EV71 induces VCAM-1 expression via PDGF receptor, PI3-K/Akt, p38 MAPK, JNK and NF-kappaB in vascular smooth muscle cells

Enterovirus 71 (EV71) is a widespread virus that causes severe and fatal diseases in patients, including circulation failure. The mechanisms underlying EV71-initiated intracellular signaling pathways to influence host cell functions remain unknown. In this study, we identified a requirement for PDGFR, PI3-K/Akt, p38 MAPK, JNK, and NF-kappaB in the regulation of VCAM-1 expression by rat vascular smooth muscle cells (VSMCs) in response to viral infection. EV71 induced VCAM-1 expression in a time- and viral concentration-dependent manner. Infection of VSMCs with EV71 stimulated VCAM-1 expression and phosphorylation of PDGFR, Akt, and p38 MAPK which were attenuated by AG1296, wortmannin, and SB202190, respectively. The phosphorylation of JNK stimulated by EV71 was not detected under present conditions. In contrast, JNK inhibitor SP600125 inhibited EV71-induced VCAM-1 expression. Furthermore, VCAM-1 expression induced by EV71 was significantly attenuated by a selective NF-kappaB inhibitor (helenalin). Consistently, EV71-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha as well as VCAM-1 mRNA expression was blocked by helenalin, AG1296, SB202190, SP600125, wortmannin, and LY294002. Moreover, the involvement of p38 MAPK, PI3-K/Akt, and NF-kappaB in EV71-induced VCAM-1 expression was reveled by that transfection with dominant negative plasmids of p38 MAPK, p85, Akt, NIK, IKK-alpha, and IKK-beta attenuated these responses. These findings suggest that in VSMCs, EV71-induced VCAM-1 expression was mediated through activation of PDGFR, PI3-K/Akt, p38 MAPK, JNK, and NF-kappaB pathways.     

Cutting edge: FADD is not required for antigen receptor-mediated NF-kappaB activation

Recently, it has been demonstrated that stimulated T cells bearing defects in caspase-8 fail to promote nuclear shuttling of NF-kappaB complexes. Such cells display strikingly similar proliferative and survival defects as T cells lacking Fas-associated death domain protein (FADD) function. We characterized NF-kappaB signaling in T cells bearing a dominant-negative FADD transgene (FADDdd). Whereas FADDdd T cells displayed proliferative defects following activation, these were not a consequence of aberrant NF-kappaB signaling, as measured by IKK/IkappaB phosphorylation and IkappaB degradation. There were no appreciable defects in nuclear translocation of p65/Rel using ImageStream, a flow-based imaging cytometer. Pretreatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a potent caspase inhibitor, also failed to impede canonical NF-kappaB signaling. Secretion of IL-2 and up-regulation of various activation markers occurred normally. Thus, FADD does not play an essential role in NF-kappaB activation, suggesting an alternative route by which this adaptor promotes the clonal expansion of T cells.     

Tumor necrosis factor alpha induction of NF-kappaB requires the novel coactivator SIMPL

A myriad of stimuli including proinflammatory cytokines, viruses, and chemical and mechanical insults activate a kinase complex composed of IkappaB kinase beta (IKK-beta), IKK-alpha, and IKK-gamma/N, leading to changes in NF-kappaB-dependent gene expression. However, it is not clear how the NF-kappaB response is tailored to specific cellular insults. Signaling molecule that interacts with mouse pelle-like kinase (SIMPL) is a signaling component required for tumor necrosis factor alpha (TNF-alpha)-dependent but not interleukin-1-dependent NF-kappaB activation. Herein we demonstrate that nuclear localization of SIMPL is required for type I TNF receptor-induced NF-kappaB activity. SIMPL interacts with nuclear p65 in a TNF-alpha-dependent manner to promote endogenous NF-kappaB-dependent gene expression. The interaction between SIMPL and p65 enhances p65 transactivation activity. These data support a model in which TNF-alpha activation of NF-kappaB dependent-gene expression requires nuclear relocalization of p65 as well as nuclear relocalization of SIMPL, generating a TNF-alpha-specific induction of gene expression.     

A pyrrolidinone derivative inhibits cytokine-induced iNOS expression and NF-kappaB activation by preventing phosphorylation and degradation of IkappaB-alpha

