Opposing roles for TRAF1 in the alternative versus classical NF-κB pathway in T cells

T cells lacking TRAF1 hyperproliferate in response to T cell receptor signaling but have impaired signaling downstream of specific TNFR family members such as 4-1BB. Here we resolve this paradox by showing that while TRAF1 is required for maximal activation of the classical NF-κB pathway downstream of 4-1BB in primary T cells, TRAF1 also restricts the constitutive activation of NIK in anti-CD3-activated T cells. Activation of the alternative NF-κB pathway is restricted in unstimulated cells by a cIAP1/2:TRAF2:TRAF3:NIK complex. Using knockdown of NIK by siRNA we show that in activated CD8 T cells TRAF1 is also involved in this process and that constitutive activation of the alternative NF-κB pathway is responsible for costimulation independent hyperproliferation and excess cytokine production in TRAF1-deficient CD8 T cells compared with WT CD8 T cells. The T cell costimulatory molecule 4-1BB critically regulates the survival of activated and memory CD8 T cells. We demonstrate that stimulation through 4-1BB induces cIAP1-dependent TRAF3 degradation and activation of the alternative NF-κB pathway. We also show that while both TRAF1 and cIAP1 have non-redundant roles in suppressing the alternative NF-κB pathway in T cells activated in the absence of costimulation, activation of the classical NF-κB pathway downstream of 4-1BB requires TRAF1, whereas cIAP1 plays a redundant role with cIAP2. Collectively these results demonstrate that TRAF1 plays a critical role in regulating T cell activation both through restricting the costimulation independent activation of NIK in activated T cells and by promoting the 4-1BB-induced classical NF-κB pathway.     

Angiotensin II promotes differentiation of mouse embryonic stem cells to smooth muscle cells through PI3-kinase signaling pathway and NF-κB

Embryonic stem cells (ES cells), the pluripotent derivatives of the inner cell mass from blastocysts, have the capacity for unlimited growth, self-renewal and differentiation toward all types of somatic cells. Angiotensin II (Ang II), the most important effector peptide of the renin–angiotensin system, is also an angiogenesis factor. However, the potential impact of Ang II on ES cell differentiation is still unknown. In the present study, we have successfully induced the differentiation of ES cells into smooth muscle cells (SMCs) on collagen IV. Interestingly, incubation of ES cells with Ang II further promoted SMC differentiation from ES cells, which was abolished by prior treatment with Ang II type 1 (AT1) receptor antagonist losartan, but not Ang II type 2 (AT2) receptor antagonist PD123319. Moreover, we found that, in parallel with SMC specific-marker induction, the expression levels of phosphoAkt and NF-Kappa B (NF-κB) p50 were up-regulated by Ang II. Importantly, addition of phosphoinositide-3 kinase (PI3K) inhibitor LY294002 led to a marked inhibition of Ang II induced SMC specific markers, phosphoAkt and NF-κB p50 expression. Furthermore, NF-κB inhibitor BAY11-7082 can inhibit Ang II induced expression of SMC specific markers. Thus, we demonstrate for the first time that Ang II plays a promotive role in the stage of ES cell differentiation to SMCs through AT1 receptor. We further confirmed that PI3K/Akt signaling pathway and NF-κB play key roles in this process.     

Role of the adipocyte-specific NF-κB activity in the regulation of IP-10 and T cell migration

