Bone Abstracts

Neuropeptide Y Y₁ receptor (Y₁R) signalling has been shown to play a key role in bone homeostasis, emerging as a novel therapeutic target in bone diseases. Y₁R knockout mice (Y₁^−/−) display a high-bone mass phenotype that has been mainly attributed to increased osteoblast activity. Nevertheless, the Y₁R regulatory role on osteoclastogenesis and matrix resorption remains largely unknown. To clarify this, we proposed to investigate the effects of Y₁R in osteoclast function and matrix demineralization/resorption, using bone marrow-derived osteoclasts retrieved from Y₁^−/− mice and compared to their wild-type (WT) counterparts (n=6 per genotype).

The number of TRAP-positive multinucleated cells was significantly elevated in Y₁^−/− cultures, when compared to WT (P<0.001). Moreover, Y₁^−/− osteoclasts surface area and number of nuclei (N>8) were also significantly increased (P<0.01), suggesting enhanced formation and osteoclast fusion, possibly due to an overexpression of monocyte chemoattractant protein-1 (MCP-1) in Y₁^-/- cultures (P<0.01). Moreover, osteoblast/osteoclast direct co-cultures demonstrated that Y₁^−/− osteoblasts expressed higher-levels of RANKL (P<0.01), leading to increased RANKL/OPG ratio (P<0.001).

Paradoxically, functional studies using dentine discs demonstrated that in Y₁^−/− cultures there is an inhibition in matrix demineralization. Scanning-electron-microscopy images revealed that Y₁^−/− osteoclasts produce poorly demineralized resorption pits. To quantify these differences we have developed a novel computational tool (BonePit program), which allowed us to generate tridimensional (3D) reconstructions of resorption pits and to analyse pits morphological features. 3D analyses showed that Y₁^−/− resorption pits displayed a marked reduction in area, volume and depth (P<0.001), when compared to WT. In fact, the gene expression of matrix resorption markers, matrix metalloproteinase-9 and cathepsin K, was downregulated in Y₁^−/− cultures, suggesting the presence of non-resorbing osteoclasts in Y₁^−/− mice.

Together, these data disclosed an important role for Y₁R in osteoclastogenesis, essentially in bone matrix resorption, which may unravel new potential therapies for the treatment of osteoclastic bone diseases.