We previously showed that 1-[3-(3-pyridyl)-acryloyl]-2-pyrrolidinone hydrochloride (N2733) inhibits lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-alpha secretion and improves the survival of endotoxemic mice. Since overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) in vascular smooth muscle cells (VSMCs) is largely responsible for the development of endotoxemic shock, and iNOS gene expression is mainly regulated by LPS and inflammatory cytokines, we studied whether or not N2733 affects interleukin (IL)-1beta-induced iNOS gene expression, NF-kappaB activation, and NF-kappaB inhibitor (IkappaB)-alpha degradation in cultured rat VSMCs. N2733 dose-dependently (10-100 microM) inhibited IL-1beta-stimulated NO production, and decreased IL-1beta-induced iNOS mRNA and protein expression, as found on Northern and Western blot analyses, respectively. Gel shift assay and an immunocytochemical study showed that N2733 inhibited IL-1beta-induced NF-kappaB activation and its nuclear translocation. Western blot analyses involving anti-IkappaB-alpha and anti-phospho IkappaB-alpha antibodies showed that IL-1beta induced transient degradation of IkappaB-alpha preceded by the rapid appearance of phosphorylated IkappaB-alpha, both of which were markedly blocked by N2733. N2733 blocked IL-1beta-induced phosphorylated IkappaB-alpha even in the presence of a proteasome inhibitor (MG115). Immunoblot analysis involving anti-IkappaB kinase (IKK)-alpha and anti-phosphoserine antibodies revealed that N2733 inhibited IL-1beta-induced IKK-alpha phosphorylation, whereas N2733 had no inhibitory effect on IL-1beta-stimulated p42/p44 MAP kinase or p38 MAP kinase activity. Our results suggest that the inhibitory action of N2733 toward IL-1beta-induced NF-kappaB activation and iNOS expression is due to its blockade of the upstream signal(s) leading to IKK-alpha activation, and subsequent phosphorylation and degradation of IkappaB-alpha in rat VSMCs.     

CD40-mediated activation of NF-kappa B in airway epithelial cells

We have reported previously that airway epithelial cells (AEC) express CD40 and that activation of this molecule stimulates the expression of inflammatory mediators, including the chemokine RANTES (regulated on activation normal T cell expressed and secreted). Because NF-kappaB regulates the expression of many inflammatory mediators, such as RANTES, we utilized CD40-mediated induction of RANTES expression to investigate the mechanisms that underlie CD40-mediated activation of NF-kappaB in AEC. Results demonstrate that, in AEC, intact NF-kappaB sites were required for CD40-mediated activation of the RANTES promoter. To examine activation of NF-kappaB binding directly, electrophoretic mobility shift analyses were performed. These analyses revealed that CD40 ligation stimulated NF-kappaB binding and that the activated NF-kappaB complexes were composed of p65 subunits. Additional studies focused on the CD40-triggered signaling pathways that facilitate NF-kappaB activation. Findings show that CD40 engagement activated the IkappaB kinases IKK-alpha and IKK-beta and stimulated IkappaBalpha phosphorylation. Analyses also examined the role of tumor necrosis factor-associated factor (TRAF) molecules in CD40-mediated NF-kappaB activation within AEC. Stable transfectants expressing wild-type or mutant forms of the cytoplasmic domain of CD40 suggested that TRAF3, but not TRAF2, binding was essential for CD40-mediated RANTES expression. Further studies indicated that exogenous expression of wild-type TRAF3 enhanced activation of the RANTES promoter, whereas exogenous expression of wild-type TRAF2 inhibited this activation; TRAF3-mediated enhancement was dependent upon NF-kappaB. Together, these findings suggest that, in AEC, ligation of CD40 regulates the expression of inflammatory mediators, such as RANTES, via activation of NF-kappaB. Moreover, these results suggest that CD40-mediated signaling in AEC differs with previously reported findings observed in other cell models, such as B lymphocytes.     

Suppression of NF-kappaB activation by Entamoeba histolytica in intestinal epithelial cells is mediated by heat shock protein 27

Little is known about the pathogenesis of Entamoeba histolytica and how epithelial cells respond to the parasite. Herein, we characterized the interactions between E. histolytica and colonic epithelial cells and the role macrophages play in modulating epithelial cell responses. The human colonic epithelial cell lines Caco-2 and T84 were grown either as monoculture or co-cultured in transwell plates with differentiated human THP-1 macrophages for 24 h before stimulation with soluble amebic proteins (SAP). In naive epithelial cells, prolonged stimulation with SAP reduced the levels of heat shock protein (Hsp) 27 and 72. However in THP-1 conditioned intestinal epithelial cells SAP enhanced Hsp27 and Hsp72, which was dependent on the activation of ERK MAP kinase. Hsp synthesis induced by SAP conferred protection against oxidative and apoptotic injuries. Treatment with SAP inhibited NF-kappaB activation induced by interleukin-1beta; specifically, the NF-kappaB-DNA binding, nuclear translocation of p65 subunit, and phosphorylation of IkappaB-alpha were reduced. Gene silencing by small interfering RNA confirmed the role of Hsp27 in suppressing NF-kappaB activation at IkappaB kinase (IKK) level. By co-immunoprecipitation studies, we found that Hsp27 interacts with IKK-alpha and IKK-beta, and this association was increased in SAP-treated conditioned epithelial cells. Overexpression of wild type Hsp27 amplified the effects of SAP, whereas a phosphorylation-deficient mutant of Hsp27 abrogated SAP-induced NF-kappaB inhibition. In conditioned epithelial cells, Hsp27 was phosphorylated at serine 15 after prolonged exposure to SAP. This mechanism may explain the absence of colonic inflammation seen in the majority of individuals infected with E. histolytica.     

