Interleukin-8 induces nuclear transcription factor-kappaB through a TRAF6-dependent pathway

Considering the potential role of interleukin-8 (IL-8) in inflammation, angiogenesis, tumorigenesis, and metastasis, we investigated the molecular mechanism involved in IL-8-mediated signaling. In this report we provide evidence that like TNF, an inducer of NF-kappaB and also a NF-kappaB-dependent gene product, IL-8 induces NF-kappaB in a unique pathway. IL-8 induces NF-kappaB activation in a dose-dependent manner in different cell types as detected by a DNA-protein binding assay. IL-8 induces NF-kappaB-dependent reporter gene expression as well as ICAM-1, VCAM-1, and Cox-2 expression. IL-8 also induces IkappaBalpha phosphorylation followed by degradation and p65 translocation. IL-8 induces c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) in a dose- and time-dependent manner. IL-8-induced NF-kappaB activation is for the most part unaltered when cells are transfected with dominant-negative TRADD, FADD, or TRAF2, but is inhibited with dominant-negative TRAF6-, NIK-, IKK-, or IkappaBalpha-transfected cells. The data suggest that IL-8-induced NF-kappaB activation proceeds through a TRAF2-independent but TRAF6-dependent pathway, followed by recruitment of IRAK and activation of IKK. IL-8-induced NF-kappaB activation is not observed in a cell-permeable peptide that has TRAF6 binding motif-treated cells or IRAK-deficient cells. IL-8-induced NF-kappaB activation proceeds mostly through interaction with TRAF6 and partially through the Rho-GTPase pathways. This is the first report that IL-8 induces NF-kappaB in a distinct pathway, and activation of NF-kappaB and its dependent genes may be one of the pathways of IL-8-induced inflammation and angiogenesis.     

p38 MAPK-induced nuclear factor-kappaB activity is required for skeletal muscle differentiation: role of interleukin-6

p38 MAPK and nuclear factor-kappaB (NF-kappaB) signaling pathways have been implicated in the control of skeletal myogenesis. However, although p38 is recognized as a potent activator of myoblast differentiation, the role of NF-kappaB remains controversial. Here, we show that p38 is activated only in differentiating myocytes, whereas NF-kappaB activity is present both in proliferation and differentiation stages. NF-kappaB activation was found to be dependent on p38 activity during differentiation, being NF-kappaB an effector of p38, thus providing a novel mechanism for the promyogenic effect of p38. Activation of p38 in C2C12 cells induced the activity of NF-kappaB, in a dual way: first, by reducing IkappaBalpha levels and inducing NF-kappaB-DNA binding activity and, second, by potentiating the transactivating activity of p65-NF-kappaB. Finally, we show that interleukin (IL)-6 expression is induced in C2C12 differentiating myoblasts, in a p38- and NF-kappaB-dependent manner. Interference of IL-6 mRNA reduced, whereas its overexpression increased, the extent of myogenic differentiation; moreover, addition of IL-6 was able to rescue significantly the negative effect of NF-kappaB inhibition on this process. This study provides the first evidence of a crosstalk between p38 MAPK and NF-kappaB signaling pathways during myogenesis, with IL-6 being one of the effectors of this promyogenic mechanism.     

Syntenin negatively regulates TRAF6-mediated IL-1R/TLR4 signaling

Toll-like receptors are involved in host defense against invading pathogens. The two members of this superfamily, IL-1R and TLR4, activate overlapping NF-kappaB activate signaling pathway mediated by TRAF6. In this study, we identified syntenin as a negative regulator of IL-1R and TLR4 mediated NF-kappaB activation. Overexpressed syntenin inhibited IL-1- or LPS-, but not TNF- induced NF-kappaB activation and IL-8 mRNA expression in a dose dependent manner. Syntenin specifically interacted with TRAF6 in human 293 cells, and inhibited TRAF6 induced NF-kappaB and AP-1 activation. Syntenin also associated with TRAF6 under physiological condition, and dissociated from TRAF6 upon IL-1 stimulation. This might be due to a competition between syntenin and IRAK1, as overexpression of IRAK1 disrupted the interaction of syntenin with TRAF6, and rescued syntenin induced reduction of TRAF6 ubiquitination. Moreover, knockdown of syntenin potentiated IL-1- or LPS- triggered NF-kappaB activation and IL-8 mRNA expression. These findings suggest that syntenin is a physiological suppressor of TRAF6 and plays an inhibitory role in IL-1R- and TLR4- mediated NF-kappaB activation pathways.     

