Aminosalicylic acid inhibits IkappaB kinase alpha phosphorylation of IkappaBalpha in mouse intestinal epithelial cells

Tumor necrosis factor alpha (TNFalpha)-stimulated nuclear factor (NF) kappaB activation plays a key role in the pathogenesis of inflammatory bowel disease (IBD). Phosphorylation of NFkappaB inhibitory protein (IkappaB) leading to its degradation and NFkappaB activation, is regulated by the multimeric IkappaB kinase complex, including IKKalpha and IKKbeta. We recently reported that 5-aminosalicylic acid (5-ASA) inhibits TNFalpha-regulated IkappaB degradation and NFkappaB activation. To determine the mechanism of 5-ASA inhibition of IkappaB degradation, we studied young adult mouse colon (YAMC) cells by immunodetection and in vitro kinase assays. We show 5-ASA inhibits TNFalpha-stimulated phosphorylation of IkappaBalpha in intact YAMC cells. Phosphorylation of a glutathione S-transferase-IkappaBalpha fusion protein by cellular extracts or immunoprecipitated IKKalpha isolated from cells treated with TNFalpha is inhibited by 5-ASA. Recombinant IKKalpha and IKKbeta autophosphorylation and their phosphorylation of glutathione S-transferase-IkappaBalpha are inhibited by 5-ASA. However, IKKalpha serine phosphorylation by its upstream kinase in either intact cells or cellular extracts is not blocked by 5-ASA. Surprisingly, immunodepletion of cellular extracts suggests IKKalpha is predominantly responsible for IkappaBalpha phosphorylation in intestinal epithelial cells. In summary, 5-ASA inhibits TNFalpha-stimulated IKKalpha kinase activity toward IkappaBalpha in intestinal epithelial cells. These findings suggest a novel role for 5-ASA in the management of IBD by disrupting TNFalpha activation of NFkappaB.     

Nuclear factor-inducing kinase plays a crucial role in osteopontin-induced MAPK/IkappaBalpha kinase-dependent nuclear factor kappaB-mediated promatrix metalloproteinase-9 activation

We have recently demonstrated that osteopontin (OPN) induces nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 activation through IkappaBalpha/IkappaBalpha kinase (IKK) signaling pathways. However, the molecular mechanism(s) by which OPN regulates promatrix metalloproteinase-9 (pro-MMP-9) activation, MMP-9-dependent cell motility, and tumor growth and the involvement of upstream kinases in regulation of these processes in murine melanoma cells are not well defined. Here we report that OPN induced alpha(v)beta(3) integrin-mediated phosphorylation and activation of nuclear factor-inducing kinase (NIK) and enhanced the interaction between phosphorylated NIK and IKKalpha/beta in B16F10 cells. Moreover, NIK was involved in OPN-induced phosphorylations of MEK-1 and ERK1/2 in these cells. OPN induced NIK-dependent NFkappaB activation through ERK/IKKalpha/beta-mediated pathways. Furthermore OPN enhanced NIK-regulated urokinase-type plasminogen activator (uPA) secretion, uPA-dependent pro-MMP-9 activation, cell motility, and tumor growth. Wild type NIK, IKKalpha/beta, and ERK1/2 enhanced and kinase-negative NIK (mut NIK), dominant negative IKKalpha/beta (dn IKKalpha/beta), and dn ERK1/2 suppressed the OPN-induced NFkappaB activation, uPA secretion, pro-MMP-9 activation, cell motility, and chemoinvasion. Pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibody drastically inhibited the OPN-induced cell migration and chemoinvasion, whereas cells pretreated with anti-MMP-2 antibody had no effect on OPN-induced pro-MMP-9 activation suggesting that OPN induces pro-MMP-2 and pro-MMP-9 activations through two distinct pathways. The level of active MMP-9 in the OPN-induced tumor was higher compared with control. To our knowledge, this is the first report that NIK plays a crucial role in OPN-induced NFkappaB activation, uPA secretion, and pro-MMP-9 activation through MAPK/IKKalpha/beta-mediated pathways, and all of these ultimately control the cell motility, invasiveness, and tumor growth.     

