NF-��B Functions in Stromal Fibroblasts to Regulate Early Postnatal Muscle Development

Classical NF-κB activity functions as an inhibitor of the skeletal muscle myogenic program. Recent findings reveal that even in newborn RelA/p65^−/− mice, myofiber numbers are increased over that of wild type mice, suggesting that NF-κB may be a contributing factor in early postnatal skeletal muscle development. Here we show that in addition to p65 deficiency, repression of NF-κB with the IκBα-SR transdominant inhibitor or with muscle-specific deletion of IKKβ resulted in similar increases in total fiber numbers as well as an up-regulation of myogenic gene products. Upon further characterization of early postnatal muscle, we observed that NF-κB activity progressively declines within the first few weeks of development. At birth, the majority of this activity is compartmentalized to muscle fibers, but by neonatal day 8 NF-κB activity from the myofibers diminishes, and instead, stromal fibroblasts become the main cellular compartment within the muscle that contains active NF-κB. We find that NF-κB functions in these fibroblasts to regulate inducible nitric-oxide synthase expression, which we show is important for myoblast fusion during the growth and maturation process of skeletal muscle. Together, these data broaden our understanding of NF-κB during development by showing that in addition to its role as a negative regulator of myogenesis, NF-κB also regulates nitric-oxide synthase expression within stromal fibroblasts to stimulate myoblast fusion and muscle hypertrophy.     

miR-101 is down-regulated by the hepatitis B virus x protein and induces aberrant DNA methylation by targeting DNA methyltransferase 3A

The hepatitis B virus x (HBx) protein has been implicated in HBV-related hepatocellular carcinoma (HCC) pathogenesis. However, whether HBx regulates miRNA expression that plays important roles in gene regulation during hepatocarcinogenesis remains unknown. The expression of microRNA-101 (miR-101) in HBV-related HCC tissues and HCC cells was evaluated by real-time PCR. The direct target of miR-101, DNA methyltransferase 3A (DNMT3A), was identified in silico and validated using a 3′-UTR reporter assay. miR-101 was functionally characterized in cells with transiently altered miR-101 expression. HBx expression was found to have a significant inverse correlation with miR-101 expression in HBx-expressing HepG2 compared to control HepG2 cells. miR-101 expression was frequently down-regulated in HBV-related HCC tissues compared to adjacent noncancerous hepatic tissues and had a significant inverse correlation with DNMT3A expression in HBV-related HCCs. Further characterization of miR-101 revealed that it negatively regulated DNA methylation partly through targeting DNMT3A. HBx-mediated miR-101 down-regulation and DNMT3A up-regulation supported the enhanced DNA methylation of several tumor-suppressor genes in HBx-expressing cells. Our studies demonstrating the deregulation of miR-101 expression by HBx may provide novel mechanistic insights into HBV-mediated hepatocarcinogenesis and identify a potential miRNA-based targeted approach for treating HBV-related HCC.     

Ubiquitin-specific Peptidase 21 Inhibits Tumor Necrosis Factor ��-induced Nuclear Factor ��B Activation via Binding to and Deubiquitinating Receptor-interacting Protein 1

Ubiquitination and deubiquitination of receptor-interacting protein 1 (RIP1) play an important role in the positive and negative regulation of the tumor necrosis factor α (TNFα)-induced nuclear factor κB (NF-κB) activation. Using a combination of functional genomic and proteomic approaches, we have identified ubiquitin-specific peptidase 21 (USP21) as a deubiquitinase for RIP1. USP21 is constitutively associated with RIP1 and deubiquitinates RIP1 in vitro and in vivo. Notably, knockdown of USP21 in HeLa cells enhances TNFα-induced RIP1 ubiquitination, IκB kinase β (IKKβ), and NF-κB phosphorylation, inhibitor of NF-κB α (IκBα) phosphorylation and ubiquitination, as well as NF-κB-dependent gene expression. Therefore, our results demonstrate that USP21 plays an important role in the down-regulation of TNFα-induced NF-κB activation through deubiquitinating RIP1.     

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.     

Prolyl Hydroxylase EGLN3 Regulates Skeletal Myoblast Differentiation through an NF-��B-dependent Pathway

The egg-laying abnormal-9 (EGLN) prolyl hydroxylases have been shown to regulate the stability and thereby the activity of the α subunits of hypoxia-inducible factor (HIF) through its ability to catalyze their hydroxylation. We have previously shown that EGLN3 promotes differentiation of C2C12 skeletal myoblasts. However, the mechanism underlying this effect remains to be fully elucidated. Here, we report that exposure of C2C12 cells to dimethyl oxalylglycine (DMOG), desferrioxamine, and hypoxia, all inhibitors of prolyl hydroxylase activity, led to repression of C2C12 myogenic differentiation. Inactivation of HIF by expression of a HIF dominant-negative mutant or deletion of HIF-1α by RNA interference did not affect the inhibitory effect of DMOG, suggesting that the effect of DMOG is HIF-independent. Pharmacologic inactivation of EGLN3 hydroxylase resulted in activation of the canonical NF-κB pathway. The inhibitory effect of DMOG on myogenic differentiation was markedly impaired in C2C12 cells expressing a dominant-negative mutant of IκBα. Exogenous expression of wild-type EGLN3, but not its catalytically inactive mutant, significantly inhibited NF-κB activation induced by overexpressed TRAF2 or IκB kinase 2. In contrast, deletion of EGLN3 by small interfering RNAs led to activation of NF-κB. These data suggest that EGLN3 is a negative regulator of NF-κB, and its prolyl hydroxylase activity is required for this effect. Furthermore, wild-type EGLN3, but not its catalytically inactive mutant, potentiated myogenic differentiation. This study demonstrates a novel role for EGLN3 in the regulation of NF-κB and suggests that it is involved in mediating myogenic differentiation, which is HIF-independent.     

Proinflammatory Cytokine and Nitric Oxide Production by Human Macrophages Stimulated with Trichomonas vaginalis

Trichomonas vaginalis commonly causes vaginitis and perhaps cervicitis in women and urethritis in men and women. Macrophages are important immune cells in response to T. vaginalis infection. In this study, we investigated whether human macrophages could be involved in inflammation induced by T. vaginalis. Human monocyte-derived macrophages (HMDM) were co-cultured with T. vaginalis. Live, opsonized-live trichomonads, and T. vaginalis lysates increased proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6 by HMDM. The involvement of nuclear factor (NF)-κB signaling pathway in cytokine production induced by T. vaginalis was confirmed by phosphorylation and nuclear translocation of p65 NF-κB. In addition, stimulation with live T. vaginalis induced marked augmentation of nitric oxide (NO) production and expression of inducible NO synthase (iNOS) levels in HMDM. However, trichomonad-induced NF-κB activation and TNF-α production in macrophages were significantly inhibited by inhibition of iNOS levels with L-NMMA (NO synthase inhibitor). Moreover, pretreatment with NF-κB inhibitors (PDTC or Bay11-7082) caused human macrophages to produce less TNF-α. These results suggest that T. vaginalis stimulates human macrophages to produce proinflammatory cytokines, such as IL-1, IL-6, and TNF-α, and NO. In particular, we showed that T. vaginalis induced TNF-α production in macrophages through NO-dependent activation of NF-κB, which might be closely involved in inflammation caused by T. vaginalis.