Antioxidants Abrogate Alpha-Tocopherylquinone-Mediated Down-Regulation of the Androgen Receptor in Androgen-Responsive Prostate Cancer Cells

Tocopherylquinone (TQ), the oxidation product of alpha-tocopherol (AT), is a bioactive molecule with distinct properties from AT. In this study, AT and TQ are investigated for their comparative effects on growth and androgenic activity in prostate cancer cells. TQ potently inhibited the growth of androgen-responsive prostate cancer cell lines (e.g., LAPC4 and LNCaP cells), whereas the growth of androgen-independent prostate cancer cells (e.g., DU145 cells) was not affected by TQ. Due to the growth inhibitory effects induced by TQ on androgen-responsive cells, the anti-androgenic properties of TQ were examined. TQ inhibited the androgen-induced activation of an androgen-responsive reporter and inhibited the release of prostate specific antigen from LNCaP cells. TQ pretreatment was also found to inhibit AR activation as measured using the Multifunctional Androgen Receptor Screening assay. Furthermore, TQ decreased androgen-responsive gene expression, including TM4SF1, KLK2, and PSA over 5-fold, whereas AT did not affect the expression of androgen-responsive genes. Of importance, the antiandrogenic effects of TQ on prostate cancer cells were found to result from androgen receptor protein down-regulation produced by TQ that was not observed with AT treatment. Moreover, none of the androgenic endpoints assessed were affected by AT. The down-regulation of androgen receptor protein by TQ was abrogated by co-treatment with antioxidants. Overall, the biological actions of TQ were found to be distinct from AT, where TQ was found to be a potent inhibitor of cell growth and androgenic activity in androgen-responsive prostate cancer cells.     

Advanced glycation end products increases matrix metalloproteinase-1, -3, and -13, and TNF-alpha in human osteoarthritic chondrocytes

We investigated the effects of advanced glycation end products (AGE) which accumulate in articular cartilage with age in human osteoarthritic chondrocytes. We found AGE-BSA significantly increased MMP-1, -3, and -13, and TNF-alpha in a dose-dependent manner. AGE-BSA-stimulated JNK, p38, and ERK and NF-kappaB activity. The stimulatory effect of AGE-BSA on MMP-1, -3, and -13 were reversed by treatment with specific JNK, p38 inhibitors, suggesting JNK and p38 are involved in AGE-BSA-induced MMPs and TNF-alpha. We also observed that NF-kappaB is involved in AGE-BSA-induced TNF-alpha. Pretreatment with soluble receptor for AGE (sRAGE) also reduced AGE-stimulated MMPs and TNF-alpha, implicating the involvement of receptor for AGE (RAGE). In conclusion, accumulation of AGE may have a role in the development of osteoarthritis by increasing MMP-1, -3, and -13, and TNF-alpha.     

Angiotensin II augments advanced glycation end product-induced pericyte apoptosis through RAGE overexpression

Advanced glycation end product (AGE)-their receptor (RAGE) and angiotensin II (AII) are implicated in diabetic retinopathy. However, a crosstalk between the two is not fully understood. In vivo, AGE injection stimulated RAGE expression in the eye of spontaneously hypertensive rats, which was blocked by an AII-type 1 receptor blocker, telmisartan. In vitro, AII-type 1 receptor-mediated reactive oxygen species generation elicited RAGE gene expression in pericytes through NF-kappaB activation. Further, AII augmented AGE-induced pericyte apoptosis, the earliest hallmark of diabetic retinopathy. Our present study may implicate a crosstalk between AGE-RAGE system and AII in diabetic retinopathy.     

