Tumor necrosis factor-alpha-induced IKK phosphorylation of NF-kappaB p65 on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway

The activation of NF-kappaB has been shown to be regulated by multiple phosphorylations of IkappaBs and the NF-kappaB p65 subunit. Here, we characterized the intracellular signaling pathway leading to phosphorylation of p65 on Ser-536 using a novel anti-phospho-p65 (Ser-536) antibody. The Ser-536 of endogenous p65 was rapidly phosphorylated in response to a wide variety of NF-kappaB stimulants including TNF-alpha in the cytoplasm and rapidly dephosphorylated in the nucleus. The TNF-alpha-but not IL-1beta-induced Ser-536 phosphorylation was severely impaired in murine embryonic fibroblasts derived from traf2-/-traf5-/- mice. Bay 11-7082, an inhibitor of IkappaB phosphorylation, inhibited the TNF-alpha-induced phosphorylation in vivo. In addition, overexpression of TGF-beta-activated kinase 1 (TAK1), IKKalpha and IKKbeta stimulated the phosphorylation, and their dominant negative mutants blocked the TNF-alpha-induced phosphorylation. Moreover, small interfering RNAs (siRNAs) against TAK1, IKKalpha and IKKbeta blocked the phosphorylation of endogenous p65. On the other hand, calyculin-A, a protein phosphatase inhibitor, blocked the dephosphorylation in the nucleus in vivo. These results indicate that similar signaling pathways were utilized for the phosphorylations of IkappaBalpha and p65, which further support the idea that both IkappaB and NF-kappaB are substrates for the IKK complex in the activation of NF-kappaB.     

Arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1 beta-stimulated Caco-2 cells independent of the heat shock response

Recent studies suggest that sodium arsenite downregulates NF-kappaB activity by inhibiting phosphorylation and subsequent degradation of IkappaBalpha. Many effects of sodium arsenite are secondary to induction of heat shock proteins. The role of the heat shock response in arsenite-induced inhibition of NF-kappaB, however, is not known. We examined the involvement of the heat shock response in arsenite-induced inhibition of NF-kappaB activity in IL-1beta-stimulated Caco-2 cells, a human colorectal adenocarcinoma cell line with enterocytic properties. Treatment of the cells with IL-1beta resulted in increased IkappaB kinase activity, reduced levels of IkappaBalpha and increased NF-kappaB DNA binding activity. Sodium arsenite blocked all of these responses to IL-1beta without inducing changes in heat shock factor activity or heat shock protein levels. Results from additional experiments showed that the protective effect of sodium arsenite on IkappaBalpha was not influenced by the oxygen radical scavenger catalase or by inhibitors of the MAP-kinase signaling pathway. The present results suggest that sodium arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1beta-stimulated Caco-2 cells independent of the heat shock response. In addition, stabilization of IkappaBalpha by sodium arsenite does not require oxygen radical formation or activation of the MAP kinase signaling pathway.     

Theileria-induced constitutive IKK activation is independent of functional Hsp90

The intracellular parasite Theileria induces uncontrolled proliferation and host cell transformation. Parasite-induced transformation is accompanied by constitutive activation of IkappaB kinase (IKK), resulting in permanently high levels of activated nuclear factor (NF)-kappaB. IKK activation pathways normally require heat shock protein 90 (Hsp90), a chaperone that regulates the stability and activity of signalling molecules and can be blocked by the benzoquinone ansamycin compound geldanamycin (GA). In Theileria-transformed cells, IkappaBalpha and p65 phosphorylation, NF-kappaB nuclear translocation and DNA binding activity are largely resistant to GA and also NF-kappaB-dependent reporter gene expression is only partly affected. Our findings indicate that parasite-induced IKK activity does not require functional Hsp90.     

Distinct roles of IkappaB proteins in regulating constitutive NF-kappaB activity

The inhibitor of NF-kappaB (IkappaB) family of proteins is believed to regulate NF-kappaB activity by cytoplasmic sequestration. We show that in cells depleted of IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins, a small fraction of p65 binds DNA and leads to constitutive activation of NF-kappaB target genes, even without stimulation, whereas most of the p65 remains cytoplasmic. These results indicate that although IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins could be dispensable for cytoplasmic retention of NF-kappaB, they are essential for preventing NF-kappaB-dependent gene expression in the basal state. We also show that in the absence of IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins, cytoplasmic retention of NF-kappaB by other cellular proteins renders the pathway unresponsive to activation.     