Interleukin-1 (IL-1)-induced TAK1-dependent Versus MEKK3-dependent NFkappaB activation pathways bifurcate at IL-1 receptor-associated kinase modification

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is phosphorylated after it is recruited to the receptor, subsequently ubiquitinated, and eventually degraded upon IL-1 stimulation. Although a point mutation changing lysine 134 to arginine (K134R) in IRAK abolished IL-1-induced IRAK ubiquitination and degradation, mutations of serines and threonines adjacent to lysine 134 to alanines ((S/T)A (131-144)) reduced IL-1-induced IRAK phosphorylation and abolished IRAK ubiquitination. Through the study of these IRAK modification mutants, we uncovered two parallel IL-1-mediated signaling pathways for NFkappaB activation, TAK1-dependent and MEKK3-dependent, respectively. These two pathways bifurcate at the level of IRAK modification. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classical NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through IkappaBalpha phosphorylation and subsequent dissociation from NFkappaB but without IkappaBalpha degradation. These results provide significant insight to our further understanding of NFkappaB activation pathways.     

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.     

The kinase activity of IL-1 receptor-associated kinase 4 is required for interleukin-1 receptor/toll-like receptor-induced TAK1-dependent NFkappaB activation

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.     

Detection of a novel parasite kinase activity at the Toxoplasma gondii parasitophorous vacuole membrane capable of phosphorylating host IkappaBalpha

Toxoplasma gondii activates the NF-kappaB pathway in the infected host cell resulting in upregulation of pro-survival genes and prevention of apoptosis. Manipulation of the NF-kappaB cascade by T. gondii correlates with the localization of phosphorylated IkappaB at the parasitophorous vacuole membrane (PVM). This suggests a parasite-mediated event, involving the recruitment and activation of the host IkappaB kinase (IKK) complex, as has been observed with the related protozoan Theileria parva. In contrast to Theileria, confocal microscopy studies showed no apparent hijacking of IKKalpha, IKKbeta, or their activated phosphorylated forms at the T. gondii PVM. Remarkably, phosphorylation of IkappaBalpha at Ser 32/36 was observed at the PVM of T. gondii-infected IKKalpha-/-, IKKbeta-/- and IKKalpha/beta double-knockout (IKKalpha/beta-/-) fibroblasts, suggesting the involvement of a parasite kinase activity independent of host IKK. The presence of a putative T. gondii IkappaB kinase was examined by in vitro kinase assays using GST-IkappaBalpha constructs and protein extracts from both extracellular parasites and PVM fractions. Interestingly, an activity capable of phosphorylating IkappaBalpha at the critical Ser 32/36 sites was identified in parasite extracts, a property restricted to the IKK signalosome. Taken together, our data support the role for a T. gondii kinase involved in phosphorylation of host cell IkappaBalpha and suggest an unusual mechanism utilized by an intracellular pathogen capable of manipulating the NF-kappaB pathway.     

AMP-activated protein kinase is involved in COX-2 expression in response to ultrasound in cultured osteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. Cyclooxygenase-2 (COX-2) is a crucial mediator in mechanically induced bone formation. AMP-activated protein kinase (AMPK) has reported to sense and regulate the cellular energy status in various cell types. Here we found that US-mediated COX-2 expression was attenuated by LKB1 and AMPKalpha1 small interference RNA (siRNA) in human osteoblasts. Pretreatment of osteoblasts with AMPK inhibitor (araA and compound C), p38 inhibitor (SB203580), NF-kappaB inhibitor (PDTC), IkappaB protease inhibitor (TPCK) and NF-kappaB inhibitor peptide also inhibited the potentiating action of US. US increased the kinase activity and phosphorylation of LKB1, AMPK and p38. Stimulation of osteoblasts with US activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. US-mediated an increase of IKKalpha/beta activity, kappaB-luciferase activity and p65 and p50 binding to the NF-kappaB element was inhibited by araA, SB203580 and LKB1 siRNA. Our results suggest that US increased COX-2 expression in osteoblasts via the LKB1/AMPKalpha1/p38/IKKalphabeta and NF-kappaB signaling pathway.     

Hyperosmotic stress activates p65/RelB NFkappaB in cultured cardiomyocytes with dichotomic actions on caspase activation and cell death

