HIV-1 Tat protein induces IL-10 production in monocytes by classical and alternative NF-kappaB pathways

The human immunodeficiency virus (HIV) transactivating Tat protein is not only critical for viral replication but also affects the host immune system by inducing the production of cytokines such as IL-10. This anti-inflammatory cytokine is upregulated during the course of HIV infection, representing an important pathway by which HIV may induce immunodeficiency. Here, we show that, by acting at the membrane, Tat induces IL-10 expression in primary monocytes and promonocytic U937 cells by NF-kappaB-dependent pathways. The trans-dominant negative mutants of NF-kappaB-inducing kinase (NIK), IKKalpha and IKKbeta expressed in our transactivation model, in accordance with the nuclear binding of p65 and p52 NF-kappaB subunits to the IL-10 promoter, suggest the involvement of both classical and alternative NF-kappaB pathways. In inactivated cells, IKKalpha is localized predominantly in the cytoplasm. Interestingly, Tat stimulates IKKalpha translocation from the cytoplasm to the nucleus in monocytes. Chromatin immunoprecipitation (ChIP) assay experiments, after Tat treatment, revealed IKKalpha and CBP/p300 recruitment to the IL-10 promoter and histone H3 phosphorylation (Ser 10) and acetylation (Lys 14) in this region, presumably leading to chromatin remodeling. We demonstrate that, upstream of NF-kappaB, PKC, ERK1/2 and p38 MAP kinases are involved in Tat-induced IKKalpha nuclear translocation and histone H3 modifications on the IL-10 promoter in accordance with the role of these three kinases in IL-10 production. As a whole, the study demonstrates that Tat activates at least three signaling pathways concurrently, including the classical, alternative and IKKalpha pathways, to promote production of IL-10.     

Respiratory syncytial virus influences NF-kappaB-dependent gene expression through a novel pathway involving MAP3K14/NIK expression and nuclear complex formation with NF-kappaB2

A member of the Paramyxoviridae family of RNA viruses, respiratory syncytial virus (RSV), is a leading cause of epidemic respiratory tract infection in children. In children, RSV primarily replicates in the airway mucosa, a process that alters epithelial cell chemokine expression, thereby inducing airway inflammation. We investigated the role of the mitogen-activated protein kinase kinase kinase 14/NF-kappaB-inducing kinase (NIK) in the activation of NF-kappaB-dependent genes in alveolus-like A549 cells. RSV infection induces a time dependent increase of NIK mRNA and protein expression that peaks 12 to 24 h after viral exposure. Immunoprecipitation kinase assays indicate that NIK kinase activity is activated even more rapidly (within 6 h of RSV adsorption) associated with an endogenous approximately 50-kDa NF-kappaB2 substrate. Because NIK associates with IKKalpha to mediate processing of the 100-kDa NF-kappaB2 precursor into its 52-kDa DNA binding isoform ("p52"), the effects of RSV on NIK complex formation with IKKalpha and NF-kappaB2 were determined by coimmunoprecipitation assay. We find that NIK, IKKalpha, and both 100 kDa- and 52-kDa NF-kappaB2 isoforms strongly complex 15 h after exposure to RSV at times subsequent to NIK kinase activation. Western immunoblot and microaffinity DNA pull-down assays showed a parallel increase in nuclear translocation and DNA binding of the NF-kappaB2-Rel B complex. Interestingly, we make the novel observations that NIK also transiently translocates into the nucleus complexed with 52-kDa NF-kappaB2. Small interfering RNA-mediated NIK "knock-down" blocked RSV-inducible 52-kDa NF-kappaB2 processing and interfered with the early activation of a subset of NF-kappaB-dependent genes, indicating the importance of this activation pathway in the genomic NF-kappaB response to RSV. Together, these data indicate that RSV infection rapidly activates the noncanonical NF-kappaB activation pathway prior to the more potent canonical pathway activation. This appears to be through a novel mechanism involving induction of NIK kinase activity, expression, and nuclear translocation of a ternary complex with IKKalpha and processed NF-kappaB2.     

