ATP Release Mediates Fluid Flow–Induced Proliferation of Human Bone Marrow Stromal Cells

Abstract

Oscillatory fluid flow induced the vesicular release of ATP from human BMSCs that directly contributes to the induction of BMSC proliferation. Degrading extracellular nucleotides prevents fluid flow-induced increases in intracellular calcium concentration, the activation of calcineurin, and the nuclear translocation of NFAT. Regulation of bone cell activity by autocrine/paracrine factors is a well-established mechanism by which skeletal homeostasis is regulated by mechanical signals. The release of extracellular nucleotides in particular has been shown to induce many of the responses thought to be necessary for load-induced bone formation. In these studies, we examined the effect of oscillatory fluid flow on the release of ATP from bone marrow stromal cells (BMSCs) and the effect of ATP release on BMSC proliferation and intracellular calcium signaling pathways. BMSCs were exposed to oscillatory fluid flow, and the concentration of ATP in conditioned media samples was determined using a luciferin:luciferase-based reaction. Western blot analysis was used to examine the expression of purinergic receptors. Using pharmacological antagonists of gap junction hemichannels and vesicular trafficking, we studied the mechanism of ATP release from BMSCs. Apyrase was used to study the effect of extracellular nucleotides on intracellular calcium concentration, calcineurin activity, and nuclear factor of activated T cells (NFAT) nuclear translocation. Fluid flow exposure induced the flow rate-dependent release of ATP from BMSCs that was attenuated by treatment with monensin and N-ethylmaleimide, suggesting a vesicular mechanism. Treating BMSCs with ATP, but not other nucleotides, increased cellular proliferation. Moreover, extracellular ATP was a prerequisite for fluid flow-induced increases in intracellular calcium concentration, activation of calcineurin, the nuclear translocation of NFATc1, and proliferation. These data indicate that ATP regulates not only osteoblastic and osteocytic cell behavior but also that of mesenchymal precursors and support our hypothesis that similar mechanotransduction mechanisms are activated by fluid flow in these cell types.