NFkappaB is a participant in the process whereby cells adapt to stress. We have evaluated the activation of NFkappaB pathway by hyperosmotic stress in cultured cardiomyocytes and its role in the activation of caspase and cell death. Exposure of cultured rat cardiomyocytes to hyperosmotic conditions induced phosphorylation of IKKalpha/beta as well as degradation of IkappaBalpha. All five members of the NFkappaB family were identified in cardiomyocytes. Analysis of the subcellular distribution of NFkappaB isoforms in response to hyperosmotic stress showed parallel migration of p65 and RelB from the cytosol to the nucleus. Measurement of the binding of NFkappaB to the consensus DNA kappaB-site binding by EMSA revealed an oscillatory profile with maximum binding 1, 2 and 6h after initiation of the hyperosmotic stress. Supershift analysis revealed that p65 and RelB (but not p50, p52 or cRel) were involved in the binding of NFkappaB to DNA. Hyperosmotic stress also resulted in activation of the NFkappaB-lux reporter gene, transient activation of caspases 9 and 3 and phosphatidylserine externalization. The effect on cell viability was not prevented by ZVAD (a general caspase inhibitor). Blockade of NFkappaB with AdIkappaBalpha, an IkappaBalpha dominant negative overexpressing adenovirus, prevented activation of caspase 9 (more than that caspase 3) but did not affect cell death in hyperosmotically stressed cardiomyocytes. We conclude that hyperosmotic stress activates p65 and RelB NFkappaB isoforms and NFkappaB mediates caspase 9 activation in cardiomyocytes. However cell death triggered by hyperosmotic stress was caspase- and NFkappaB-independent.     

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.     

Stress-induced inhibition of the NF-kappaB signaling pathway results from the insolubilization of the IkappaB kinase complex following its dissociation from heat shock protein 90

Activation of the stress response attenuates proinflammatory responses by suppressing cytokine-stimulated activation of the NF-kappaB signaling pathway. In this study, we show that the activation of the cellular stress response, either by heat shock treatment or after exposure to sodium arsenite, leads to a transient inhibition of IkappaBalpha phosphorylation. Inhibition of IkappaBalpha phosphorylation after stress was associated with the detergent insolubilization of the upstream kinases, IkappaB kinase alpha (IKKalpha) and IkappaB kinase beta, components involved in IkappaBalpha phosphorylation. Pretreatment of cells with glycerol, a chemical chaperone that reduces the extent of stress-induced protein denaturation, reduced the stress-dependent detergent insolubility of the IKK complex and restored the cytokine-stimulated phosphorylation of IkappaB. The stress-dependent insolubility of the IKK complex appeared reversible; as the cells recovered from the heat shock treatment, the IKK complex reappeared within the soluble fraction of cells and was again capable of mediating the phosphorylation of IkappaBalpha in response to added cytokines. Treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) function, also resulted in IKK detergent insolubility and proteasome-mediated degradation of the IKK complex. Furthermore, while IKKalpha coprecipitated with Hsp90 in control cells, coprecipitation of the two proteins was greatly reduced in those cells early after stress or following exposure to geldanamycin. Stress-induced transient insolubilization of the IkappaB kinase complex following its dissociation from Hsp90 represents a novel mechanism by which the activation of the stress response inhibits the NF-kappaB signaling pathway in response to proinflammatory stimuli.     

Interleukin-1-induced NF-kappaB activation is NEMO-dependent but does not require IKKbeta

Activation of NF-kappaB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IkappaB kinase (IKK) complex, which contains two kinases named IKKalpha and IKKbeta and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKbeta is required for TNF-induced NF-kappaB activation. To determine whether NEMO and IKKalpha can form a functional IKK complex capable of activating the classical NF-kappaB pathway in the absence of IKKbeta, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IkappaBalpha degradation absolutely requires NEMO and IKKbeta. In contrast, we consistently observed intact IkappaBalpha degradation and NF-kappaB activation in response to IL-1 in two separate cell lines lacking IKKbeta. Furthermore, exogenously expressed, catalytically inactive IKKbeta blocked TNF- but not IL-1-induced IkappaBalpha degradation in wild-type MEFs, and reconstitution of IKKalpha/beta double knockout cells with IKKalpha rescued IL-1- but not TNF-induced NF-kappaB activation. Finally, we have shown that incubation of IKKbeta-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-kappaB activation. Our results therefore demonstrate that NEMO and IKKalpha can form a functional IKK complex that activates the classical NF-kappaB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways.     