Nuclear factor kappa B-inducing kinase and Ikappa B kinase-alpha signal skeletal muscle cell differentiation

Nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK), IkappaB kinase (IKK)-alpha and -beta, and IkappaBalpha are common elements that signal NF-kappaB activation in response to diverse stimuli. In this study, we analyzed the role of this pathway during insulin-like growth factor II (IGF-II)-induced myoblast differentiation. L6E9 myoblasts differentiated with IGF-II showed an induction of NF-kappaB DNA-binding activity that correlated in time with the activation of IKKalpha, IKKbeta, and NIK. Moreover, the activation of IKKalpha, IKKbeta, and NIK by IGF-II was dependent on phosphatidylinositol 3-kinase, a key regulator of myogenesis. Adenoviral transduction with the IkappaBalpha(S32A/S36A) mutant severely impaired both IGF-II-dependent NF-kappaB activation and myoblast differentiation, indicating that phosphorylation of IkappaBalpha at Ser-32 and Ser-36 is an essential myogenic step. Adenoviral transfer of wild-type or kinase-deficient forms of IKKalpha or IKKbeta revealed that IKKalpha is required for IGF-II-dependent myoblast differentiation, whereas IKKbeta is not essential for this process. Finally, overexpression of kinase-proficient wild-type NIK showed that the activation of NIK is sufficient to generate signals that trigger myogenin expression and multinucleated myotube formation in the absence of IGF-II.     

Induction of p100 processing by NF-kappaB-inducing kinase involves docking IkappaB kinase alpha (IKKalpha) to p100 and IKKalpha-mediated phosphorylation

The processing of the nfkappab2 gene product p100 to generate p52 is a regulated event, which is important for the instrumental function of NF-kappaB. We previously demonstrated that this tightly controlled event is regulated positively by NF-kappaB-inducing kinase (NIK) and its downstream kinase, IkappaB kinase alpha (IKKalpha). However, the precise mechanisms by which NIK and IKKalpha induce p100 processing remain unclear. Here, we show that, besides activating IKKalpha, NIK also serves as a docking molecule recruiting IKKalpha to p100. This novel function of NIK requires two specific amino acid residues, serine 866 and serine 870, of p100 that are known to be essential for inducible processing of p100. We also show that, after being recruited into p100 complex, activated IKKalpha phosphorylates specific serines located in both N- and C-terminal regions of p100 (serines 99, 108, 115, 123, and 872). The phosphorylation of these specific serines is the prerequisite for ubiquitination and subsequent processing of p100 mediated by the beta-TrCP ubiquitin ligase and 26 S proteasome, respectively. These results highlight the critical but different roles of NIK and IKKalpha in regulating p100 processing and shed light on the mechanisms mediating the tight control of p100 processing. These data also provide the first evidence for explaining why overexpression of IKKalpha or its activation by many other stimuli such as tumor necrosis factor and mitogens fails to induce p100 processing.     

beta-TrCP binding and processing of NF-kappaB2/p100 involve its phosphorylation at serines 866 and 870

Processing of the NF-kappaB2 precursor protein p100 is a major step in noncanonical NF-kappaB signaling. This signaling step requires the NF-kappaB inducing kinase (NIK) and its downstream kinase, IkappaB kinase alpha (IKKalpha). We show here that p100 undergoes phosphorylation at serines 866, 870, and possibly 872, in cells stimulated with noncanonical NF-kappaB stimuli or transfected with NIK and IKKalpha. Phosphorylation of this serine cluster creates a binding site for beta-TrCP, the receptor subunit of the beta-TrCP(SCF) ubiquitin ligase. Mutation of either serine 866 or serine 870 abolishes the beta-TrCP recruitment and ubiquitination of p100. The functional significance of p100 phosphorylation is further supported by the finding that this molecular event occurs in a NIK- and IKKalpha-dependent manner. Additionally, induction of p100 phosphorylation can be blocked by a protein synthesis inhibitor, suggesting the requirement of de novo protein synthesis. These data suggest that p100 processing involves its phosphorylation at specific terminal serines, which form a binding site for beta-TrCP thereby regulating p100 ubiquitination.     

Respiratory syncytial virus induces RelA release from cytoplasmic 100-kDa NF-kappa B2 complexes via a novel retinoic acid-inducible gene-I{middle dot}NF- kappa B-inducing kinase signaling pathway

Respiratory syncytial virus (RSV) is a primary cause of severe lower respiratory tract infection in children worldwide. RSV infects airway epithelial cells, where it activates inflammatory genes via the NF-kappaB pathway. NF-kappaB is controlled by two pathways, a canonical pathway that releases sequestered RelA complexes from the IkappaBalpha inhibitor, and a second, the noncanonical pathway, that releases RelB from the 100-kDa NF-kappaB2 complex. Recently we found that the retinoic acid-inducible gene I (RIG-I) is a major intracellular RSV sensor upstream of the canonical pathway. In this study, we surprisingly found that RIG-I silencing also inhibited p100 processing to 52-kDa NF-kappaB2 ("p52"), suggesting that RIG-I was functionally upstream of the noncanonical regulatory kinase complex composed of NIK.IKKalpha subunits. Co-immunoprecipitation experiments not only demonstrated that NIK associated with RIG-I and its downstream adaptor, mitochondrial antiviral signaling (MAVS), but also showed the association between IKKalpha and MAVS. To further understand the role of the NIK.IKKalpha pathway, we compared RSV-induced NF-kappaB activation using wild type, Ikkgamma(-/-), Nik(-/-), and Ikkalpha(-/-)-deficient MEF cells. Interestingly, we found that in canonical pathway-defective Ikkgamma(-/-) cells, RSV induced RelA by liberation from p100 complexes. RSV was still able to activate IP10, Rantes, and Grobeta gene expression in Ikkgamma(-/-) cells, and this induction was inhibited by small interfering RNA-mediated RelA knockdown but not RelB silencing. These data suggest that part of the RelA activation in response to RSV infection was induced by a "cross-talk" pathway involving the noncanonical NIK.IKKalpha complex downstream of RIG-I.MAVS. This pathway may be a potential target for RSV treatment.     

