Extracellular NO signalling from a mechanically stimulated osteocyte

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Abstract

Bone remodelling is a dynamic process that requires the coordinated interaction of osteocytes, osteoblasts, and osteoclasts, collaborating in basic multicellular units (BMUs). Communication between these cells can be by extracellular soluble molecules as well as directly propagating intercellular signalling molecules. Key to the understanding of bone remodelling is osteocyte mechanosensing and chemical signalling to the surrounding cells, since osteocytes are believed to be the mechanosensors of bone, responding to mechanical stresses. Nitric oxide (NO) is an important parameter to study osteocyte activation following mechanical loading. It is a small short-lived molecule, which makes its real-time, quantitative monitoring difficult. However, recently we demonstrated that DAR-4M AM chromophore can be used for real-time quantitative monitoring of intracellular NO production in individual cells following mechanical loading. Here we studied if a single mechanically stimulated osteocyte communicates with, and thus activates its surrounding cells via extracellular soluble factors. We monitored quantitatively intracellular NO production in the stimulated osteocyte and in its surrounding osteocytes, which were not interconnected. Mechanical stimulation by microneedle of a single-MLO-Y4 osteocyte-like cell upregulated the average intracellular NO production by 94% in the stimulated cell, and by 31–150% in the surrounding osteocytes. In conclusion, a single osteocyte can disseminate a mechanical stimulus to its surrounding osteocytes via extracellular soluble signalling factors. This reinforces the putative mechanosensory role of osteocytes, and demonstrates a possible mechanism by which a single mechanically stimulated osteocyte can communicate with other cells in a BMU, which might help to better understand the intricacies of intercellular interactions in BMUs and thus bone remodelling.

Introduction

Osteocytes are widely accepted as the mechanosensors in bone (Cowin et al., 1991; Burger and Klein-Nulend, 1999). Anatomically, osteocytes are housed in the lacunar spaces within the mineralised matrix and form a network with surrounding osteocytes and surface lining cells via their cell processes that run through the canaliculi (Doty, 1981; Palumbo et al., 1990). Bone remodelling involves osteocytes, osteoblasts, and osteoclasts, which interact in basic multicellular units (BMUs). The concerted activity of osteoblasts and osteoclasts orchestrated by the mechanosensing osteocytes likely leads to bone remodelling (Smit et al., 2002).

Mechanical loading activates the osteocytes, which produce signalling molecules like nitric oxide (NO) (Klein-Nulend et al., 1995, Klein-Nulend et al., 1998; Pitsillides et al., 1995; Vatsa et al., 2006) that modulates the activity of bone-forming osteoblasts (Chow et al., 1998; Vezeridis et al., 2006) and bone-resorbing osteoclasts (MacIntyre et al., 1991). This intercellular communication via signalling molecules like NO plays a key role in bone remodelling (Smit et al., 2002; Burger et al., 2003). NO is ubiquitously present in human physiological systems (Davis et al., 2001). It functions as a messenger molecule in many signalling processes through the cellular guanisine 3′,5′-monophosphate (cGMP) pathway and through the nitrosylation of regulatory thiols (Davis et al., 2001). NO is a small and short-lived molecule which can readily cross the cell membrane, and thus functions as a paracrine second messenger. NO is essential for mechanically induced bone remodelling (Turner et al., 1996; Chow et al., 1998). The precise mechanism of action of NO in bone remodelling is not fully elucidated, but upregulation of NO production following mechanical stimulation has been used as a marker for the activation of osteocytes (Klein-Nulend et al., 1995).

Osteocytes form a gap-junction-coupled network, interconnecting osteocytes and bone lining cells in bone (Doty, 1981; Palumbo et al., 1990). Mechanical stimulation has been shown to modulate the activity of gap junctional proteins (Cherian et al., 2003) and gap-junction-dependent propagation of Ca2+ waves in osteoblasts (Jorgensen et al., 2000), showing the presence of direct intercellular signalling through intercellular connections. Both direct intercellular signalling and indirect extracellular signalling likely contribute to the communication between the mechanosensing osteocytes and the effector cells, the osteoblasts and osteoclasts, in a BMU. Little is known about the capacity of a single mechanically stimulated osteocyte to disseminate information of local mechanical stimulation to the surrounding cells in a BMU. Insight into interactions between the different cells in a BMU might shed light on the intricacies of bone remodelling.

Recently, we have shown that localised mechanical stimulation of a single osteocyte upregulates intracellular NO production (Vatsa et al., 2006). This site-specific mechanical loading enables to study the effect of mechanical stimulation of a single osteocyte on the intracellular NO production in the surrounding cells. Moreover, absolute concentrations of intracellular NO production can be quantitatively determined in situ at a single-cell level using the chromophore DAR-4M AM (Vatsa et al., 2005).

In this study, we applied direct single cell mechanical stimulation and intracellular NO detection to investigate whether a mechanically stimulated single osteocyte communicates with, and thus activates its surrounding cells via soluble extracellular factors, and hence propagates the mechanical signal to its surrounding cells. This may contribute to uncovering the complexities of intercellular interactions in BMUs and to better understand bone remodelling.

Section snippets

Bone cell culture

MLO-Y4 osteocyte-like cells (a kind gift from Dr. Bonewald, San Antonio, TX, USA) (Bonewald, 1999) were cultured in 25 cm2 flasks (Nunc, Roskilde, Denmark) in Dulbecco's modified Eagle's medium (DMEM; Gibco, Paisley, UK) at 37 °C in a humidified atmosphere of 5% CO2 in air. DMEM was supplemented with 5% foetal bovine serum (FBS), 5% calf serum (CS), 100 U/ml penicillin (Sigma, St. Louis, MO, USA), 50 μg/ml streptomycin sulphate (Gibco), and 1.25 μg/ml fungizone (Gibco). Cells between passages 30 and

Results

Fig. 1 shows the position of individual DAR-4M AM chromophore-loaded MLO-Y4 osteocytes in relation to each other. The osteocytes did not form cell processes and hence intercellular connections during the short incubation time of 20–30 min after seeding. The representative osteocytes were chosen for their varying distances (33–175 μm) and different radial directions from the mechanically stimulated cell (Fig. 1). Osteocytes showed sufficient chromophore uptake, leading to a uniform visible

Discussion

The purpose of this study was to monitor the propagation of mechanical stimulation in single-MLO-Y4 osteocyte-like cells by studying NO signalling after mechanical stimulation of a single osteocyte. Intracellular NO production of each osteocyte was monitored quantitatively in real time by using DAR-4M AM chromophore. A single-DAR-4M AM-loaded osteocyte was mechanically stimulated by using a microneedle tip. We hypothesised that a mechanically stimulated osteocyte can disseminate the information

Acknowledgements

This study was funded by Foundation for Fundamental Research on Matter (FOM), The Netherlands (ALW/FOM/NWO Project no. 01FB28/2). The authors are grateful to Christoph Schmidt, Fred MacKintosh, and Daisuke Mizuno for helpful discussions on the quantification of NO production in single cells.

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