IkappaB kinase alpha (IKKalpha) regulation of IKKbeta kinase activity

Two related kinases, IkappaB kinase alpha (IKKalpha) and IKKbeta, phosphorylate the IkappaB proteins, leading to their degradation and the subsequent activation of gene expression by NF-kappaB. IKKbeta has a much higher level of kinase activity for the IkappaB proteins than does IKKalpha and is more critical than IKKalpha in modulating tumor necrosis factor alpha activation of the NF-kappaB pathway. These results indicate an important role for IKKbeta in activating the NF-kappaB pathway but leave open the question of the role of IKKalpha in regulating this pathway. In the current study, we demonstrate that IKKalpha directly phosphorylates IKKbeta. Moreover, IKKalpha either directly or indirectly enhances IKKbeta kinase activity for IkappaBalpha. Finally, transfection studies to analyze NF-kappaB-directed gene expression suggest that IKKalpha is upstream of IKKbeta in activating the NF-kappaB pathway. These results indicate that IKKalpha, in addition to its previously described ability to phosphorylate IkappaBalpha, can increase the ability of IKKbeta to phosphorylate IkappaBalpha.     

Simvastatin potentiates TNF-alpha-induced apoptosis through the down-regulation of NF-kappaB-dependent antiapoptotic gene products: role of IkappaBalpha kinase and TGF-beta-activated kinase-1

Numerous recent reports suggest that statins (hydroxy-3-methylglutaryl-CoA reductase inhibitors) exhibit potential to suppress tumorigenesis through a mechanism that is not fully understood. Therefore, in this article, we investigated the effects of simvastatin on TNF-alpha-induced cell signaling. We found that simvastatin potentiated the apoptosis induced by TNF-alpha as indicated by intracellular esterase activity, caspase activation, TUNEL, and annexin V staining. This effect of simvastatin correlated with down-regulation of various gene products that mediate cell proliferation (cyclin D1 and cyclooxygenase-2), cell survival (Bcl-2, Bcl-x(L), cellular FLIP, inhibitor of apoptosis protein 1, inhibitor of apoptosis protein 2, and survivin), invasion (matrix mellatoproteinase-9 and ICAM-1), and angiogenesis (vascular endothelial growth factor); all known to be regulated by the NF-kappaB. We found that simvastatin inhibited TNF-alpha-induced NF-kappaB activation, and l-mevalonate reversed the suppressive effect, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. Simvastatin suppressed not only the inducible but also the constitutive NF-kappaB activation. Simvastatin inhibited TNF-alpha-induced IkappaBalpha kinase activation, which led to inhibition of IkappaBalpha phosphorylation and degradation, suppression of p65 phosphorylation, and translocation to the nucleus. NF-kappaB-dependent reporter gene expression induced by TNF-alpha, TNFR1, TNFR-associated death domain protein, TNFR-associated factor 2, TGF-beta-activated kinase 1, receptor-interacting protein, NF-kappaB-inducing kinase, and IkappaB kinase beta was abolished by simvastatin. Overall, our results provide novel insight into the role of simvastatin in potentially preventing and treating cancer through modulation of IkappaB kinase and NF-kappaB-regulated gene products.     

Short-term modulation of interleukin-1beta signaling by hyperoxia: uncoupling of IkappaB kinase activation and NF-kappaB-dependent gene expression

We have been interested in elucidating how simultaneous stimuli modulate inflammation-related signal transduction pathways in lung parenchymal cells. We previously demonstrated that exposing respiratory epithelial cells to 95% oxygen (hyperoxia) synergistically increased tumor necrosis factor-alpha (TNF-alpha)-mediated activation of NF-kappaB and NF-kappaB-dependent gene expression by a mechanism involving increased activation of IkappaB kinase (IKK). Because the signal transduction mechanisms induced by IL-1beta are distinct to that of TNF-alpha, herein we sought to determine whether hyperoxia modulates IL-1beta-dependent signal transduction. In A549 cells, simultaneous treatment with hyperoxia and IL-1beta caused increased activation of IKK, prolonged the degradation of IkappaBalpha, and prolonged the nuclear translocation and DNA binding of NF-kappaB compared with cells treated with IL-1beta alone in room air. Hyperoxia did not affect IL-1beta-dependent degradation of the interleukin receptor-associated kinase differently from treatment with IL-beta alone. In contrast to the effects on the IKK/IkappaBalpha/NF-kappaB pathway, simultaneous treatment with hyperoxia and IL-1beta did not augment NF-kappaB-dependent gene expression compared with treatment with IL-1beta alone. Similar observations were made in a different human respiratory epithelial cell line, BEAS-2B cells. In addition, simultaneous treatment with hyperoxia and IL-1beta caused hyperphosphorlyation of the NF-kappaB p65 subunit compared with treatment with IL-1beta alone. In summary, concomitant treatment of A549 cells with hyperoxia and IL-1beta augments activation of IKK, prolongs degradation of IkappaBalpha, and prolongs nuclear translocation and DNA binding of NF-kappaB. This activation, however, is not coupled to increased expression of NF-kappaB-dependent genes, and the mechanism of this decoupling is not related to decreased phosphorylation of p65.     