NF-kappaB-dependency and consequent regulation of IL-8 in echinomycin-induced apoptosis of HT-29 colon cancer cells

The present study was to see whether echinomycin-induced apoptosis would be NF-kappaB-dependent and if so, whether echinomycin would activate or inhibit NF-kappaB as well as resultant chemokine IL-8 expression. In HT-29 cells echinomycin activated NF-kappaB in time-dependent manner. EMSA in the presence of antibodies specific for p50 and p65 subunits indicated that echinomycin-induces the translocation of p50-p65 heterodimeric subunits of NF-kappaB. Levels of IkappaB were detected at initial echinomycin treatment and thereafter decreased, faintly seen after a 6h treatment. In contrast p-IkappaB levels were clearly detected throughout 6-24h of echinomycin treatment, albeit initially fainted. To clarify the role of NF-kappaB on IL-8 expression in echinomycin-mediated apoptosis of HT-29 cells, ELISA plus RT-PCR clearly showed that IL-8 production is inducible by echinomycin treatment. Using a specific inhibitor, IL-8 regulation at echinomycin treatment in HT-29 cells occurred via both caspase-3 and NF-kappaB-dependent signal pathway. To confirm whether two different pathways (NF-kappaB and caspase) would be coupled, only NF-kappaB inhibitor (PDTC) and caspase-3 specific inhibitor (Z-DEVD-FMK) together significantly attenuated echinomycin-initiated apoptosis of HT-29 cells, pretreatment of HT-29 cells with PDTC rarely affected echinomycin-induced caspase-3 activation. So echinomycin-induced apoptosis in HT-29 cells occurs via NF-kappaB activation independent of caspase-3 activation modulating the resultant-linked key chemokine IL-8 expression and echinomycin-induced apoptosis is NF-kappaB-dependant and directly related to NF-kappaB activation, consequently regulating IL-8 expression.     

Involvement of TNF receptor-associated factor 6 in IL-25 receptor signaling

IL-25 (IL-17E) induces IL-4, IL-5, and IL-13 production from an unidentified non-T/non-B cell population and subsequently induces Th2-type immune responses such as IgE production and eosinophilic airway inflammation. IL-25R is a single transmembrane protein with homology to IL-17R, but the IL-25R signaling pathways have not been fully understood. In this study, we investigated the signaling pathway under IL-25R, especially the possible involvement of TNFR-associated factor (TRAF)6 in this pathway. We found that IL-25R cross-linking induced NF-kappaB activation as well as ERK, JNK, and p38 activation. We also found that IL-25R-mediated NF-kappaB activation was inhibited by the expression of dominant negative TRAF6 but not of dominant negative TRAF2. Furthermore, IL-25R-mediated NF-kappaB activation, but not MAPK activation, was diminished in TRAF6-deficient murine embryonic fibroblast. In addition, coimmunoprecipitation assay revealed that TRAF6, but not TRAF2, associated with IL-25R even in the absence of ligand binding. Finally, we found that IL-25R-mediated gene expression of IL-6, TGF-beta, G-CSF, and thymus and activation-regulated chemokine was diminished in TRAF6-deficient murine embryonic fibroblast. Taken together, these results indicate that TRAF6 plays a critical role in IL-25R-mediated NF-kappaB activation and gene expression.     

NF-kappaB activator Act1 associates with IL-1/Toll pathway adaptor molecule TRAF6

NF-kappaB activator 1 (Act1), also called CIKS, is a recently identified protein with NF-kappaB and AP-1 activation activities through its association with the IkappaB kinase complex. We identified and confirmed that Act1 interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6); notably, Act1 binds to TRAF6 only among TRAF family proteins. The amino-terminal half of Act1 is required for its interaction with the TRAF domain. Act1-mediated NF-kappaB activation was inhibited by a dominant-negative mutant of TRAF6 in a dose-dependent manner, and IL-1-induced NF-kappaB activation was inhibited by a high level of Act1 expression. Our results suggest that Act1 is involved in IL-1/Toll-mediated signaling through TRAF6.