Modeled gravitational unloading triggers differentiation and apoptosis in preosteoclastic cells

Abstract

Gravity acts permanently on organisms as either static or dynamic stimulation. Understanding the influence of gravitational and mechanical stimuli on biological systems is an intriguing scientific problem. More than two decades of life science studies in low g, either real or modeled by clinostats, as well as experimentation with devices simulating different types of controlled mechanical stimuli, have shown that important biological functions are altered at the single cell level. Here, we show that the human leukemic line FLG 29.1, characterized as an osteoclastic precursor model, is directly sensitive to gravitational unloading, modeled by a random positioning machine (RPM). The phenotypic expression of cytoskeletal proteins, osteoclastic markers, and factors regulating apoptosis was investigated using histochemical and immunohistochemical methods, while the expression of the corresponding genes was analyzed using RT-PCR. A quantitative bone resorption assay was performed. Autofluorescence spectroscopy and imaging were applied to gain information on cell metabolism. The results show that modeled hypogravity may trigger both differentiation and apoptosis in FLG 29.1 cells. Indeed, when comparing RPM versus 1 Â g cultures, in the former we found cytoskeletal alterations and a marked increase in apoptosis, but the surviving cells showed an osteoclastic-like morphology, overexpression of osteoclastic markers and the ability to resorb bone. In particular, the overexpression of both RANK and its ligand RANKL, maintained even after return to 1 Â g conditions, is consistent with the firing of a differentiation process via a paracrine/autocrine mechanism. J. Cell. Biochem. 98: 65-80, 2006. ß 2005 Wiley-Liss, Inc.Key words: weightlessness; preosteoclastic differentiation; apoptosis; bone resorption; osteoporosis; gene expressionThe importance of gravity in modulating some biological processes, such as plant gravitropism and the adaptation of the vertebrate skeleton in relation to its load-bearing function, has long been known. Nevertheless, the role of gravity and other mechanical factors in biological processes is far from clear. A weightless environment is a powerful tool for understanding this role and for studying the related cellular and molecular mechanisms. Several investigations conducted in space laboratories and with instruments averaging the gravity vector, and thus simulating conditions of reduced gravity, have shown that isolated cells may change their behavior under altered gravitational conditions. Loss of cell activation [Cogoli et al., 1984], perturbation of signal transduction [Schmitt et al., 1996;Hashemi et al., 1999] and modification of genetic expression [Walther et al., 1998;Hammond et al., 1999] have been described. This suggests that some of the mechanisms at the roots of the systemic effects observed in weightlessness, such as depression of the immune system [Gmü nder and Cogoli, 1996]

Effect of IR Laser on Myoblasts: Prospects of Application for Counteracting Microgravity-Induced Muscle Atrophy

“…No significant differences were found between untreated and laser-treated 1 × g controls (a, b, c, f, i, j). The samples exposed to modelled microgravity and then to the laser treatment showed a cytoskeletal structure restored and similar to the 1 × g controls (d, h, l) increase in apoptosis, which often occurs in cell cultures exposed to weightless conditions (Uva et al 2002;Monici et al 2006), a microgravity-induced decrease in myoblast proliferation, which has been recently found by other authors (Pache et al 2010), altered protein synthesis/degradation (Moriggi et al 2010), or a combination of these effects.…”

Simulated microgravity induce apoptosis and down-regulation of erythropoietin receptor of UT-7/EPO cells

“…Yang et al found the apoptosis in luteal cells induced by clinostat rotation was related to the mitochondrial dysfunction (Yang et al, 2002). Simulated microgravity-induced apoptosis was related to both extrinsic and intrinsic pathways by the evidences of activation of caspase-3 and increases of Fas and Bax in normal thyroid cells and papillary thyroid carcinoma cells (Kossmehl et al, 2003), as well as human endothelial cells (Infanger et al, 2006a) and preosteoclastic FLG 29.1 cells (Monici et al, 2006). Simulated microgravity activates caspase-8 and -3 and the transcription factor NF-jB necessary to stimulate the apoptotic pathway in mice testis (Sharma et al, 2008).…”

“…The data represent the mean ± SD from three independent experiments (*P < 0.05). (Monici et al, 2006). Yang et al found the apoptosis in luteal cells induced by clinostat rotation was related to the mitochondrial dysfunction (Yang et al, 2002).…”

Impact of Osteoclast Precursors Subjected to Random Positioning Machine on Osteoblasts

“…The simulated weightlessness was produced by averaging the gravity vector via the independent rotation of the two frames. 14 For culturing RAW264.7 cells in the RPM, cultures were seeded in 25 mL culture°a sks and cultured overnight. Then the°asks were completely¯lled with medium to eliminate air bubbles, and diminish turbulence and shear forces.…”