Interleukin-20 Promotes Migration of Bladder Cancer Cells through Extracellular Signal-regulated Kinase (ERK)-mediated MMP-9 Protein Expression Leading to Nuclear Factor (NF-κB) Activation by Inducing the Up-regulation of p21WAF1 Protein Expression

The role of inflammatory cytokine interleukin-20 (IL-20) has not yet been studied in cancer biology. Here, we demonstrated up-regulation of both IL-20 and IL-20R1 in muscle-invasive bladder cancer patients. The expressions of IL-20 and IL-20R1 were observed in bladder cancer 5637 and T-24 cells. We found that IL-20 significantly increased the expression of matrix metalloproteinase (MMP)-9 via binding activity of NF-κB and AP-1 in bladder cancer cells and stimulated the activation of ERK1/2, JNK, p38 MAPK, and JAK-STAT signaling. Among the pathways examined, only ERK1/2 inhibitor U0126 significantly inhibited IL-20-induced migration and invasion. Moreover, siRNA knockdown of IL-20R1 suppressed migration, invasion, ERK1/2 activation, and NF-κB-mediated MMP-9 expression induced by IL-20. Unexpectedly, the cell cycle inhibitor p21^WAF1 was induced by IL-20 treatment without altering cell cycle progression. Blockade of p21^WAF1 function by siRNA reversed migration, invasion, activation of ERK signaling, MMP-9 expression, and activation of NF-κB in IL-20-treated cells. In addition, IL-20 induced the activation of IκB kinase, the degradation and phosphorylation of IκBα, and NF-κB p65 nuclear translocation, which was regulated by ERK1/2. IL-20 stimulated the recruitment of p65 to the MMP-9 promoter region. Finally, the IL-20-induced migration and invasion of cells was confirmed by IL-20 gene transfection and by addition of anti-IL-20 antibody. This is the first report that p21^WAF1 is involved in ERK1/2-mediated MMP-9 expression via increased binding activity of NF-κB, which resulted in the induction of migration in IL-20/IL-20R1 dyad-induced bladder cancer cells. These unexpected results might provide a critical new target for the treatment of bladder cancer.     

Differential regulation of membrane type 1-matrix metalloproteinase activity by ERK 1/2- and p38 MAPK-modulated tissue inhibitor of metalloproteinases 2 expression controls transforming growth factor-beta1-induced pericellular collagenolysis

Acquisition of matrix metalloproteinase-2 (MMP-2) activity is temporally associated with increased migration and invasiveness of cancer cells. ProMMP-2 activation requires multimolecular complex assembly involving proMMP-2, membrane type 1-MMP (MT1-MMP, MMP-14), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because transforming growth factor-beta1 (TGF-beta1) promotes tumor invasion in advanced squamous cell carcinomas, the role of TGF-beta1 in the regulation of MMP activity in a cellular model of invasive oral squamous cell carcinoma was examined. Treatment of oral squamous cell carcinoma cells with TGF-beta1 promoted MMP-dependent cell scattering and collagen invasion, increased expression of MMP-2 and MT1-MMP, and enhanced MMP-2 activation. TGF-beta1 induced concomitant activation of ERK1/2 and p38 MAPK, and kinase inhibition studies revealed a negative regulatory role for ERK1/2 in modulating acquisition of MMP-2 activity. Thus, a reciprocal effect on proMMP-2 activation was observed whereupon blocking ERK1/2 phosphorylation promoted proMMP-2 activation and MT1-MMP activity, whereas inhibiting p38 MAPK activity decreased proteolytic potential. The cellular mechanism for the control of MT1-MMP catalytic activity involved concurrent reciprocal modulation of TIMP-2 expression by ERK1/2 and p38 MAPKs, such that inhibition of ERK1/2 phosphorylation decreased TIMP-2 production, and down-regulation of p38 MAPK activity enhanced TIMP-2 synthesis. Further, p38 MAPK inhibition promoted ERK1/2 phosphorylation, providing additional evidence for cross-talk between MAPK pathways. These observations demonstrate the complex reciprocal effects of ERK1/2 and p38 MAPK in the regulation of MMP activity, which could complicate the use of MAPK-specific inhibitors as therapeutic agents to down-regulate the biologic effects of TGF-beta1 on pericellular collagen degradation and tumor invasion.     

Selective activation of p38 mitogen-activated protein kinase cascade in human neutrophils stimulated by IL-1beta.

We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1beta. IL-1beta induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1beta for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1beta. IL-1beta primed neutrophils for enhanced release of superoxide (O(2)(-)) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1beta also induced O(2)(-) release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1beta and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1beta induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1beta and activation of this cascade mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.     

