Involvement of p42/p44 MAPK, JNK, and NF-kappaB in IL-1beta-induced ICAM-1 expression in human pulmonary epithelial cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells and contributes to inflammatory responses. However, the mechanisms regulating ICAM-1 expression by IL-1beta in human A549 cells was not completely understood. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB pathways for IL-1beta-induced ICAM-1 expression were investigated in A549 cells. IL-1beta induced expression of ICAM-1 protein and mRNA in a time- and concentration-dependent manner. The IL-1beta induction of ICAM-1 mRNA and protein were partially inhibited by U0126 and PD98059 (specific inhibitors of MEK1/2) and SP600125 [a specific inhibitor of c-Jun-N-terminal kinase (JNK)]. U0126 was more potent than other inhibitors to attenuate IL-1beta-induced ICAM-1 expression. Consistently, IL-1beta stimulated phosphorylation of p42/p44 MAPK and JNK which was attenuated by pretreatment with U0126 or SP600125, respectively. Moreover, transfection with dominant negative mutants of MEK1/2 (MEK K97R) or ERK2 (ERK2 K52R) also attenuated IL-1beta-induced ICAM-1 expression. The combination of PD98059 and SP600125 displayed an additive effect on IL-1beta-induced ICAM-1 gene expression. IL-1beta-induced ICAM-1 expression was almost completely blocked by a specific NF-kappaB inhibitor helenalin. Consistently, IL-1beta stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha which was blocked by helenalin, U0126, or SP600125. Taken together, these results suggest that activation of p42/p44 MAPK and JNK cascades, at least in part, mediated through NF-kappaB pathway is essential for IL-1beta-induced ICAM-1 gene expression in A549 cells. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in the airway disease.     

Reciprocal role of ERK and NF-kappaB pathways in survival and activation of osteoclasts

To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-kappaB) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (Ras(DN)), constitutively active MEK1 (MEK(CA)), dominant negative IkappaB kinase 2 (IKK(DN)), and constitutively active IKK2 (IKK(CA)). Inhibiting ERK activity by Ras(DN) overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEK(CA) remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-kappaB pathway with IKK(DN) virus suppressed the pit-forming activity of OCLs and NF-kappaB activation by IKK(CA) expression upregulated it without affecting their survival. Interleukin 1alpha (IL-1alpha) strongly induced ERK activation as well as NF-kappaB activation. Ras(DN) virus partially inhibited ERK activation, and OCL survival promoted by IL-1alpha. Inhibiting NF-kappaB activation by IKK(DN) virus significantly suppressed the pit-forming activity enhanced by IL-1alpha. These results indicate that ERK and NF-kappaB regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-kappaB regulates osteoclast activation for bone resorption.     

Tumor necrosis factor alpha-induced activation of downstream NF-kappaB site of the promoter mediates epithelial ICAM-1 expression and monocyte adhesion. Involvement of PKCalpha, tyrosine kinase, and IKK2, but not MAPKs, pathway

TNF-alpha induced an increase in intercellular adhesion molecule-1 (ICAM-1) expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. The enhanced ICAM-1 expression was shown to increase the adhesion of U937 cells to A549 cells. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D 609) attenuated TNF-alpha-induced ICAM-1 expression. TNF-alpha produced an increase in protein kinase C (PKC) activity and this effect was inhibited by D 609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited TNF-alpha-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression, this effect was inhibited by genistein or tyrphostin 23. Treatment of cells with TNF-alpha resulted in stimulation of p44/42 MAPK, p38, and JNK. However, TNF-alpha-induced ICAM-1 expression was not affected by either MEK inhibitor, PD 98059, or p38 inhibitor, SB 203580. A cell-permeable ceramide analog, C(2) ceramide, also stimulated the activation of these three MAPKs, but had no effect on ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by TNF-alpha and these effects were inhibited by D 609, calphostin C, or tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity, these effects being inhibited by genistein or tyrphostin 23. TNF-alpha- or TPA-induced ICAM-1 promoter activity was inhibited by dominant negative PKCalpha or IKK2, but not IKK1 mutant. IKK activity was stimulated by both TNF-alpha and TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. These data suggest that, in A549 cells, TNF-alpha activates PC-PLC to induce activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression and neutrophil adhesion. However, activation of p44/42 MAPK, p38, and JNK is not involved in this event.     

