Hsp72对类风湿关节炎滑膜细胞IL-6、IL-8表达及NFκ-B活化的影响

目的:探讨热休克蛋白(Hsp)72对类风湿关节炎患者滑膜细胞IL-6、IL-8表达的影响,从NFκ-B信号通路活化的角度阐明其作用机制。方法:原代培养类风湿关节炎患者的滑膜细胞;采用酶联免疫吸附试验(ELISA)法检测细胞培养上清中IL-6和IL-8的含量;采用Western blot检测滑膜细胞NFκ-B和ΙκBα蛋白的表达变化;采用免疫荧光技术检测NFκ-B核移位的变化。结果:Hsp72抑制TNFα-所诱导的IL-6和IL-8的生成;Hsp72抑制TNFα-所诱导NFκ-B在核内的表达和移位;Hsp72抑制TNFα-所诱导ΙκBα蛋白降解。结论:Hsp72可能通过抑制滑膜细胞IL-6、IL-8表达及抑制NF-κB信号通路活化而对类风湿关节炎发挥抗炎作用。     

I-κBα depletion by transglutaminase 2 and μ-calpain occurs in parallel with the ubiquitin-proteasome pathway

Transglutaminase 2 (TGase2) is a calcium-dependent, cross-linking enzyme that catalyzes iso-peptide bond formation between peptide-bound lysine and glutamine residues. TGase 2 can activate NF-κB through the polymerization-mediated depletion of I-κBα without IKK activation. This NF-κB activation mechanism is associated with drug resistance in cancer cells. However, the polymers cannot be detected in cells, while TGase 2 over-expression depletes free I-κBα, which raises the question of how the polymerized I-κBα can be metabolized in cells. Among proteasome, lysosome and calpain systems, calpain inhibition was found to effectively increase the accumulation of I-κBα polymers in MCF7 cells transfected with TGase 2, and induced high levels of I-κBα polymers as well in MDA-MB-231 breast cancer cells that naturally express a high level of TGase 2. Inhibition of calpain also boosted the level of I-κBα polymers in HEK-293 cells in case of TGase 2 transfection either with I-κBα or I-κBα mutant (S32A, S36A). Interestingly, the combined inhibition of calpain and the proteasome resulted in an increased accumulation of both I-κBα polymers and I-κBα, concurrent with an inhibition of NF-κB activity in MDA-MB-231 cells. This suggests that μ-calpain proteasome-dependent I-κBα polymer degradation may contribute to cancer progression through constitutive NF-κB activation.     

Aspirin Prevents Apoptosis and NF-κB Activation Induced by H2O2 in HeLa Cells

The classical pathway of nuclear factor-kappa B (NF-κB) activation by several inducers mainly involves the phosphorylation of IκBα by a signalsome complex composed of IκBα kinases (IKKα and IKKβ). However, in some cell types hydrogen peroxide (H²O²) has been shown to activate an alternative pathway that does not involve the classical signalsome activation process. In this study, we demonstrate that H²O² induced NF-κB activation in HeLa cells through phosphorylation and degradation of IκB proteins as shown by immunblot analysis. Our studies reveal that a commonly used non-steroid anti-inflammatory drug, acetylsalicylic acid (aspirin) prevents H²O²-induced NF-κB activation in a dose-dependent manner through inhibition of phosphorylation and degradation of IκBα and IκBβ. Differential staining and DNA fragmentation analysis also show that aspirin preloading of HeLa cells also prevents H²O²-induced apoptosis in a dose-dependent manner with maximum efficiency at 10?mM concentration. Additionally, aspirin effectively prevents caspase-3 and caspase-9 (cysteinyl aspartate-specific proteases) activation by H²O². These results suggest that NF-κB activation is involved in H²O²-induced apoptosis and aspirin may inhibit both processes simultaneously.     

