity was inhibited by the pan-PKC inhibitor Ro-31-8425, but not by the PKCα-specific inhibitor Gö6976. The knockdown of individual PKC isoforms including PKCα, δ and ? showed only marginal effects on the NF-κB activity. However, the knockdown of both PKCδ and PKC?, together with treatment with a PKCα-specific inhibitor, depressed the NF-κB activity. VK2 suppressed the PKCα kinase activity and the phosphorylation of PKC? after TPA treatment, but neither the activation nor the enzyme activity of PKCδ was affected. The knockdown of PKC? abolished the TPA-induced phosphorylation of PKD1, and the effects of PKD1 knockdown on NF-κB activation were similar to those of PKC? knockdown. Collectively, all of the PKCs, including α, δ and ?, and PKD1 are involved in the TPA-mediated activation of NF-κB. VK2 inhibited the NF-κB activation through the inhibition of PKCα and ? kinase activities, as well as subsequent inhibition of PKD1 activation.     

Functional characterization of the NF-κB transcription factor gene REL2 from Anopheles gambiae

The REL2 gene plays an important role in innate immunity against both Gram （＋） and Gram （-） bacteria and malaria parasites in Anopheles gambiae, the main vector of malaria in Africa. Through alternative splicing, REL2 produces two protein products, REL2F （with a Rel-homology domain as well as an inhibitory ankyrin repeat region） and REL2S （without the ankyrin repeats）. In the immune-competent cell line SualB from An. gambiae, REL2 has been shown to be a key regulator for cecropin A （or CEC1）. The high level expression of CEC1 in SualB was postulated to be the result of constitutive activation of REL2F. Here we showed that REL2F is indeed processed, albeit at a low level, in the SualB cell line. The primary cleavage requires residue 678 （an aspartic acid）. Proteolytic cleavage of REL2F can be enhanced by challenge with bacteria Escherichia coli and Bacillus subtilis, but not with fungus Beauveria bassiana. The inducible cleavage can be substantially reduced by RNA interference against PGRP-LC and CASPL1. Over-expression of REL2S or a constitutively active form of REL2F （REL2F380C or REL2F678） in An. gambiae cell line can further increase expression of CEC1 and other antimicrobial peptide genes. Over-expression of these constitutive active proteins in an immune naive cell line, MSQ43, from Anopheles stephensi, results in even more dramatic increased expression of antimicrobial peptides.     

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.     

Triglyceride down-regulates matrix metalloprotease-12 expression in THP-1 macrophages through activation of the NF-κB pathway

Triglyceride (TG) is known to be associated with inflammatory diseases including atherosclerosis. At the cellular level, TG can act as an immunomodulatory stimulus for macrophages. In this study we show that TG treatment of PMA-differentiated macrophages resulted in down-regulation of MMP-12 expression in a time- and dose-dependent manner. MMP-19 expression was unaffected by TG treatment. Using a variety of chemical inhibitors for cell signaling pathways we demonstrate that TG-induced down-regulation of MMP-12 occurs in part by activation of the NF-κB pathway. Finally, TG treatment of PMA-differentiated macrophages decreased cell migration in a wound healing assay. Taken together our data suggests that one function of TG is to modulate macrophage migration in tissues.     

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.     

Inactivation of GSK3β and activation of NF-κB pathway via Axl represents an important mediator of tumorigenesis in esophageal squamous cell carcinoma

The receptor tyrosine kinase Axl has been described as an oncogene, and its deregulation has been implicated in the progression of several human cancers. While the role of Axl in esophageal adenocarcinoma has been addressed, there is no information about its role in esophageal squamous cell carcinoma (OSCC). In the current report, we identified, for the first time, deregulation of Axl expression in OSCC. Axl is consistently overexpressed in OSCC cell lines and human tumor samples, mainly in advanced stages of the disease. Blockage of Axl gene expression by small interfering RNA inhibits cell survival, proliferation, migration, and invasion in vitro and esophageal tumor growth in vivo. Additionally, repression of Axl expression results in Akt-dependent inhibition of pivotal genes involved in the nuclear factor-kappaB (NF-κB) pathway and in the induction of glycogen synthase kinase 3β (GSK3β) activity, resulting in loss of mesenchymal markers and induction of epithelial markers. Furthermore, treatment of esophageal cancer cells with the Akt inhibitor wortmannin inhibits NF-κB signaling, induces GSK3β activity, and blocks OSCC cell proliferation in an Axl-dependent manner. Taken together, our results establish a clear role for Axl in OSCC tumorigenesis with potential therapeutic implications.