Ablation of Y1 receptor impairs osteoclast bone-resorbing activity

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.

Sensory neurons sprouting is dependent on osteoclast-derived extracellular vesicles involving the activation of epidermal growth factor receptors

“…Conditioned medium from osteoclasts precursors was collected, centrifuged at 140 g, 4°C, 5 min, and stored at −80°C. Adherent cells were then detached with a cell scrapper and seeded at a density of 5 × 10 4 cells/cm 2 in the presence of 30 ng/mL M-CSF and 100 ng/mL receptor activator of nuclear factor kappa-B ligand (RANKL, PeproTech) (Bartell et al, 2014; Sousa et al, 2016). Cells were also seeded on top of bone slices (boneslices.com, Denmark) to collect the secretome from active resorbing osteoclasts.…”

The lack of neuropeptide Y‐Y 1 receptor signaling modulates the chemical and mechanical properties of bone matrix

“…Genotyping was performed using a PCR-based method to detect WT and knockout alleles. 9,10 On the day before the culling, mice were fasted overnight. In the next morning, mice were anesthetized with a Ketamine (150 mg/Kg) + Medetomidine (2 mg/Kg) solution and blood was collected by cardiac puncture until complete exsanguination.…”

“…The sympathetic neurons of the peripheral nervous system that project to the bone microenvironment release sympathetic neurotransmitters, such as the Neuropeptide Y (NPY), that activate receptors expressed by bone resident cells. [7][8][9] In this regard, NPY Y 1 receptor (Y 1 R) has been the major NPY signaling pathway shown to play a powerful regulatory action in bone remodeling, 10,11 fracture healing, 12 and bone repair. 13 Important insights from functional and genetic studies revealed that the absence of NPY Y 1 R sympathetic pathway promotes an increase in the bone mass and mineral apposition, which were consistent in femoral, tibial, and vertebral bones.…”

“…19,20 Although the high bone mass phenotype exhibited by Y 1 R knockout (Y 1 R −/− ) mice has been mainly attributed to enhanced osteoblastic activity, 14 our previous functional studies reported that the osteoclastic function and activity is also impaired. 9 The Y 1 R sympathetic signaling deficiency in male mice stimulates the formation of giant multinucleated osteoclasts, with reduced capacity to resorb bone matrix as appreciated by the formation of poorly demineralized eroded pits, and markedly smaller pits in terms of area, volume, and depth, as determined by quantitative 3D analysis using a computational tool. 9 As abovementioned, such defective matrix degradation can lead to substantial structural changes, namely in bone matrix composition, and might compromise bone biomechanical properties.…”

“…9 The Y 1 R sympathetic signaling deficiency in male mice stimulates the formation of giant multinucleated osteoclasts, with reduced capacity to resorb bone matrix as appreciated by the formation of poorly demineralized eroded pits, and markedly smaller pits in terms of area, volume, and depth, as determined by quantitative 3D analysis using a computational tool. 9 As abovementioned, such defective matrix degradation can lead to substantial structural changes, namely in bone matrix composition, and might compromise bone biomechanical properties. In this order of knowledge, we propose to characterize the bone ECM upon Y 1 R sympathetic pathway disruption, by analyzing the bone physical, chemical and biomechanical properties of germline Y 1 R −/− male mice, and compare to their wild-type (WT) littermates.…”