JAK2 is an important signal transducer in IL-33-induced NF-κB activation

IL-33, a member of the IL-1 family of cytokines, has been shown to activate NF-κB and MAP kinase family through the IL-1 receptor-related protein, ST2L. In this study, we found that IL-33 rapidly activated a tyrosine kinase, JAK2. Interestingly, we demonstrated the functional involvement of JAK2 in IL-33-induced IκBα degradation and NF-κB activation, since a JAK2 inhibitor, AG490, effectively inhibited this signaling pathway. Furthermore, IL-33 failed to induce IκBα degradation and NF-κB activation in JAK2-deficient MEFs expressing ST2L, compared with wild-type MEFs expressing ST2L. In addition, the introduction of wild-type JAK2 but not kinase dead JAK2 mutant (K882R) restored the IL-33-induced efficient activation of NF-κB in JAK2-deficient MEFs expressing ST2L, resulting in the induction of IL-6, CCL2/MCP-1 and CXCL1/KC expression. On the other hand, the activation of ERK, JNK and p38 was unaffected by JAK2 inhibition and JAK2 deficiency. Thus, these data demonstrate that JAK2 plays an important role in regulating IL-33-induced NF-κB activation.     

A vaccinia virus deletion mutant reveals the presence of additional inhibitors of NF-kappaB

The classical nuclear factor kappa B (NF-κB) signaling pathway is an important regulator of inflammation and innate immunity that is activated by a wide variety of stimuli, including virus infection, tumor necrosis factor alpha (TNF-α), and interleukin 1β (IL-1β). Poxviruses, including vaccinia virus (VV) and ectromelia virus, encode multiple proteins that function in immune evasion. Recently, a growing number of genes encoded by poxviruses have been shown to target and disrupt the NF-κB signaling pathway. To determine if additional gene products that interfere with NF-κB signaling existed, we used a vaccinia virus deletion mutant, VV811, which is missing 55 open reading frames lacking all known inhibitors of TNF-α-induced NF-κB activation. Immunofluorescence analysis of HeLa cells treated with TNF-α and IL-1β revealed that NF-κB translocation to the nucleus was inhibited in VV811-infected cells. This was further confirmed through Western blotting of cytoplasmic and nuclear extracts for NF-κB. Additionally, VV811 infection inhibited TNF-α-induced IκBα degradation. In contrast to vaccinia virus strain Copenhagen (VVCop)-infected cells, VV811 infection resulted in the dramatic accumulation of phosphorylated IκBα. Correspondingly, coimmunoprecipitation assays demonstrated that the NF-κB-inhibitory IκBα-p65-p50 complex was intact in VV811-infected cells. Significantly, cells treated with 1-β-d-arabinofuranosylcytosine, an inhibitor of poxvirus late gene expression, demonstrated that an additional vaccinia virus late gene was involved in the stabilization of IκBα. Overall, this work indicates that unidentified inhibitors of NF-κB exist in vaccinia virus. The complex inhibition of NF-κB by vaccinia virus illustrates the importance of NF-κB activation in the antiviral response.     

NEMO differentially regulates TCR and TNF-α induced NF-κB pathways and has an inhibitory role in TCR-induced NF-κB activation

NF-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase (IKK) complex, is an essential adaptor both for inflammation stimuli and TCR-induced NF-κB activation. However, the exact mechanism of its function has not been fully understood. Here, we report that knockdown of NEMO by RNA interference in Jurkat E6.1 cells enhanced TCR-induced NF-κB report gene activity and IL-2 production by promotion of IκBα degradation and p65 nuclear translocation, whereas inhibited TNF-α and LPS-induced IκBα degradation without influencing the phosphorylation of MAPKs. In human primary T and Jurkat E6.1 cells, both CD3/CD28 and PMA/Ionomycin induced NF-κB activation showed a para-curve correlation with the dosage of small interfering RNA targeting NEMO (siNEMO): the NF-κB report gene activity was increased along with ascending doses of transfected siNEMO and reached the highest activity when knockdown about 70% of NEMO, then turned to decline and gradually be blocked once almost thoroughly knockdown of NEMO. Meanwhile, TNF-α induced NF-κB was always inhibited no matter how much NEMO was knockdown. Subcellular fractionation results suggested that upon CD3/CD28 costimulation, NEMO and IKKβ may not cotranslocate to cytoskeleton fraction as a conventional NEMO/IKK complex with a static stoichiometric ratio, instead the ratio of NEMO: IKKβ continuously shift from high to low. Depletion of NEMO accelerated TCR-induced cytoskeleton translocation of IKKβ. Altogether, this study suggests that NEMO may function as a rheostat exerting a negative action on TCR-induced NF-κB activation and differentially regulates TNF-α and TCR-induced NF-κB pathways.     