Artemisolide is a typical inhibitor of IkappaB kinase beta targeting cysteine-179 residue and down-regulates NF-kappaB-dependent TNF-alpha expression in LPS-activated macrophages

Nuclear factor (NF)-kappaB regulates a central common signaling for immunity and cell survival. Artemisolide (ATM) was previously isolated as a NF-kappaB inhibitor from a plant of Artemisia asiatica. However, molecular basis of ATM on NF-kappaB activation remains to be defined. Here, we demonstrate that ATM is a typical inhibitor of IkappaB kinase beta (IKKbeta), resulting in inhibition of lipopolysaccharide (LPS)-induced NF-kappaB activation in RAW 264.7 macrophages. ATM inhibited the kinase activity of highly purified IKKbeta and also LPS-induced IKK activity in the cells. Moreover, the effect of ATM on IKKbeta activity was completely abolished by substitution of Cys-179 residue of IKKbeta to Ala residue, indicating direct targeting site of ATM. ATM could inhibit IkappaBalpha phosphorylation in LPS-activated RAW 264.7 cells and subsequently prevent NF-kappaB activation. Further, we demonstrate that ATM down-regulates NF-kappaB-dependent TNF-alpha expression. Taken together, this study provides a pharmacological potential of ATM in NF-kappaB-dependent inflammatory disorders.     

Acute alcohol exposure exerts anti-inflammatory effects by inhibiting IkappaB kinase activity and p65 phosphorylation in human monocytes

Acute alcohol use is associated with impaired immune responses and decreased proinflammatory cytokine production. Our earlier studies have shown that acute alcohol intake inhibits NF-kappaB DNA binding in an IkappaBalpha-independent manner. We report using human peripheral blood monocytes and Chinese hamster ovary cells transfected with CD14 cells that acute alcohol treatment in vitro exerts NF-kappaB inhibition by disrupting phosphorylation of p65. Immunoprecipitation of p65 and IkappaBalpha revealed that acute alcohol exposure for 1 h decreased NF-kappaB-IkappaBalpha complexes in the cytoplasm. Phosphorylation of p65 at Ser(536) is mediated by IkappaB kinase (IKK)beta and is required for NF-kappaB-dependent cellular responses. We show that acute alcohol treatment decreased LPS-induced IKKalpha and IKKbeta activity resulting in decreased phosphorylation of p65 at Ser(536). Furthermore, nuclear expression of IKKalpha increased after alcohol treatment, which may contribute to inhibition of NF-kappaB. Decreased phosphorylation of nuclear p65 at Ser(276) was likely not due to alcohol-induced inhibition of protein kinase A and mitogen- and stress-activated protein kinase-1 activity. Although decreased IkappaBalpha phosphorylation after acute alcohol treatment was attributable to reduced IKKbeta activity, degradation of IkappaBalpha during alcohol exposure was IKKbeta-independent. Alcohol-induced degradation of IkappaBalpha in the presence of a 26S proteasome inhibitor suggested proteasome-independent IkappaBalpha degradation. Collectively, our studies suggest that acute alcohol exposure modulates IkappaBalpha-independent NF-kappaB activity primarily by affecting phosphorylation of p65. These findings further implicate an important role for IKKbeta in the acute effects of alcohol in immune cells.     