Interleukin-1beta-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKS,and NF-kappaB pathways in canine tracheal smooth muscle cells

Interleukin-beta (IL-1beta) was found to induce inflammatory responses in the airways, which exerted a potent stimulus for PG synthesis. This study was to determine the mechanisms of IL-1beta-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). IL-1beta markedly increased COX-2 expression and PGE(2) formation in a time- and concentration-dependent manner in TSMCs. Both COX-2 expression and PGE(2) formation in response to IL-1beta were attenuated by a tyrosine kinase inhibitor, genistein, a phosphatidylcholine-phospholipase C inhibitor, D609, a phosphatidylinositol-phospholipase C inhibitor, U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. IL-1beta-induced activation of NF-kappaB correlated with the degradation of IkappaB-alpha in TSMCs. IL-1beta-induced NF-kappaB activation, COX-2 expression, and PGE(2) synthesis were inhibited by the dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. IL-1beta-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 inhibitor), but these two inhibitors had no effect on IL-1beta-induced NF-kappaB activation, indicating that activation of p42/44 and p38 MAPK and NF-kappaB signalling pathways were independently required for these responses. These findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from IL-1beta-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways in canine TSMCs. IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.     

Interleukin-8 production by THP-1 cells stimulated by Salmonella enterica serovar Typhimurium porins is mediated by AP-1, NF-kappaB and MAPK pathways

Interleukin-8 (IL-8) is released in response to inflammatory stimuli, such as bacterial products. Either porins or lipopolysaccharide (LPS) stimulated THP-1 cells to release IL-8 after 24 h. We have previously reported that stimulation of monocytic cells with Salmonella enterica serovar Typhimurium porins led to the activation of mitogen-activated protein kinase cascades and of protein tyrosine kinases (PTKs). In this report, we demonstrate, using two potent and selective inhibitors of MEK activation by Raf-1 (PD-098059) and p38 (SB-203580), that both ERK1/2 and p38 pathways play a key role in the production of IL-8 by porins and LPS. Porin-stimulated expression of activating protein 1 (AP-1) and correlated IL-8 release is also inhibited by PD-098059 or SB-203580 indicating that the Raf-1/MEK1-MEK2/MAPK cascade is required for their activation. Also PTKs modulate the pathway that control IL-8 gene expression, in fact its expression is abolished by tyrphostin. By using N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) an inhibitor of nuclear factor-kappaB (NF-kappaB) activity, we also observed IL-8 release modulation. Our results elucidate some of the molecular mechanisms by which AP-1 and NF-kappaB regulate IL-8 release and open new strategies for the design of specific molecules that will modulate IL-8 effects in various infectious diseases.     

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.     

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.     

The mitogen-activated protein kinase p38 is necesssary for interleukin 1beta-induced monocyte chemoattractant protein 1 expression by human mesangial cells

Mitogen-activated protein (MAP) kinases have been suggested as potential mediators for interleukin 1beta (IL-1beta)-induced gene activation. This study investigated the role of the MAP kinases p38 and ERK2 in IL-1beta-mediated expression of the chemokine MCP-1 by human mesangial cells. Phosphorylation of p38 kinase, which is necessary for activation, increased significantly after IL-1beta treatment. p38 kinase immunoprecipitated from IL-1beta-treated cells phosphorylated target substrates to a greater extent than p38 kinase from controls. SB 203580, a selective p38 kinase inhibitor, was used to examine the role of p38 kinase in MCP-1 expression. SB 203580 decreased IL-1beta-induced MCP-1 mRNA and protein levels, but did not affect MCP-1 mRNA stability. Because NF-kappaB is necessary for MCP-1 gene expression, the effect of p38 kinase inhibition on IL-1beta induction of NF-kappaB was measured. SB 203580 (up to 25 microM) had no effect on IL-1beta-induced NF-kappaB nuclear translocation or DNA binding activity. Our previous work showed that IL-1beta also activates the MAP kinase ERK2 in human mesangial cells. PD 098059, a selective inhibitor of the ERK activating kinase MEK1, had no effect on IL-1beta-induced MCP-1 mRNA or protein levels, or on IL-1beta activation of NF-kappaB. These data indicate that p38 kinase is necessary for the induction of MCP-1 expression by IL-1beta, but is not involved at the level of cytoplasmic activation of NF-kappaB. In contrast, ERK2 does not mediate IL-1beta induced MCP-1 gene expression.     