Testes-specific protease 50 (TSP50) promotes cell proliferation through the activation of the nuclear factor κB (NF-κB) signalling pathway

TSP50 (testes-specific protease 50) is a testis-specific expression protein, which is expressed abnormally at high levels in breast cancer tissues. This makes it an attractive molecular marker and a potential target for diagnosis and therapy; however, the biological function of TSP50 is still unclear. In the present study, we show that overexpression of TSP50 in CHO (Chinese-hamster ovary) cells markedly increased cell proliferation and colony formation. Mechanistic studies have revealed that TSP50 can enhance the level of TNFα (tumour necrosis factor α)- and PMA-induced NF-κB (nuclear factor κB)-responsive reporter activity, IκB (inhibitor of NF-κB) α degradation and p65 nuclear translocation. In addition, the knockdown of endogenous TSP50 in MDA-MB-231 cells greatly inhibited NF-κB activity. Co-immunoprecipitation studies demonstrated an interaction of TSP50 with the NF-κB-IκBα complex, but not with the IKK (IκB kinase) α/β-IKKγ complex, which suggested that TSP50, as a novel type of protease, promoted the degradation of IκBα proteins by binding to the NF-κB-IκBα complex. Our results also revealed that TSP50 can enhance the expression of NF-κB target genes involved in cell proliferation. Furthermore, overexpression of a dominant-negative IκB mutant that is resistant to proteasome-mediated degradation significantly reversed TSP50-induced cell proliferation, colony formation and tumour formation in nude mice. Taken together, the results of the present study suggest that TSP50 promotes cell proliferation, at least partially, through activation of the NF-κB signalling pathway.     

Hepatocyte growth factor preferentially activates the anti-inflammatory arm of NF-κB signaling to induce A20 and protect renal proximal tubular epithelial cells from inflammation

Inflammation induces the NF-κB dependent protein A20 in human renal proximal tubular epithelial cells (RPTEC), which secondarily contains inflammation by shutting down NF-κB activation. We surmised that inducing A20 without engaging the pro-inflammatory arm of NF-κB could improve outcomes in kidney disease. We showed that hepatocyte growth factor (HGF) increases A20 mRNA and protein levels in RPTEC without causing inflammation. Upregulation of A20 by HGF was NF-κB/RelA dependent as it was abolished by overexpressing IκBα or silencing p65/RelA. Unlike TNFα, HGF caused minimal IκBα and p65/RelA phosphorylation, with moderate IκBα degradation. Upstream, HGF led to robust and sustained AKT activation, which was required for p65 phosphorylation and A20 upregulation. While HGF treatment of RPTEC significantly increased A20 mRNA, it failed to induce NF-κB dependent, pro-inflammatory MCP-1, VCAM-1, and ICAM-1 mRNA. This indicates that HGF preferentially upregulates protective (A20) over pro-inflammatory NF-κB dependent genes. Upregulation of A20 supported the anti-inflammatory effects of HGF in RPTEC. HGF pretreatment significantly attenuated TNFα-mediated increase of ICAM-1, a finding partially reversed by silencing A20. In conclusion, this is the first demonstration that HGF activates an AKT-p65/RelA pathway to preferentially induce A20 but not inflammatory molecules. This could be highly desirable in acute and chronic renal injury where A20-based anti-inflammatory therapies are beneficial.