Ablation of Y1 receptor impairs osteoclast bone-resorbing activity.

Daniela M Sousa,Inês S Alencastre,Francisco Conceição,Herbert Herzog,Paulo Aguiar,Diana I Silva,Luís Leitão,Estrela Neto,Cecília J Alves,Meriem Lamghari

doi:10.1038/srep33470

Abstract

Y1 receptor (Y1R)-signalling pathway plays a pivotal role in the regulation of bone metabolism. The lack of Y1R-signalling stimulates bone mass accretion that has been mainly attributed to Y1R disruption from bone-forming cells. Still, the involvement of Y1R-signalling in the control of bone-resorbing cells remained to be explored. Therefore, in this study we assessed the role of Y1R deficiency in osteoclast formation and resorption activity. Here we demonstrate that Y1R germline deletion (Y1R−/−) led to increased formation of highly multinucleated (n > 8) osteoclasts and enhanced surface area, possibly due to monocyte chemoattractant protein-1 (MCP-1) overexpression regulated by RANKL-signalling. Interestingly, functional studies revealed that these giant Y1R−/− multinucleated cells produce poorly demineralized eroded pits, which were associated to reduce expression of osteoclast matrix degradation markers, such as tartrate-resistant acid phosphatase-5b (TRAcP5b), matrix metalloproteinase-9 (MMP-9) and cathepsin-K (CTSK). Tridimensional (3D) morphologic analyses of resorption pits, using an in-house developed quantitative computational tool (BonePit), showed that Y1R−/− resorption pits displayed a marked reduction in surface area, volume and depth. Together, these data demonstrates that the lack of Y1Rs stimulates the formation of larger multinucleated osteoclasts in vitro with reduced bone-resorbing activity, unveiling a novel therapeutic option for osteoclastic bone diseases based on Y1R-signalling ablation.

Highlights

Anabolic effects have been exclusively attributed to increased osteoblast activity, given that Y1Rs are expressed in differentiated osteoblasts[11] and that the specific deletion of receptors from mature osteoblasts augmented osteoblast differentiation and activity[12]
These results provided the first indication that Y1Rs might play a regulatory function in osteoclast activity
Taking into account that Y1R seems to stimulate the formation of larger multinucleated cells, we have proposed to evaluate the expression of key markers of osteoclast precursors migration and fusion[17] that might be upregulated in Y1R−/− mice (Fig. 2K)

Summary

Introduction

Anabolic effects have been exclusively attributed to increased osteoblast activity, given that Y1Rs are expressed in differentiated osteoblasts[11] and that the specific deletion of receptors from mature osteoblasts augmented osteoblast differentiation and activity[12]. The involvement of Y1R signalling in bone turnover via osteoclast activity remains to be fully addressed. Y1R has been shown to be robustly expressed by immune cells (e.g. T-cells, monocytes and macrophages)[13] and to play a pivotal role in macrophage function[14]. Since both osteoclasts and macrophages share a common progenitor lineage, these studies suggest a direct role for Y1Rs in osteoclast progenitors activity. The aim of this study was to investigate the functional role of Y1R signalling in osteoclastogenesis and bone matrix resorption, using bone marrow-derived osteoclasts retrieved from wild-type (WT) and Y1R germ line knockout (Y1R−/−) mice. We have developed a novel computational tool – BonePit – that allowed to perform a detailed morphologic analysis of the resorption pits produced by osteoclasts in a 3D space

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Conclusion