UTP-induced ATP release is a fine-tuned signalling pathway in osteocytes

Abstract

Osteocytes reside as a cellular network throughout the mineralised matrix of bone and are considered the primary mechanosensors of this tissue. They sense mechanical stimulation such as fluid flow and are able to regulate osteoblast and osteoclast functions on the bone surface. Previously, we found that ATP is released load-dependently from osteocytes from the onset of mechanical stimulation. Therefore, the aim of the present study was to investigate whether and how ATP release can be evoked in osteocytes via purinergic receptor activation. ATP release was quantified by real-time determination using the luciferin-luciferase assay and the release pathway was investigated using pharmacological inhibition. The P2Y receptor profile was analysed using gene expression analysis by reverse transcription polymerase chain reaction, while functional testing was performed using measurements of intracellular calcium responses to P2 receptor agonists. These investigations demonstrated that MLO-Y4 osteocytes express functional P2Y(2), P2Y(4), P2Y(12) and P2Y(13) receptors in addition to the previously reported P2X receptors. Further, we found that osteocytes respond to nucleotides such as ATP, UTP and ADP by increasing the intracellular calcium concentration and that they release ATP dose-dependently upon stimulation with 1-10 μM UTP. In addition to this, osteocytes release large amounts of ATP upon cell rupture, which might also be a source for other nucleotides, such as UTP. These findings indicate that mechanically induced ATP signals may be propagated by P2 receptor activation and further ATP release in the osteocyte network and implicate purinergic signalling as a central signalling pathway in osteocyte mechanotransduction.

ATP release, generation and hydrolysis in exocrine pancreatic duct cells

“…Previous studies on Schwann cells and osteocytes have shown that UTP can enhance the ATP e concentration [33,34]. UTP could be released together with ATP from acinar zymogen granules or from duct cells, as it has been shown for airway epithelia and astrocytoma cells [35].…”

The role of purinergic signalling in the musculoskeletal system

“…An early study found that the calcium signals induced by fluid flow were decreased by suramin and thapsigargin, suggesting involvement of ATP acting via P2Y receptors (Huo et al, 2008). Expression of functional P2X2, P2X7, P2Y2, P2Y4, P2Y12 and P2Y13 receptors on MLO-Y4 osteocyte-like cells has recently been described (Kringelbach et al, 2014) Several studies have also reported that osteocytes release ATP in a controlled manner (Genetos et al, 2007;Kringelbach et al, 2014;Thompson et al, 2011). This release is enhanced by UTP and mechanical stimulation and is thought to be mediated primarily by vesicular exocytosis (Kringelbach et al, 2014).…”

“…Expression of functional P2X2, P2X7, P2Y2, P2Y4, P2Y12 and P2Y13 receptors on MLO-Y4 osteocyte-like cells has recently been described (Kringelbach et al, 2014) Several studies have also reported that osteocytes release ATP in a controlled manner (Genetos et al, 2007;Kringelbach et al, 2014;Thompson et al, 2011). This release is enhanced by UTP and mechanical stimulation and is thought to be mediated primarily by vesicular exocytosis (Kringelbach et al, 2014). Mechanically induced ATP release can also be regulated by the α2δ1 auxillary subunit of Ttype voltage sensitive calcium channels (Thompson et al, 2011).…”

“…Controlled ATP release has been demonstrated from numerous excitatory and non-excitatory cells. In the bone microenvironment, osteoblasts (Buckley et al, 2003;Genetos et al, 2005;Orriss et al, 2009;Romanello et al, 2001;Rumney et al, 2012), osteoclasts and MLO-Y4 osteocyte-like cells (Genetos et al, 2007;Kringelbach et al, 2014) have all been shown to constitutively release ATP.…”

Pyrophosphate: a key inhibitor of mineralisation

“…Since osteocytes are embedded within bone they must be capable of preventing over-mineralisation of their lacunae (which could potentially compromise cell viability and function). ATP, which is released by all bone cells including osteocytes [22,25,[60][61][62][63][64][65], is an important source of pyrophosphate in bone [17,66]. Previous work has shown that endogenous ATP released by osteoblasts acts as an important local brake on mineralisation, an effect mediated by both purinergic signalling and the breakdown to produce pyrophosphate [38, 67,68].…”