IL-27 induces Th1 differentiation via p38 MAPK/T-bet- and intercellular adhesion molecule-1/LFA-1/ERK1/2-dependent pathways

IL-27, a novel member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in positive and negative regulations of immune responses. We recently demonstrated that IL-27 induces Th1 differentiation through ICAM-1/LFA-1 interaction in a STAT1-dependent, but T-bet-independent mechanism. In this study, we further investigated the molecular mechanisms by focusing on p38 MAPK and ERK1/2. IL-27-induced Th1 differentiation was partially inhibited by lack of T-bet expression or by blocking ICAM-1/LFA-1 interaction with anti-ICAM-1 and/or anti-LFA-1, and further inhibited by both. Similarly, the p38 MAPK inhibitor, SB203580, or the inhibitor of ERK1/2 phosphorylation, PD98059, partially suppressed IL-27-induced Th1 differentiation and the combined treatment completely suppressed it. p38 MAPK was then revealed to be located upstream of T-bet, and SB203580, but not PD98059, inhibited T-bet-dependent Th1 differentiation. In contrast, ERK1/2 was shown to be located downstream of ICAM-1/LFA-1, and PD98059, but not SB203580, inhibited ICAM-1/LFA-1-dependent Th1 differentiation. Furthermore, it was demonstrated that STAT1 is important for IL-27-induced activation of ERK1/2, but not p38 MAPK, and that IL-27 directly induces mRNA expression of growth arrest and DNA damage-inducible 45gamma, which is known to mediate activation of p38 MAPK. Finally, IL-12Rbeta2 expression was shown to be up-regulated by IL-27 in both T-bet- and ICAM-1/LFA-1-dependent mechanisms. Taken together, these results suggest that IL-27 induces Th1 differentiation via two distinct pathways, p38 MAPK/T-bet- and ICAM-1/LFA-1/ERK1/2-dependent pathways. This is in contrast to IL-12, which induces it via only p38 MAPK/T-bet-dependent pathway.     

S100A8 and S100A9 promotes invasion and migration through p38 mitogen-activated protein kinase-dependent NF-κB activation in gastric cancer cells

S100A8 and S100A9 (S100A8/A9) are low-molecular weight members of the S100 family of calcium-binding proteins. Recent studies have reported S100A8/A9 promote tumorigenesis. We have previously reported that S100A8/A9 is mostly expressed in stromal cells and inflammatory cells between gastric tumor cells. However, the role of environmental S100A8/A9 in gastric cancer has not been defined. We observed in the present study the effect of S100A8/A9 on migration and invasion of gastric cancer cells. S100A8/ A9 treatment increased migration and invasionat lower concentrations that did not affect cell proliferation and cell viability. S100A8/A9 caused activation of p38 mitogenactivated protein kinase (MAPK) and nuclear factor-κB (NF-κB). The phosphorylation of p38 MAPK was not affected by the NF-κB inhibitor Bay whereas activation of NF-κB was blocked by p38 MAPK inhibitor SB203580, indicating that S100A8/A9-induced NF-κB activation is mediated by phosphorylation of p38 MAPK. S100A8/A9-induced cell migration and invasion was inhibited by SB203580 and Bay, suggesting that activation of p38 MAPK and NF-κB is involved in the S100A8/A9 induced cell migration and invasion. S100A8/A9 caused an increase in matrix metalloproteinase 2 (MMP2) and MMP12 expression, which were inhibited by SB203580 and Bay. S100A8/A9-induced cell migration and invasion was inhibited by MMP2 siRNA and MMP12 siRNA, indicating that MMP2 and MMP12 is related to the S100A8/A9 induced cell migration and invasion. Taken together, these results suggest that S100A8/A9 promotes cell migration and invasion through p38 MAPKdependent NF-κB activation leading to an increase of MMP2 and MMP12 in gastric cancer.     

CD23-mediated transport of IgE/immune complexes across human intestinal epithelium: role of p38 MAPK

We previously reported that CD23/FcepsilonRII (low-affinity IgE receptor) is expressed on human intestinal epithelial cells and is responsible for transepithelial transport of IgE. In this study, we compared the transport of IgE with that of immune complexes in both the apical-to-serosal and the serosal-to-apical directions across HT29 epithelial cell layers and examined the effects of two p38 MAPK inhibitors, SKF86002 and SB203580, on the expression and function of CD23. Our study showed that both p38 MAPK inhibitors at 10 microM significantly inhibited constitutive and IL-4-upregulated CD23 protein expression in epithelial cells. Both inhibitors, in a concentration-dependent manner, also significantly reduced IgE binding and uptake into cells. Transepithelial transport of IgE and immune complexes across the epithelial barrier were similarly inhibited. IL-4 upregulated the phosphorylation and activity of p38 MAPK and the phosphorylation of the downstream substrate MAPKAPK-2 (MK-2). The inhibitors exerted effects in the pathway post the p38 MAPK; SB203580 significantly inhibited the phosphorylation of MK-2. Our results indicate that CD23 expression in these human intestinal epithelial cells is mediated through the p38 MAPK pathway and that inhibition of p38 MAPK consequently interferes with the transport of IgE and immune complexes across the intestinal epithelial barrier.     