Involvement of MAPKs and NF-kappaB in LPS-induced VCAM-1 expression in human tracheal smooth muscle cells

Lipopolysaccharide (LPS) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for LPS-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in HTSMCs. LPS-induced expression of VCAM-1 protein and mRNA in a time-dependent manner, was significantly inhibited by inhibitors of MEK1/2 (U0126), p38 (SB202190), and c-Jun-N-terminal kinase (JNK; SP600125). The involvement of p42/p44 MAPK and p38 in these responses was further confirmed by that transfection with small interference RNAs (siRNA) direct against MEK, p42, and p38 significantly attenuated LPS-induced VCAM-1 expression. Consistently, LPS-stimulated phosphorylation of p42/p44 MAPK and p38 was attenuated by pretreatment with U0126 or SB202190, and transfection with these siRNAs, respectively. In addition, LPS-induced VCAM-1 expression was significantly blocked by a specific NF-kappaB inhibitor helenalin. LPS-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha was blocked by helenalin, U0126, SB202190, or SP600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to monolayer of HTSMCs which was blocked by pretreatment with helenalin, U0126, or SP600125 prior to LPS exposure. Taken together, these results suggest that in HTSMCs, activation of p42/p44 MAPK, p38, and JNK pathways, at least in part, mediated through NF-kappaB, is essential for LPS-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of LPS action that bacterial toxins may promote inflammatory responses in the airway disease.     

Role of adapter function in oncoprotein-mediated activation of NF-kappaB. Human T-cell leukemia virus type I Tax interacts directly with IkappaB kinase gamma.

Mechanisms by which the human T-cell leukemia virus type I Tax oncoprotein activates NF-kappaB remain incompletely understood. Although others have described an interaction between Tax and a holo-IkappaB kinase (IKK) complex, the exact details of protein-protein contact are not fully defined. Here we show that Tax binds to neither IKK-alpha nor IKK-beta but instead complexes directly with IKK-gamma, a newly characterized component of the IKK complex. This direct interaction with IKK-gamma correlates with Tax-induced IkappaB-alpha phosphorylation and NF-kappaB activation. Thus, our findings establish IKK-gamma as a key molecule for adapting an oncoprotein-specific signaling to IKK-alpha and IKK-beta.     

NF-kappaB activation and IkappaB alpha dynamism involved in iNOS and chemokine induction in astroglial cells

This review will discuss the recent literature on the molecular mechanism of NF-kappaB activation, with special focus on IkappaB alpha dynamism involved in iNOS- and chemokine-induction in glial cells. NF-kappaB, a heterotrimer composed of p50, p65 (Rel A) and IkappaB alpha, has been shown to be activated by elimination of the regulatory subunit IkappaB alpha from the heterotrimer. The elimination of IkappaB alpha (formation of active NF-kappaB, p50-p65) is due to phosplorylation of serines 32 and 36 of IkappaB alpha, followed by polyubiquitination and 26S proteasomal degradation of IkappaB alpha. Experiments using stable clones of rat C6 glioma cells transfected with dominant negative IkappaB alpha (serines 32 and 36 replaced by alanine) suggest that NF-kappaB activation (phosphorylation of IkappaB alpha) is involved in LPS/IFNgamma- or IL-1beta/IFNgamma-induced iNOS expression. Furthermore, the time courses of phosphorylation, ubiquitination of IkappaB alpha and proteasome activity after IL-1beta treatment also suggest that 26S proteasomal degradation of IkappaB alpha is more crucial for chemokine expression in glial cells.     

Surfactant protein A activation of atypical protein kinase C zeta in IkappaB-alpha-dependent anti-inflammatory immune regulation

The pulmonary collectin surfactant protein (SP)-A has a pivotal role in anti-inflammatory modulation of lung immunity. The mechanisms underlying SP-A-mediated inhibition of LPS-induced NF-kappaB activation in vivo and in vitro are only partially understood. We previously demonstrated that SP-A stabilizes IkappaB-alpha, the primary regulator of NF-kappaB, in alveolar macrophages (AM) both constitutively and in the presence of LPS. In this study, we show that in AM and PBMC from IkappaB-alpha knockout/IkappaB-beta knockin mice, SP-A fails to inhibit LPS-induced TNF-alpha production and p65 nuclear translocation, confirming a critical role for IkappaB-alpha in SP-A-mediated LPS inhibition. We identify atypical (a) protein kinase C (PKC) zeta as a pivotal upstream regulator of SP-A-mediated IkappaB-alpha/NF-kappaB pathway modulation deduced from blocking experiments and confirmed by using AM from PKCzeta-/- mice. SP-A transiently triggers aPKCThr(410/403) phosphorylation, aPKC kinase activity, and translocation in primary rat AM. Coimmunoprecipitation experiments reveal that SP-A induces aPKC/p65 binding under constitutive conditions. Together the data indicate that anti-inflammatory macrophage activation via IkappaB-alpha by SP-A critically depends on PKCzeta activity, and thus attribute a novel, stimulus-specific signaling function to PKCzeta in SP-A-modulated pulmonary immune response.