Research Containing: Gene Expression

Purpose. Bone mass loss is a major consequence of extended periods of weightlessness. Many studies performed on astronauts and animals have shown that impaired maturation of osteoblast cells as well as a decrease of their bone-synthesising activity play key roles in microgravity-dependent bone mass loss. Several experiments on single cells and tissues showed that weightlessness can also influence cells cultured in vitro. Many molecular mechanisms are affected, among which the cytoskeleton, signal transduction cascades and gene expression. However, the underlying mechanisms of these changes and their molecular consequences are far from being fully understood. In contrast to weightlessness, dynamic mechanical loading increases bone density and strength and promotes osteoblast proliferation, differentiation and matrix production. A growing body of evidence points to extracellular nucleotides (i.e. ATP and UTP) as soluble factors that are released by several cell types in response to mechanical stimulation and that eventually trigger an intracellular signal. We have recently demonstrated that ATP and UTP, as well as mechanical stimulation, can activate two fundamental transcription factors, Runx2 and Egr-1, in the human HOBIT osteoblast cell line [Pines A. et al.,Biochem J. 2003; Costessi A. et al., Bone, 2005]. The purpose of the present study was to investigate the possible role(s) of extracellular nucleotides in the molecular response of osteoblast cells to weightlessness conditions. We focused on two aspects: 1. whether administration of ATP could stimulate osteoblast cells in weightlessness, possibly balancing or overcoming its known negative effects; 2. An analysis of the proteome of osteoblast cells exposed to weightlessness by means of two dimension electrophoresis (2-DE) coupled to mass spectrometry, to identify new molecular targets. Methodology – The BOP experiment. BOP was selected in the Success 2002 Student Contest organized by ESA and awarded to A.C. We developed and produced a new and low-cost dedicated hardware to support the experiment. We decided to use the human osteoblast cell line MG-63, since they had been studied in previous space experiments. The cells were grown in space for about five days in three chambers (control cells and cells treated with ATP for 20’ or 3 hours) and eventually lysed. A parallel ground experiment was performed. BOP flew during the Italian Soyuz Taxi flight in April 2005. Results and conclusions. We developed in less than nine months a new hardware that provided 70 cm2 per chamber and a total of more than 200 cm2. Preliminary analysis indicate that administration of ATP to MG-63 cells cultured in weightlessness conditions is able to increase ERK phosphorylation. Analysis of 2D gels revealed several differentially regulated proteins in response to ATP treatment. The identification of these proteins is in progress. To the best of our knowledge, BOP is the first proteomic study on mammalian cells cultured in space. The conclusion of the analysis will reveal new aspects of osteoblast biology and provide new insights into the molecular responses of human cells to weightlessness.

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