Mechanical perturbation has been shown to modulate a wide variety of changes in second message signals and patterns of gene expression in osteoblasts. Embryonic chick osteoblasts were subjected to a dynamic spatially uniform biaxial strain (1.3% applied strain) at 0.25 Hz for a single 2-h period, and osteopontin (OPN), an Arg-Gly-Asp (RGD)-containing protein, was shown to be a mechanoresponsive gene. Expression of opn mRNA reached a maximal 4-fold increase 9 h after the end of the mechanical perturbation that was not inhibited by cycloheximide, thus demonstrating that mechanoinduction of opn expression is a primary response through the activation of pre-existing transcriptional factors. The signal transduction pathways, which mediated the increased expression of opn in response to mechanical stimuli, were shown to be dependent on the activation of a tyrosine kinase(s) and protein kinase A (PKA) or a PKA-like kinase. Selective inhibition of protein kinase C (PKC) had no effect on the mechanoinduction of osteopontin even though opn has been demonstrated to be an early response gene to phorbol 12-myristate 13-acetate (PMA) stimulation. Mechanotransduction was dependent on microfilament integrity since cytochalasin-D blocked the up-regulation of the opn expression; however, microfilament disruption had no effect on the PMA induction of the gene. The microtubule component of the cytoskeleton was not related to the mechanism of signal transduction involved in controlling opn expression in response to mechanical stimulation since colchicine did not block opn expression. Mechanical stimulus was shown to activate focal adhesion kinase (FAK), which specifically became associated with the cytoskeleton after mechanical perturbation, and its association with the cytoskeleton was dependent on tyrosine kinase activity. In conclusion, these results demonstrate that the signal transduction pathway for mechanical activation of opn is uniquely dependent on the structural integrity of the microfilament component of the cytoskeleton. In contrast, the PKC pathway, which also activates this gene in osteoblasts, acts independently of the cytoskeleton in the transduction of its
Conclusion.These data demonstrate the presence of TLRs in human articular cartilage. The suppressive effects of LPS on cartilage biosynthetic activity are dependent on the presence of TLR-4, are governed, at least in part, by an up-regulation of IL-1, and are mediated by p38 kinase. These in vitro data indicate an anti-anabolic effect of TLR-4 in articular chondrocytes that may hamper cartilage repair in various joint diseases.
Based on neoadjuvant chemotherapy, the prognosis of osteosarcoma patients has improved dramatically. However, due to therapy resistance in patient subgroups, the development of new treatment strategies is still of utmost importance. The aim of our study was to test the effects of the nitrogen-containing bisphosphonate zoledronic acid (ZOL) on osteosarcoma cell lines (N ¼ 9). Exposure to ZOL at low micromolar concentrations induced a dose-and time-dependent block of DNA synthesis and cell cycle progression followed by microfilament breakdown and apoptosis induction. The ZOL-induced cell cycle accumulation in S phase was accompanied by significant changes in the expression of cyclins and cyclin-dependent kinase inhibitors with a prominent loss of cyclin E and D1. ZOL not only inhibited growth but also migration of osteosarcoma cells. The mevalonate pathway intermediary geranyl-geraniol (GGOH) but not farnesol (FOH) significantly inhibited the anticancer effects of ZOL against osteosarcoma cells. Correspondingly, ZOL sensitivity correlated with the blockade of protein geranylgeranylation indicated by unprenylated Rap1. Overexpression of even high levels of P-glycoprotein, as frequently present in therapy-resistant osteosarcomas, did not impair the anticancer activity of ZOL. Summarizing, our data suggest that ZOL, which selectively accumulates in the bone, represents a promising agent to improve osteosarcoma therapy. ß
Background: Despite the increasing promotion of alcohol-based hand rubs and the worldwide use of ethanol-based hand rubs in hospitals only few studies have specifically addressed the issue of ethanol absorption when repeatedly applied to human skin. The aim of this study was to assess if ethanol absorption occurs during hygienic and surgical hand disinfection using three different alcohol-based hand-rubs, and to quantify absorption levels in humans.
The structural integrity of microfilaments has been shown to be necessary for the signal transduction of mechanical stimuli within osteoblasts. Qualitative and quantitative changes within the cytoskeleton of osteoblasts may therefore be crucial components of the signal transduction processes of these cells in response to mechanical stimulation. Avian osteoblasts were strained with a device that deforms a flexible, cell-laden membrane at a defined frequency and intensity in a uniform biaxial manner. We examined the effects of mechanical strain on the accumulation of protein and the expression of the major cytoskeletal elements and specific integrin-binding (arginine-glycine-aspartic acid) proteins of these cells. Mechanical strain increased the level of total extracellular matrix-accumulated fibronectin by approximately 150% and decreased that of osteopontin by approximately 60% but had no quantifiable effect on the accumulation of beta1 integrin subunit or collagen type I. An examination of the major elements of the cytoskeleton demonstrated that neither the level of actin nor that of the intermediate filament protein vimentin changed; however, the amount of tubulin decreased by approximately 75% and the amount of vinculin, a major protein of focal adhesion complexes, increased by approximately 250%. An analysis of protein synthesis by two-dimensional gel electrophoresis of [35S]methionine-labeled cytoskeletal proteins demonstrated that the changes in the accumulation of vinculin and tubulin resulted from their altered synthesis. Messenger RNA analysis confirmed that the changes in accumulation and protein synthesis observed for vinculin, fibronectin, and osteopontin were controlled at a pretranslational level. Immunofluorescent microscopy demonstrated that mechanical strain led to increased formation and thickening of actin stress fibers, with a commensurate dissociation in microtubules and a clear increase in levels of vinculin at the peripheral edges of the cells. In conclusion, the elevated rate of synthesis and the increased accumulation of vinculin and fibronectin, as well as the increase in the number and size of stress fibers and focal adhesion complexes, suggest that mechanical strain leads to a coordinated change both in the cytoskeleton and in extracellular matrix proteins that will facilitate tighter adhesion of an osteoblast to its extracellular matrix.
The results of this study indicate that Bc1-2 and Livin are highly expressed in osteosarcoma cells and that possibly, the evaluation of nuclear Livin expression might be a useful prognostic marker in osteosarcoma.
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