Thalidomide suppresses NF-kappa B activation induced by TNF and H2O2, but not that activated by ceramide, lipopolysaccharides, or phorbol ester

Thalidomide ([+]-alpha-phthalimidoglutarimide), a psychoactive drug that readily crosses the blood-brain barrier, has been shown to exhibit anti-inflammatory, antiangiogenic, and immunosuppressive properties through a mechanism that is not fully established. Due to the central role of NF-kappaB in these responses, we postulated that thalidomide mediates its effects through suppression of NF-kappaB activation. We investigated the effects of thalidomide on NF-kappaB activation induced by various inflammatory agents in Jurkat cells. The treatment of these cells with thalidomide suppressed TNF-induced NF-kappaB activation, with optimum effect occurring at 50 microg/ml thalidomide. These effects were not restricted to T cells, as other hematopoietic and epithelial cell types were also inhibited. Thalidomide suppressed H(2)O(2)-induced NF-kappaB activation but had no effect on NF-kappaB activation induced by PMA, LPS, okadaic acid, or ceramide, suggesting selectivity in suppression of NF-kappaB. The suppression of TNF-induced NF-kappaB activation by thalidomide correlated with partial inhibition of TNF-induced degradation of an inhibitory subunit of NF-kappaB (IkappaBalpha), abrogation of IkappaBalpha kinase activation, and inhibition of NF-kappaB-dependent reporter gene expression. Thalidomide abolished the NF-kappaB-dependent reporter gene expression activated by overexpression of TNFR1, TNFR-associated factor-2, and NF-kappaB-inducing kinase, but not that activated by the p65 subunit of NF-kappaB. Overall, our results clearly demonstrate that thalidomide suppresses NF-kappaB activation specifically induced by TNF and H(2)O(2) and that this may contribute to its role in suppression of proliferation, inflammation, angiogenesis, and the immune system.     

Carbachol activates IkappaB kinase in isolated canine gastric parietal cells.

IkappaB kinase (IKK) is a recently discovered kinase complex composed of the kinases IKKalpha and beta, which plays a crucial role in the activation of NF-kappaB. In this study we examined the regulation of IKK by carbachol in isolated gastric parietal cells. IKKalpha and beta activities were measured by immune complex kinase assay. Carbachol induced both IKK alpha and beta in a time-dependent fashion, with a maximal stimulatory effect detected after 5 min of incubation. The action of carbachol was inhibited by the intracellular Ca(++) chelator BAPTA-AM, the PKC inhibitor GF109203X, and the NF-kappaB inhibitor PDTC. Carbachol also induced degradation of IkappaBalpha, which was reversed by addition of both GF109203X and PDTC and stimulated the activity of a NF-kappaB-luciferase reporter gene plasmid in COS-7 cells stably expressing the human M3 muscarinic receptor. In conclusion, carbachol induces IKK in the parietal cells via intracellular Ca(++)- and PKC-dependent signaling pathways. This observation represents a novel mechanism for the regulation of NF-kappaB through the activation of seven transmembrane G-protein-coupled receptors.     

CK2 Is a C-Terminal IkappaB Kinase Responsible for NF-kappaB Activation during the UV Response

NF-kappaB is activated in response to proinflammatory stimuli, infections, and physical stress. While activation of NF-kappaB by many stimuli depends on the IkappaB kinase (IKK) complex, which phosphorylates IkappaBs at N-terminal sites, the mechanism of NF-kappaB activation by ultraviolet (UV) radiation remained enigmatic, as it is IKK independent. We now show that UV-induced NF-kappaB activation depends on phosphorylation of IkappaBalpha at a cluster of C-terminal sites that are recognized by CK2 (formerly casein kinase II). Furthermore, CK2 activity toward IkappaB is UV inducible through a mechanism that depends on activation of p38 MAP kinase. Inhibition of this pathway prevents UV-induced IkappaBalpha degradation and increases UV-induced cell death. Thus, the p38-CK2-NF-kappaB axis is an important component of the mammalian UV response.     

Inhibition of hepatitis B virus replication by MyD88 is mediated by nuclear factor-kappaB activation

In our previous paper, we reported that myeloid differential primary response protein (MyD88), a key adaptor in the signaling cascade of the innate immune response, inhibits hepatitis B virus (HBV) replication. The MyD88 activated nuclear factor-kappaB (NF-kappaB) signaling pathway and the intracellular upregulation of NF-kappaB signaling can induce an antiviral effect. Therefore, the association between the inhibition of HBV replication by MyD88 and NF-kappaB activation was investigated further. The results show that NF-kappaB activation was moderately increased after MyD88 expression. The strong activation of NF-kappaB by the IkappaB kinase complex IKKalpha/IKKbeta dramatically suppressed HBV replication; the MyD88 dominant negative mutant that abrogated NF-kappaB activity did not inhibit HBV replication. Furthermore, the IkappaBalpha dominant negative mutant restored the inhibition of HBV replication by MyD88. These results support a role for NF-kappaB activation in the inhibition of HBV replication and suggest a novel mechanism for the inhibition of HBV replication by MyD88 protein.