Effects of the NIK aly mutation on NF-kappaB activation by the Epstein-Barr virus latent infection membrane protein, lymphotoxin beta receptor, and CD40

Homozygosity for the aly point mutation in NF-kappaB-inducing kinase (NIK) results in alymphoplasia in mice, a phenotype similar to that of homozygosity for deletion of the lymphotoxin beta receptor (LTbetaR). We now find that NF-kappaB activation by Epstein-Barr virus latent membrane protein 1 (LMP1) or by an LMP1 transmembrane domain chimera with the LTbetaR signaling domain in human embryonic kidney 293 cells is selectively inhibited by a wild type dominant negative NIK comprised of amino acids 624-947 (DN-NIK) and not by aly DN-NIK. In contrast, LMP1/CD40 is inhibited by both wild type (wt) and aly DN-NIK. LMP1, an LMP1 transmembrane domain chimera with the LTbetaR signaling domain, and LMP1/CD40 activate NF-kappaB in wt or aly murine embryo fibroblasts. Although wt and aly NIK do not differ in their in vitro binding to tumor necrosis factor receptor-associated factor 1, 2, 3, or 6 or in their in vivo association with tumor necrosis factor receptor-associated factor 2 and differ marginally in their very poor binding to IkappaB kinase beta (IKKbeta), only wt NIK is able to bind to IKKalpha. These data are compatible with a model in which activation of NF-kappaB by LMP1 and LTbetaR is mediated by an interaction of NIK or a NIK-like kinase with IKKalpha that is abrogated by the aly mutation. On the other hand, CD40 mediates NF-kappaB activation through a kinase that interacts with a different component of the IKK complex.     

Essential role of IkappaB kinase alpha in thymic organogenesis required for the establishment of self-tolerance

IkappaB kinase (IKK) alpha exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-kappaB activation pathway. The in vivo function of IKKalpha, however, still remains elusive. Because a natural strain of mice with mutant NF-kappaB-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKKalpha axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKKalpha-deficient mice. The thymic microenvironment that caused autoimmunity in an IKKalpha-dependent manner was associated with defective processing of NF-kappaB2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKKalpha in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.     

Tyrosine phosphorylation of I-kappa B kinase alpha/beta by protein kinase C-dependent c-Src activation is involved in TNF-alpha-induced cyclooxygenase-2 expression

The signaling pathway involved in TNF-alpha-induced cyclooxygenase-2 (COX-2) expression was further studied in human NCI-H292 epithelial cells. A protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or a Src kinase inhibitor (PP2) attenuated TNF-alpha- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity. TNF-alpha- or TPA-induced I-kappaB kinase (IKK) activation was also blocked by these inhibitors, which reversed I-kappaBalpha degradation. Activation of c-Src and Lyn kinases, two Src family members, was inhibited by the PKC, tyrosine kinase, or Src kinase inhibitors. The dominant-negative c-Src (KM) mutant inhibited induction of COX-2 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKCalpha (PKCalpha A/E) or wild-type c-Src plasmids induced COX-2 promoter activity, and these effects were inhibited by the dominant-negative c-Src (KM), NF-kappaB-inducing kinase (NIK) (KA), or IKKbeta (KM) mutant. The dominant-negative PKCalpha (K/R) or c-Src (KM) mutant failed to block induction of COX-2 promoter activity caused by wild-type NIK overexpression. In coimmunoprecipitation experiments, IKKalpha/beta was found to be associated with c-Src and to be phosphorylated on its tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr(188) and Tyr(199), near the activation loop of IKKbeta, were identified to be crucial for NF-kappaB activation. Substitution of these residues with phenylalanines attenuated COX-2 promoter activity and c-Src-dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA. These data suggest that, in addition to activating NIK, TNF-alpha also activates PKC-dependent c-Src. These two pathways cross-link between c-Src and NIK and converge at IKKalpha/beta, and go on to activate NF-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate COX-2 expression.