Ikappakappa mediates NF-kappaB activation in human immunodeficiency virus-infected cells

Human monocytes and macrophages are persistent reservoirs of human immunodeficiency virus (HIV) type-1. Persistent HIV infection of these cells results in increased levels of NF-kappaB in the nucleus secondary to increased IkappaBalpha, IkappaBbeta, and IkappaBepsilon degradation, a mechanism postulated to regulate viral persistence. To characterize the molecular mechanisms regulating HIV-mediated degradation of IkappaB, we have sought to identify the regulatory domains of IkappaBalpha targeted by HIV infection. Using monocytic cells stably expressing different transdominant molecules of IkappaBalpha, we determined that persistent HIV infection of these cells targets the NH2 but not the COOH terminus of IkappaBalpha. Further analysis demonstrated that phosphorylation at S32 and S36 is necessary for HIV-dependent IkappaBalpha degradation and NF-kappaB activation. Of the putative N-terminal IkappaBalpha kinases, we demonstrated that the Ikappakappa complex, but not p90(rsk), is activated by HIV infection and mediates HIV-dependent NF-kappaB activation. Analysis of viral replication in cells that constitutively express IkappaBalpha negative transdominant molecules demonstrated a lack of correlation between virus-induced NF-kappaB (p65/p50) nuclear translocation and degree of viral persistence in human monocytes.     

Constitutive nuclear expression of the I kappa B kinase complex and its activation in human neutrophils

A singular feature of human neutrophils is that they constitutively express substantial amounts of NF-kappaB/Rel proteins and IkappaB-alpha in the nucleus. In this study, we show that in these cells, IkappaB kinase alpha (IKKalpha), IKKbeta, and IKKgamma also partially localize to the nucleus, whereas IKK-related kinases (IKKepsilon, TANK-binding kinase-1) are strictly cytoplasmic, and the NF-kappaB-inducing kinase is strictly nuclear. Following neutrophil activation, IKKbeta and IKKgamma become transiently phosphorylated in both the cytoplasm and nucleus, whereas IKKalpha transiently vanishes from both compartments in what appears to be an IKKbeta-dependent process. These responses are paralleled by the degradation of IkappaB-alpha, and by the phosphorylation of RelA on serine 536, in both compartments. Although both proteins can be IKK substrates, inhibition of IKK prevented IkappaB-alpha phosphorylation, while that of RelA was mostly unaffected. Finally, we provide evidence that the nuclear IKK isoforms (alpha, beta, gamma) associate with chromatin following neutrophil activation, which suggests a potential role in gene regulation. This is the first study to document IKK activation and the phosphorylation of NF-kappaB/Rel proteins in primary neutrophils. More importantly, our findings unveil a hitherto unsuspected mode of activation for the IKK/IkappaB signaling cascade within the cell nucleus.     

Okadaic acid induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha in human neutrophils

Human neutrophils differ from other cells by containing high amount of IkappaBalpha in the nucleus, and this increased nuclear IkappaBalpha accumulation is associated with the inhibition of NFkappaB activity and increased apoptosis. However, the mechanisms regulating NFkappaB activation and IkappaBalpha degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFkappaB activity and IkappaBalpha degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-delta (PKCdelta) and IkappaB kinase (IKK) inhibit the OA-induced activation of NFkappaB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFkappaB is regulated by a continuous phosphorylation and degradation of the nuclear IkappaBalpha.     

Inhibition of IkappaB kinase by vaccinia virus virulence factor B14

The IkappaB kinase (IKK) complex is a key regulator of signal transduction pathways leading to the induction of NF-kappaB-dependent gene expression and production of pro-inflammatory cytokines. It therefore represents a major target for the development of anti-inflammatory therapeutic drugs and may be targeted by pathogens seeking to diminish the host response to infection. Previously, the vaccinia virus (VACV) strain Western Reserve B14 protein was characterised as an intracellular virulence factor that alters the inflammatory response to infection by an unknown mechanism. Here we demonstrate that ectopic expression of B14 inhibited NF-kappaB activation in response to TNFalpha, IL-1beta, poly(I:C), and PMA. In cells infected with VACV lacking gene B14R (vDeltaB14) there was a higher level of phosphorylated IkappaBalpha but a similar level of IkappaBalpha compared to cells infected with control viruses expressing B14, suggesting B14 affects IKK activity. Direct evidence for this was obtained by showing that B14 co-purified and co-precipitated with the endogenous IKK complex from human and mouse cells and inhibited IKK complex enzymatic activity. Notably, the interaction between B14 and the IKK complex required IKKbeta but not IKKalpha, suggesting the interaction occurs via IKKbeta. B14 inhibited NF-kappaB activation induced by overexpression of IKKalpha, IKKbeta, and a constitutively active mutant of IKKalpha, S176/180E, but did not inhibit a comparable mutant of IKKbeta, S177/181E. This suggested that phosphorylation of these serine residues in the activation loop of IKKbeta is targeted by B14, and this was confirmed using Ab specific for phospho-IKKbeta.