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Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment and play critical roles in tumorigenesis. CAFs consists of multiple subpopulations, which have diverse functions. The detailed mechanism, including the role of NF-κB, a critical transcription factor for inflammation and cell survival, in CAFs has not been adequately explored. In this study, we examined the roles of IKKβ, a key kinase for NF-κB activation, in activated CAFs by using mice (KO mice) with deletion of IKKβ in activated fibroblasts (aFbs). We found that melanoma cells implanted in KO mice showed significantly more growth than those implanted in control mice. To exclude the effects of deletion of IKKβ in cells other than aFbs, we implanted a mixture of melanoma cells and IKKβ-deleted aFbs in wild-type mice and observed that the mixture showed greater growth than a mixture of melanoma cells and normal aFbs. In exploring the mechanisms, we found that conditioned medium from IKKβ-deleted aFbs promotes the proliferation of melanoma cells, and the expression of growth arrest-specific 6 (GAS6) and hepatocyte growth factor (HGF), which are major tumor-promoting factors, was upregulated in IKKβ-deleted aFbs. These results indicated the tumor-suppressing function of IKKβ in activated CAFs.

NF-κB is a major transcription factor regulating cell survival, organ development and inflammation, but its role in cardiac development has been inadequately explored. To examine this function, we generated mice in which IKKβ, an essential kinase for NF-κB activation, was constitutively activated in embryonic cardiomyocytes. For this purpose, we used smooth muscle-22α (SM22α)-Cre mice, which are frequently used for gene recombination in embryonic cardiomyocytes. Embryonic hearts of SM22αCre-CA (constitutively active) IKKβ

Aspirin has been widely used as analgesic, antipyretic and anti-inflammatory medicine for long. In addition to these traditional effects, clinical studies suggest that aspirin can protect against cancer, but its mechanism has not been explored. To unveil it, we identified the proteins up- or down-regulated after incubation with aspirin by using proteomics analysis with Nano-flow LC/MALDI-TOF system. Interestingly, the analysis identified the protein of Adenomatous Polyposis Coli (APC) as one of the most up-regulated protein. APC regulates cell proliferation or angiogenesis, and is widely known as a tumor-suppressing gene which can cause colorectal cancer when it is mutated. Western blots confirmed this result, and real-time PCR indicated it is transcriptionally regulated. We further tried to elucidate the molecular mechanism with focusing on IKKβ. IKKβ is the essential kinase in activation of nuclear factor-kappa B (NF-κB), major transcriptional factors that regulate genes responsible for inflammation or immune response. Previous reports indicated that aspirin specifically inhibits IKKβ activity, and constitutively active form of IKKβ accelerates APC loss. We found that aspirin suppressed the expression of IKKβ, and the deletion of IKKβ by siRNA increases the expression of APC in HEK294 cells. Finally, we observed similar effects of aspirin in human umbilical vein endothelial cells. Taken together, these results reveal that aspirin up-regulates the expression of APC via the suppression of IKKβ. This can be a mechanism how aspirin prevents cancer at least in part, and a novel link between inflammatory NF-κB signaling and cancer.

In the downstream regions of stenotic vessels, cells are subjected to a vortex motion under low shear forces, and atherosclerotic plaques tend to be localized. It has been reported that such a change of shear force on endothelial cells has an atherogenic effect by inducing the expression of adhesion molecules. However, the effect of vortex-induced mechanical stress on leukocytes has not been investigated. In this study, to elucidate whether vortex flow can affect the cell adhesive property, we have examined the effect of vortex-mediated mechanical stress on integrin activation in THP-1 cells, a monocytic cell line, and its signaling mechanisms. When cells are subjected to vortex flow at 400-2,000 rpm, integrin-dependent cell adhesion to vascular cell adhesion molecule-1 or fibronectin increased in a speed- and time-dependent manner. Next, to examine the role of Ca(2+) in this integrin activation, various pharmacological inhibitors involved in Ca(2+) signaling were tested to inhibit the cell adhesion. Pretreatment of cells with BAPTA-AM, thapsigargin +NiCl(2), or U-73122 (a phospholipase C inhibitor) inhibited cell adhesion induced by vortex-mediated mechanical stress. We also found that W7 (a calmodulin inhibitor) blocked the cell adhesion. However, pretreatment of cells with GdCl(3), NiCl(2), or ryanodine did not affect the cell adhesion. These data indicate that vortex-mediated mechanical stress induces integrin activation through calmodulin and inositol 1,4,5-trisphosphate-mediated Ca(2+) releases from intracellular Ca(2+) stores in THP-1 cells.

Monocyte chemoattractant protein 1 (MCP-1) has a crucial role in atherogenesis and inflammation. However, MCP-1-mediated signalling pathways in monocytes have not been fully elucidated. In the present study we investigated the role of tyrosine kinases such as proline-rich tyrosine kinase 2 (Pyk2) in MCP-1-mediated signal transduction in the monocytic cell line THP-1. Pyk2 was tyrosine phosphorylated very quickly after stimulation with MCP-1. We found that Lyn, Shc and paxillin were also tyrosine phosphorylated by MCP-1. We examined the association of these molecules by immunoprecipitation and immunoblot analysis. The association of Pyk2 with Lyn was dependent on stimulation with MCP-1 and on tyrosine phosphorylation of Pyk2. Phosphorylation of p38 was also dependent on tyrosine phosphorylation of Pyk2. However, the association of Pyk2 with paxillin and Grb2 was not affected by stimulation with MCP-1. Phosphorylation of ERK (extracellular-signal-regulated protein kinase) was not affected by overexpression of kinase-negative Pyk2. Our results indicate that Pyk2 forms a complex with paxillin, Grb2 and Lyn in THP-1 cells. However, Pyk2 is not always involved in MCP-1-mediated signalling pathways.

Transmigration of monocytes to the subendothelial space is the initial step of atherosclerotic plaque formation and inflammation. Integrin activation and chemotaxis are two important functions involved in monocyte transmigration. To delineate the signaling cascades leading to integrin activation and chemotaxis by monocyte chemoattractant protein-1 (MCP-1), we have investigated the roles of MAPK and Rho GTPases in THP-1 cells, a monocytic cell line. MCP-1 stimulated beta1 integrin-dependent, but not beta2 integrin-dependent cell adhesion in a time-dependent manner. MCP-1-mediated cell adhesion was inhibited by a MEK inhibitor but not by a p38-MAPK inhibitor. In contrast, MCP-1-mediated chemotaxis was inhibited by the p38-MAPK inhibitor but not by the MEK inhibitor. The inhibitor of Rho GTPase, C3 exoenzyme, and a Rho kinase inhibitor abrogated MCP-1-dependent chemotaxis but not integrin-dependent cell adhesion. Further, C3 exoenzyme and the Rho kinase inhibitor blocked MCP-1-dependent p38-MAPK activation. These data indicate that ERK is responsible for integrin activation, that p38-MAPK and Rho are responsible for chemotaxis mediated by MCP-1, and that Rho and the Rho kinase are upstream of p38-MAPK in MCP-1-mediated signaling. This study demonstrates that two distinct MAPKs regulate two dependent signaling cascades leading to integrin activation and chemotaxis induced by MCP-1 in THP-1 cells.