Osteopontin mediates tumorigenic transformation of a preneoplastic murine cell line by suppressing anoikis: An Arg‐Gly‐Asp‐dependent‐focal adhesion kinase‐caspase‐8 axis

Osteopontin (OPN), an adhesive, matricellular glycoprotein, is a rate‐limiting factor in tumor promotion of skin carcinogenesis. With a tumor promotion model, the JB6 Cl41.5a cell line, we have shown that suppressing 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced OPN expression markedly inhibits TPA‐induced colony formation in soft agar, an assay indicative of tumorigenic transformation. Further, the addition of exogenous OPN promotes colony formation of these cells. These findings support a function of OPN in mediating TPA‐induced neoplastic transformation of JB6 cells. In regard to the mechanism of action by OPN, we hypothesized that, for JB6 cells grown in soft‐agar, secreted OPN induced by TPA stimulates cell proliferation and/or prevents anoikis to facilitate TPA‐induced colony formation. Analyses of cell cycle and cyclin D1 expression, and direct cell counting of JB6 cells treated with OPN indicate that OPN does not stimulate cell proliferation relative to non‐treated controls. Instead, at 24 h, OPN decreases anoikis by 41%, as assessed by annexin V assays. Further, in suspended cells OPN suppresses caspase‐8 activation, which is mediated specifically through its RGD‐cell binding motif that transduces signals through integrin receptors. Transfection studies with wild‐type and mutant focal adhesion kinases (FAK) and Western blot analyses suggest that OPN suppression of caspase‐8 activation is mediated through phosphorylation of FAK at Tyr861. In summary, these studies indicate that induced OPN is a microenvironment modulator that facilitates tumorigenic transformation of JB6 cells by inhibiting anoikis through its RGD‐dependent suppression of caspase‐8 activity, which is mediated in part through the activation of FAK at Tyr861. © 2013 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111135/1/mc22108.pd

Improved bone-forming functionality on diameter-controlled TiO2 nanotube surface

The titanium dioxide (TiO2) nanotube surface enables significantly accelerated osteoblast adhesion and exhibits strong bonding with bone. We prepared various sizes (30-100 nm diameter) of titanium dioxide (TiO2) nanotubes on titanium substrates by anodization and investigated the osteoblast cellular behavior in response to these different nanotube sizes. The unique and striking result of this study is that a change in osteoblast behavior is obtained in a relatively narrow range of nanotube dimensions, with small diameter (similar to 30 nm) nanotubes promoting the highest degree of osteoblast adhesion, while larger diameter (70-100 nm) nanotubes elicit a lower population of cells with extremely elongated cellular morphology and much higher alkaline phosphatase levels. Increased elongation of nuclei was also observed with larger diameter nanotubes. By controlling the nanotopography, large diameter nanotubes, in the similar to 100 min regime, induced extremely elongated cellular shapes, with an aspect ratio of 11:1, which resulted in substantially enhanced up-regulation of alkaline phosphatase activity, suggesting greater bone-forming ability than nanotubes with smaller diameters. Such nanotube structures, already being a strongly osseointegrating implant material, offer encouraging implications for the development and optimization of novel orthopedics-related treatments with precise control toward desired cell and bone growth behavior. (C) 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

γδ T Cells but Not NK Cells Are Essential for Cell-Mediated Immunity against Plasmodium chabaudi Malaria ▿

Blood-stage Plasmodium chabaudi infections are suppressed by antibody-mediated immunity and/or cell-mediated immunity (CMI). To determine the contributions of NK cells and γδ T cells to protective immunity, C57BL/6 (wild-type [WT]) mice and B-cell-deficient (JH−/−) mice were infected with P. chabaudi and depleted of NK cells or γδ T cells with monoclonal antibody. The time courses of parasitemia in NK-cell-depleted WT mice and JH−/− mice were similar to those of control mice, indicating that deficiencies in NK cells, NKT cells, or CD8+ T cells had little effect on parasitemia. In contrast, high levels of noncuring parasitemia occurred in JH−/− mice depleted of γδ T cells. Depletion of γδ T cells during chronic parasitemia in B-cell-deficient JH−/− mice resulted in an immediate and marked exacerbation of parasitemia, suggesting that γδ T cells have a direct killing effect in vivo on blood-stage parasites. Cytokine analyses revealed that levels of interleukin-10, gamma interferon (IFN-γ), and macrophage chemoattractant protein 1 (MCP-1) in the sera of γδ T-cell-depleted mice were significantly (P < 0.05) decreased compared to hamster immunoglobulin-injected controls, but these cytokine levels were similar in NK-cell-depleted mice and their controls. The time courses of parasitemia in CCR2−/− and JH−/− × CCR2−/− mice and in their controls were nearly identical, indicating that MCP-1 is not required for the control of parasitemia. Collectively, these data indicate that the suppression of acute P. chabaudi infection by CMI is γδ T cell dependent, is independent of NK cells, and may be attributed to the deficient IFN-γ response seen early in γδ T-cell-depleted mice