Osteosarcoma is a mesenchymal tumour of bone common among children and young adults. Prognosis is poor if metastases are present at diagnosis. The transforming growth factor beta (TGF-β) signaling pathway plays a complex dual role in cancer. We hypothesized that alterations in the TGF-β signaling pathway are important in the tumorigenesis of osteosarcoma cell lines. Smad phosphorylation and nuclear localization, Smad4 expression, and TAZ expression were determined in the HOS osteosarcoma cell line and tumorigenic derivatives. Basal TGF-β activity and TAZ expression correlated with a tumorigenic phenotype in the KHOS cell lines as measured by Anchorage Independent Growth (AIG). In comparison, exogenous TGF-β suppressed AIG and acted as a tumour suppressor, while Smad4-deficient KHOS cells were resistant to the inhibitory TGF-β effect. In conclusion, basal TGF-β signaling and TAZ correlate with increased tumorigenic potential in osteosarcoma cell lines, whereas exogenous TGF-β acted as a tumour suppressor in a Smad4-dependent manner.

In atherosclerosis, infiltrating leukocytes and vascular smooth muscle cells (VSMCs) cause progressive vascular narrowing. Platelet-mediated thrombosis ultimately causes complete vessel occlusion, resulting in heart attack or stroke. In animal models and human patients, individually blocking these events is only partially effective. Another therapeutic strategy would be to globally target these multiple cell types. Slit proteins act as developmental neuronal repellents, and Slit2 via interaction with its receptor, Robo-1, impairs inflammatory recruitment of leukocytes and VSMCs. We detected Robo-1 expression in human and murine platelets. Using static and shear assays, we demonstrate that Slit2 impaired platelet adhesion and spreading on fibrinogen, fibronectin and collagen. Slit2 mediated these effects, in part, by suppressing activation of Akt but not Rac1, Cdc42, Erk or p38 MAPK. Slit2 also prevented ADP-mediated granular secretion. In mouse tail-bleeding experiments, Slit2 dose-dependently prolonged bleeding times in vivo. These data suggest a therapeutic role of Slit2 in atherothrombosis.

The pediatric sarcoma alveolar rhabdomyosarcoma (ARMS) is highly aggressive with a poor prognosis for diagnosed patients. Here, we demonstrate that targeting the unique oncogene integrin-linked kinase (ILK) in ARMS cells in conjunction with the common chemotherapy agent vincristine, a synergistic effect is found in the reduction of cell viability in vitro. This result was achieved by both RNAi-mediated depletion of ILK and using a small molecule kinase inhibitor specific for ILK. Both techniques were found to disrupt important protein interactions at the site of the centrosome. Combination ILK disruption and vincristine treatment of cells induced the expression of apoptotic markers and arrested cells in the G2/M stage of the cell cycle. Interestingly, protein levels of JNK and its target c-Jun were regulated with combined treatment. Altogether, these findings indicate that the use of molecular targets like ILK may further improve the clinical treatment of ARMS.

Legionella pneumophila (Lp.), the causative agent of Legionnaires’ disease, has been well known for infecting and replicating inside airway macrophages. Previous results from our laboratory indicated that Lp. attaches to non-polarized lung epithelial cells by binding and activating E-cadherin and β1-integrin. However, in the polarized alveolar epithelia these receptors are segregated from the apical cell surface by tight junctions (TJs). Our results showed that Lp. behaves as an opportunistic pathogen that efficiently targets disrupted, but not integrated epithelial cell monolayer. In such conditions, Lp. can reach its host-cell receptors and consequently invade and replicate intracellularly, producing large amount of bacterial progeny. These results and clinical evidence suggest the contribution of damaged airway epithelia to Legionellosis by providing a replication reservoir for the bacteria.

Legionella pneumophila (Lp) is the pathogen responsible for Legionnaires disease. Lp invades and survives in human macrophages to form an intracellular compartment, called the Legionella containing vacuole (LCV). Within the LCV, Lp avoids degradation and replicates, before killing its host and releasing its virulent progeny. Lp is pleomorphic, exhibiting a combination of short rod phenotypes and filaments. Filamentous Lp has been reported in patient samples, but current studies of Lp virulence are exclusively focused on rods. Our results show that filamentous Lp can invade and replicate in human and murine macrophages. Filaments are phagocytosed gradually into tubular phagocytic cups. Interestingly, the formation of the LCV starts at this stage of phagocytosis, and LCV markers were detected before the sealing of the phagosome occurred. We present evidence that the filamentous morphology acts cooperatively with Lp effectors to subvert the microbicidal activities of the macrophage, contributing to the survival of Lp.