Infiltration of immune cells into adipose tissue plays a central role in the pathophysiology of obesity-associated low-grade inflammation. The aim of this study was to analyze the role of adipocyte NF-κB signaling in the regulation of the chemokine/adipokine interferon-γ-induced protein 10 kDa (IP-10) and adipocyte-mediated T cell migration. Therefore, the regulation of IP-10 was investigated in adipose tissue of male C57BL/6J mice, primary human and 3T3-L1 preadipocytes/adipocytes. To specifically block the NF-κB pathway, 3T3-L1 cells stably overexpressing a transdominant mutant of IκBα were generated, and the chemical NF-κB inhibitor Bay117082 was used. Adipocyte-mediated T cell migration was assessed by a migration assay. It could be shown that IP-10 expression was higher in mature adipocytes compared with preadipocytes. Induced IP-10 expression and secretion were completely blocked by an NF-κB inhibitor in 3T3-L1 and primary human adipocytes. Stable overexpression of a transdominant mutant of IκBα in 3T3-L1 adipocytes led to an inhibition of basal and stimulated IP-10 expression and secretion. T cell migration was induced by 3T3-L1 adipocyte-conditioned medium, and both basal and induced T cell migration was strongly inhibited by stable overexpression of a transdominant IκBα mutant. In addition, with the use of an anti-IP-10 antibody, a significant decrease of adipocyte-induced T cell migration was shown. In conclusion, in this study, we could demonstrate that the NF-κB pathway is essential for the regulation of IP-10 in 3T3-L1 and primary human adipocytes. Adipocytes rather than preadipocytes contribute to NF-κB-dependent IP-10 expression and secretion. Furthermore, NF-κB-dependent factors and especially IP-10 represent novel signals from adipocytes to induce T cell migration.     

Identification of constitutively active interleukin 33 (IL-33) splice variant

IL-33/IL-1F11 is a new member of the IL-1 family ligand and provokes T helper-type immune responses. IL-33 is the ligand of ST2 and IL-1 receptor accessory protein (IL-1RAcP) that triggers nuclear factor-κ light chain enhancer of activated B cells (NF-κB) and MAPK signaling. We discovered a novel short splice variant of IL-33 that was termed spIL-33. The new spIL-33 lacks exon 3 containing a proposed caspase-1 cleavage site. We isolated spIL-33 cDNA from the Huh7 human hepatocarcinoma cell line and expressed the recombinant spIL-33 protein in Escherichia coli. The recombinant spIL-33 and pro-IL-33 were not cleaved by caspase-1, unlike IL-18 (IL-1F4). The recombinant spIL-33 was constitutively active, and spIL-33-induced inflammatory cytokine production was caspase-1-independent in HMC-1 and Raw 264.7 cells. The recombinant spIL-33 induced the phosphorylation of IL-1 receptor-associated kinase (IRAK1), NF-κB, p38 MAPK, p44/42 MAPK, and JNK in a time- and dose-dependent manner. Anti-ST2 monoclonal antibody specifically blocked the spIL-33-induced cytokine production. In this study, we identified and characterized a new IL-33 splice variant, which was a constitutively active IL-33 isoform. The existence of constitutively active spIL-33 suggests that the biological activity of IL-33 could be triggered by diverse stimulations during immune responses. Further investigation of the spIL-33 expression pattern may contribute to understanding the involvement of IL-33 in inflammatory disorders.     

Endothelin-1 promotes MMP-13 production and migration in human chondrosarcoma cells through FAK/PI3K/Akt/mTOR pathways

Tumor malignancy is associated with several cellular properties including proliferation and ability to metastasize. Endothelin-1 (ET-1) the most potent vasoconstrictor plays a crucial role in migration and metastasis of human cancer cells. We found that treatment of human chondrosarcoma (JJ012 cells) with ET-1 increased migration and expression of matrix metalloproteinase (MMP)-13. ET-1-mediated cell migration and MMP-13 expression were reduced by pretreatment with inhibitors of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR), as well as the NF-κB inhibitor and the IκB protease inhibitor. In addition, ET-1 treatment induced phosphorylation of FAK, PI3K, AKT, and mTOR, and resulted in increased NF-κB-luciferase activity that was inhibited by a specific inhibitor of PI3K, Akt, mTOR, and NF-κB cascades. Taken together, these results suggest that ET-1 activated FAK/PI3K/AKT/mTOR, which in turn activated IKKα/β and NF-κB, resulting in increased MMP-13 expression and migration in human chondrosarcoma cells.