Thrombin induces rapid PAR1-mediated non-classical FGF1 release

Thrombin induces cell proliferation and migration during vascular injury. We report that thrombin rapidly stimulated expression and release of the pro-angiogenic polypeptide fibroblast growth factor 1 (FGF1). Thrombin failed to induce FGF1 release from protease-activated receptor 1 (PAR1) null fibroblasts, indicating that this effect was dependent on PAR1. Similarly to thrombin, FGF1 expression and release were induced by TRAP, a specific oligopeptide agonist of PAR1. These results identify a novel aspect of the crosstalk between FGF and thrombin signaling pathways which both play important roles in tissue repair and angiogenesis.     

Regulation of notch signaling activity

Cell-cell signalling mediated by the receptor Notch is used widely across the metazoans to determine cell fate and regulate pattern formation. Notch signals via a conserved regulated intramembrane proteolysis. Recent analyses of the cell biology of the Notch receptor have identified several fundamental mechanisms that contribute to regulate Notch signaling activity in space and time.     

Regulation of Nod1-mediated signaling pathways

Nod1 is a member of the NLR/Nod/CATERPILLER family. It acts as a sensor for intracellular bacteria by recognizing specific glycopeptides derived from peptidoglycan. Nod1 activation mediates distinct cellular responses including activation of MAP kinases, IL-8 release, apoptosis and suppression of several estrogen-dependent responses in MCF-7 cells. Here we have extended these studies by identifying key regulatory steps in Nod1-dependent signaling pathways. We provide multiple lines of data showing that Nod1-dependent apoptosis is a caspase 8-mediated event and that apoptosis requires RIP2. In contrast, several lines of evidence show that Nod1-dependent JNK activation and IL-8 production did not require the presence of caspase 8 but required activation of TAK1 as well as RIP2. Thus, we have identified several key control points that lie downstream of Nod1. This work provides the basis for further studies of the biological significance and regulation of the Nod1 pathway.     

Unique signaling properties of CTAR1 in LMP1-mediated transformation

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene is considered the EBV oncogene as it is necessary for EBV-mediated transformation of B lymphocytes and itself transforms rodent fibroblasts. LMP1 activates the NF-kappaB, phosphatidylinositol 3-kinase (PI3K)-Akt, mitogen-activated protein kinase, and Jun N-terminal protein kinase signaling pathways through its two signaling domains, carboxyl-terminal activating regions 1 and 2 (CTAR1 and CTAR2). CTAR1 and CTAR2 induce signal transduction pathways through their direct (CTAR1) or indirect (CTAR2) recruitment of tumor necrosis factor receptor-associated factors (TRAFs). CTAR1 is necessary for LMP1-mediated transformation as well as activation of PI3K signaling and induction of cell cycle markers associated with G(1)/S transition. In this study, activation of PI3K-Akt signaling and deregulation of cell cycle markers were mapped to the TRAF-binding domain within CTAR1 and to the residues between CTAR1 and CTAR2. LMP1 CTAR1 also activated the MEK1/2-extracellular signal-regulated kinase 1/2 signaling pathway, and this activation was necessary for LMP1-induced transformation of Rat-1 fibroblasts. Dominant-negative forms of TRAF2 and TRAF3 inhibited but did not fully block LMP1-mediated transformation. These findings identify a new signaling pathway that is uniquely activated by the TRAF-binding domain of LMP1 and is required for transformation.     

Regulation of colon cancer cell migration and invasion by CLIC1-mediated RVD

The metastasis of colorectal cancer is one of the most common causes of death in the world. In this investigation, we used the human colon cancer cell lines LOVO and HT29 as model systems to determine the role of the chloride intracellular channel 1 (CLIC1) in the metastasis of colonic cancer. In the present study, we found that regulatory volume decrease (RVD) capacity was markedly up-regulated in LOVO cells, which are characterized by a high metastatic potential. Functionally suppressing CLIC1 using the specific chloride intracellular channel 1 blocker Indanyloxyacetic acid 94 inhibited RVD and decreased the migration and invasion of colon cancer cells. Moreover, these effects occurred in a dose-dependent manner. The migration and invasion abilities in two cell lines also were inhibited by the knockdown of CLIC1 using small interfering RNA transfection. The mRNA and protein expression of CLIC1 is up-regulated in LOVO cells. In human colon cancer cells, CLIC1 is primarily located in the plasma membrane, where it functions as a chloride channel. Taken together, the results suggest that CLIC1 modulates the metastasis of colon cancer through its RVD-mediating chloride channel function. This study demonstrates, for the first time, that CLIC1 regulates the migration and invasion of colon cancer.     

Rho signaling regulates pannexin 1-mediated ATP release from airway epithelia

ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.     

Synergistic Ca2+ and Cu2+ requirements of the FGF1-S100A13 interaction measured by quartz crystal microbalance: an initial step in amlexanox-reversible non-classical release of FGF1

It is known that fibroblast growth factor-1 (FGF1) lacking a conventional signal peptide sequence shows non-classical release independent of the endoplasmic reticulum-Golgi system. Recent studies reveal that FGF1 is co-released with S100A13, a Ca2+-binding protein that acts as an extracellular cargo molecule. Although both FGF1 and S100A13 are Cu2+-binding proteins, the role of Cu2+, as well as that of Ca2+, in non-classical release, remains to be clarified. In the present study we examined the requirements of both metal ions for the interaction between these two proteins. The addition of Ca2+ significantly increased the ka value, while decreasing the KD value, for the interaction between Strep-tagII-S100A13 and GST-FGF1; both values were obtained by use of a quartz crystal microbalance, a real-time mass-measuring device. The EC50 of Ca2+ to enhance the interaction was 10.11 microM. Although the addition of Cu2+ alone had no effect, it caused a marked potentiation of the Ca2+-enhanced interaction. The EC50 of Cu2+ for the potentiation was 50.45 nM. On the other hand, the EC50 of Ca2+ and the KD values were decreased from 11.69 to 2.07 microM and 0.75 to 0.38 x 10(-7)M, respectively, by the addition of 200 nM Cu2+. The Cu2+-induced potentiation of this interaction was abolished by amlexanox, which inhibits non-classical release of FGF1. All of these findings suggest that synergistic effects of Ca2+ and Cu2+ play a key role in the interaction between FGF1 and S100A13, which is the initial step in non-classical release of FGF1.     

Viral immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling in cell transformation and cancer

Viruses frequently co-opt host cell pathways to enhance their propagation or to enable latent infection. Certain receptors expressed by hematopoietic cells have immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domains that initiate cellular activation, proliferation and differentiation. Some viruses have evolved, or acquired from their host, genes that encode ITAM-bearing proteins. These ITAM-bearing viral proteins have been implicated in cellular transformation in virus-infected hematopoietic cells, typically B cells, but also in non-hematopoietic tissues--including endothelial and epithelial cells.