Inhibition of interleukin-1beta -induced NF-kappa B activation by calcium/calmodulin-dependent protein kinase kinase occurs through Akt activation associated with interleukin-1 receptor-associated kinase phosphorylation and uncoupling of MyD88

Calcium/calmodulin-dependent protein kinase kinase (CaMKK) and Akt are two multifunctional kinases involved in many cellular responses. Although Akt and Ca(2+) signals have been implicated in NF-kappaB activation in response to certain stimuli, these results are still controversial, and the mechanism(s) involved remains unknown. In this study, we show the roles that CaMKK and Akt play in regulating interleukin-1beta (IL-1beta)-induced NF-kappaB signaling. In human embryonic kidney 293 cells, IL-1beta induces IkappaB kinase beta (IKKbeta) activation, IkappaBalpha degradation, NF-kappaB transactivation, and weak Akt activation. A CaMKK inhibitor (KN-93) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002) do not inhibit IL-1beta-induced NF-kappaB activation. However, IL-1beta-induced NF-kappaB activity is attenuated by increased intracellular calcium in response to ionomycin, UTP, or thapsigargin or by overexpression of CaMKKc and/or Akt. Ionomycin and CaMKKc overexpression increases Akt phosphorylation on Thr(308) and enzyme activity. Under these conditions or upon overexpression of wild type Akt, IL-1beta-induced IKKbeta activity is diminished. Furthermore, a dominant negative mutant of Akt abolishes IKKbeta inhibition by CaMKKc and ionomycin, suggesting that Akt acts as a mediator of CaMKK signaling to inhibit IL-1beta-induced IKK activity at an upstream target site. We have also identified a novel interaction between CaMKK-stimulated Akt and interleukin-1 receptor-associated kinase 1 (IRAK1), which plays a key role in IL-1beta-induced NF-kappaB activation. CaMKKc and Akt overexpression decreases IRAK1-mediated NF-kappaB activity and its association with MyD88 in response to IL-1beta stimulation. Furthermore, CaMKKc and Akt overexpression increases IRAK1 phosphorylation at Thr(100), and point mutation of this site abrogates the inhibitory effect of Akt on IRAK1-mediated NF-kappaB activation. Taken together, these results indicate a novel regulatory mechanism for IL-1beta signaling and suggest that CaMKK-dependent Akt activation inhibits IL-1beta-induced NF-kappaB activation through interference with the coupling of IRAK1 to MyD88.     

Duration of alpha 1-antichymotrypsin gene activation by interleukin-1 is determined by efficiency of inhibitor of nuclear factor kappa B alpha resynthesis in primary human astrocytes

Expression of alpha1antichymotrypsin (ACT) is significantly activated by interleukin-1 (IL-1) in human astrocytes; however, it is barely affected by IL-1 in hepatocytes. This tissue-specific regulation depends upon an enhancer that contains both nuclear factor kappaB (NF-kappaB) and activating protein 1 (AP-1) elements, and is also observed for an NF-kappaB reporter but not for an AP-1 reporter. We found efficient activation of NF-kappaB binding in both cell types; however, this binding was persistent in glial cells and only transient in hepatocytes. IL-1-activated NF-kappaB complexes consisted of p65 and p50, with p65 transiently phosphorylated on serine 536 in glial cells whereas more persistently in hepatic cells. Overexpression of p65 or constitutively active IKKbeta (inhibitor of NF-kappaB kinase beta) resulted in an efficient activation of the ACT reporter in hepatic cells, indicating that a specific mechanism exists in these cells terminating IL-1 signaling. IL-1 effectively induced the degradation of inhibitor of NF-kappaBalpha (IkBalpha) and IkBepsilon in both cell types but IkBbeta was not affected. However, IkBalpha was resynthesized much more rapidly in hepatic cells in comparison to glial cells. In addition, the initial levels of IkBalpha were much lower in glial cells. We propose that the tissue-specific regulation of the ACT gene expression by IL-1 is determined by different efficiencies of IkBalpha resynthesis in glial and hepatic cells.