Stigmas of Arabidopsis thaliana carrying the exo70A1-1 mutation are defective in early pollen-pistil interactions; pollen grain adhesion to the stigma, pollen hydration, and penetration of the stigmatic surface by the pollen tube. Exo70 function in directed secretion has been linked to its ability to bind the phosphatidylinositides. To provide support that the classical, octomeric exocyst complex, which contains the Exo70 subunit, participates in compatible pollen-pistil interactions, this process was analyzed in plants deficient in Sec15, another subunit of the exocyst. Additionally, the role of phosphatidylinositol-4-phosphate (PI-4-P) in female fertility was evaluated through the use of the mutants ROOT HAIR DEFECTIVE 4-1 and PI4Kβ1β2 -/-, which have increased and decreased levels of PI-4-P respectively. Reduction of Sec15b levels or perturbation of PI-4-P in the stigma resulted in a reduced ability of of the transgenic/mutant stigmas to support pollen grain hydration; though all other stages of early pollen pistil interactions were unaffected.

Osteosarcoma is a mesenchymal tumour of bone common among children and young adults. Prognosis is poor if metastases are present at diagnosis. The transforming growth factor beta (TGF-β) signaling pathway plays a complex dual role in cancer. We hypothesized that alterations in the TGF-β signaling pathway are important in the tumorigenesis of osteosarcoma cell lines. Smad phosphorylation and nuclear localization, Smad4 expression, and TAZ expression were determined in the HOS osteosarcoma cell line and tumorigenic derivatives. Basal TGF-β activity and TAZ expression correlated with a tumorigenic phenotype in the KHOS cell lines as measured by Anchorage Independent Growth (AIG). In comparison, exogenous TGF-β suppressed AIG and acted as a tumour suppressor, while Smad4-deficient KHOS cells were resistant to the inhibitory TGF-β effect. In conclusion, basal TGF-β signaling and TAZ correlate with increased tumorigenic potential in osteosarcoma cell lines, whereas exogenous TGF-β acted as a tumour suppressor in a Smad4-dependent manner.

The intricate processes of osteoclastogenesis are highly dependent on the dynamic regulation of the actin cytoskeleton. Adseverin, a member of the gelsolin superfamily of actin binding proteins, regulates actin remodeling by severing and capping actin filaments in a calcium dependent manner. The objective of this project was to characterize the role(s) of adseverin during osteoclastogenesis, by assessing adseverin expression throughout osteoclastogenesis and through differentiation assays using a knockdown strategy. Methods: qRT-PCR and immunoblot analyses were used to examine adseverin expression during osteoclastogenesis. A stable adseverin knockdown macrophage cell line was generated using a retroviral shRNA construct. Results: Adseverin expression increased significantly in response to RANKL during the early phases of osteoclastogenesis, and adseverin was highly expressed in mature osteoclasts. Adseverin knockdown macrophages experienced a major osteoclastogenesis defect, most likely caused by a defect in pre-osteoclast fusion. Conclusion: Adseverin is a RANKL induced early and pro-fusion marker of osteoclastogenesis.

Vascular inflammation and associated leukocyte influx is a hallmark in the pathogenesis of atherosclerosis. In both animal models and human subjects, inhibiting monocyte recruitment is beneficial in preventing atherosclerosis and its clinical manifestations. The trafficking signals that recruit cells to areas of inflammation are provided by small secreted proteins called chemokines. Chemokines play a major role in the pathogenesis of inflammation, and redundancy among the chemokine signaling pathways means that blocking one pathway could result in another assuming its function. Therefore, we aim to block a cell’s response to a range of chemokine-induced directional migration signals. Slit2 treatment inhibits monocyte migration in vitro using transwell migration assays, and in vivo, using a murine peritonitis model of inflammatory cell influx. This inhibition is shown to be dose- and time- dependent. Furthermore, Slit2 inhibits monocyte adhesion to activated endothelial cell monolayers. These data may suggest a therapeutic role for Slit2 in atherosclerosis.

In atherosclerosis, infiltrating leukocytes and vascular smooth muscle cells (VSMCs) cause progressive vascular narrowing. Platelet-mediated thrombosis ultimately causes complete vessel occlusion, resulting in heart attack or stroke. In animal models and human patients, individually blocking these events is only partially effective. Another therapeutic strategy would be to globally target these multiple cell types. Slit proteins act as developmental neuronal repellents, and Slit2 via interaction with its receptor, Robo-1, impairs inflammatory recruitment of leukocytes and VSMCs. We detected Robo-1 expression in human and murine platelets. Using static and shear assays, we demonstrate that Slit2 impaired platelet adhesion and spreading on fibrinogen, fibronectin and collagen. Slit2 mediated these effects, in part, by suppressing activation of Akt but not Rac1, Cdc42, Erk or p38 MAPK. Slit2 also prevented ADP-mediated granular secretion. In mouse tail-bleeding experiments, Slit2 dose-dependently prolonged bleeding times in vivo. These data suggest a therapeutic role of Slit2 in atherothrombosis.