Acacetin, a flavonoid, inhibits the invasion and migration of human prostate cancer DU145 cells via inactivation of the p38 MAPK signaling pathway

Acacetin (5,7-dihydroxy-4′-methoxyflavone), a flavonoid compound, has anti-peroxidative and anti-inflammatory effects. The effect of acacetin on antimetastasis in human prostate cancer DU-145 cells was investigated. First, the result demonstrated acacetin could exhibit an inhibitory effect on the abilities of the adhesion, invasion, and migration by cell–matrix adhesion assay, wound-healing assay, and Boyden chamber assay. Data also showed acacetin could inhibit the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) involved in the downregulation of the expressions of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-type plasminogen activator (u-PA) at both the protein and mRNA levels. Next, acacetin significantly decreased the nuclear levels of nuclear factor kappa B (NF-κB), c-Fos, and c-Jun. Also, the treatment with acacetin to DU145 cells also leads to a dose-dependent inhibition on the binding ability of NF-κB and activator protein-1 (AP-1). Furthermore, the treatment of inhibitors specific for p38 MAPK (SB203580) to DU145 cells could cause reduced expressions of MMP-2, MMP-9, and u-PA. These results showed acacetin could inhibit the invasion and migration abilities of DU145 cells by reducing MMP-2, MMP-9, and u-PA expressions through suppressing p38 MAPK signaling pathway and inhibiting NF-κB- or AP-1-binding activity. These findings proved acacetin might be offered further application as an antimetastatic agent.     

Involvement of p38 mitogen-activated protein kinase in the cell growth inhibition by sodium arsenite.

It is well-known that p38 mitogen-activated protein kinase (p38MAPK) participates in cellular responses to mitogenic stimuli, environmental and genotoxic stresses, and apoptotic agents. Although there are several reports on p38MAPK in relation to cell growth and apoptosis, the exact mechanism of p38MAPK-mediated cell growth regulation remains obscure. Here, we examined possible roles of p38MAPK in the sodium arsenite-induced cell growth inhibition in NIH3T3 cells. Sodium arsenite induced transient cell growth delay with marked activation of p38MAPK. In addition, arsenite induced CDK inhibitor p21(CIP1/WAF1) and enhanced its binding to the CDK2, which resulted in inhibition of CDK2 activity. The levels of cyclin D1 expression and the CDK4 kinase activity were also significantly reduced. pRB was hypophosphorylated by sodium arsenite. SB203580, a specific inhibitor of p38MAPK, blocked arsenite-induced growth inhibition as well as the arsenite-induced p21(CIP1/WAF1) expression. Expression of dominant negative p38MAPK also blocked arsenite-induced p21(CIP1/WAF1) expression. Inhibited-CDK2 activity was also completely reversed by SB203580 or expression of dominant negative p38MAPK, while the decreased-cyclin D1 protein by the compound was not restored. These data demonstrate a possible link between the activation of p38MAPK and induction of p21(CIP1/WAF1), suggesting that the activation of p38MAPK is, at least in part, related to the cell growth inhibition by sodium arsenite.     

Role of TNF receptor-associated factor 2 and p38 mitogen-activated protein kinase activation during 4-1BB-dependent immune response

4-1BB is a costimulatory member of the TNFR family, expressed on activated CD4(+) and CD8(+) T cells. Previous results showed that 4-1BB-mediated T cell costimulation is CD28-independent and involves recruitment of TNFR-associated factor 2 (TRAF2) and activation of the stress-activated protein kinase cascade. Here we describe a role for the p38 mitogen-activated protein kinase (MAPK) pathway in 4-1BB signaling. Aggregation of 4-1BB alone induces p38 activation in a T cell hybridoma, whereas, in normal T cells, p38 MAPK is activated synergistically by immobilized anti-CD3 plus immobilized 4-1BB ligand. 4-1BB-induced p38 MAPK activation is inhibited by the p38-specific inhibitor SB203580 in both a T cell hybridoma and in murine T cells. T cells from TRAF2 dominant-negative mice are impaired in 4-1BB-mediated p38 MAPK activation. A link between TRAF2 and the p38 cascade is provided by the MAPK kinase kinase, apoptosis-signal-regulating kinase 1. A T cell hybrid transfected with a kinase-dead apoptosis-signal-regulating kinase 1 fails to activate p38 MAPK in response to 4-1BB signaling. To assess the role of p38 activation in an immune response, T cells were stimulated in an MLR in the presence of SB203580. In a primary MLR, SB203580 blocked IL-2, IFN-gamma, and IL-4 secretion whether the costimulatory signal was delivered via 4-1BB or CD28. In contrast, following differentiation into Th1 or Th2 cells, p38 inhibition blocked IL-2 and IFN-gamma without affecting IL-4 secretion. Nevertheless, IL-4 secretion by Th2 cells remained costimulation-dependent. Thus, critical T cell signaling events diverge following Th1 vs Th2 differentiation.