Abstract
Calcium balance is important in bone homeostasis. The transient receptor potential vanilloid (TRPV) channel is a nonselective cation channel permeable to calcium and is activated by various physiological and pharmacological stimuli. TRPV1 and TRPV4, in particular, have important roles in intracellular Ca2+ signaling and extracellular calcium homeostasis in bone cells. TRPV1 and TRPV4 separately mediate osteoclast and osteoblast differentiation, and deficiency in any of these channels leads to increased bone mass. However, it remains unknown whether bone mass increases in the absence of both TRPV1 and TRPV4. In this study, we used TRPV1 and TRPV4 double knockout (DKO) mice to evaluate their bone mass in vivo, and osteoclast and osteoblast differentiation in vitro. Our results showed that DKO mice and wild type (WT) mice had no significant difference in body weight and femur length. However, the results of dual-energy X-ray absorption, microcomputed tomography, and bone histomorphometry clearly showed that DKO mice had higher bone mass than WT mice. Furthermore, DKO mice had less multinucleated osteoclasts and had lower bone resorption. In addition, the results of cell culture using flushed bone marrow from mouse femurs and tibias showed that osteoclast differentiation was suppressed, whereas osteoblast differentiation was promoted in DKO mice. In conclusion, our results suggest that the increase in bone mass in DKO mice was induced not only by the suppression of osteoclast differentiation and activity but also by the augmentation of osteoblast differentiation and activity. Our findings reveal that both the single deficiency of TRPVs and the concurrent deficiency of TRPVs result in an increase in bone mass. Furthermore, our data showed that DKO mice and single KO mice had varying approaches to osteoclast and osteoblast differentiation in vitro, and therefore, it is important to conduct further studies on TRPVs regarding the increase in bone mass to explore not only individual but also a combination of TRPVs.
Highlights
The skeletal system is affected by both intracellular Ca2+ signaling and external Ca2+ balance in bone cells
Transient receptor potential (TRP) channels are transmembrane protein channels composed of six transmembrane segments and are Abbreviations: TRP vanilloid (TRPV), transient receptor potential vanilloid; V1KO, TRPV1 knock out; V4KO, TRPV4 knock out; double knockout (DKO), double knock out; WT, wild type; PCR, polymerase chain reaction; dual-energy X-ray absorptiometry (DXA), dual-energy X-ray absorption; CT, computed tomography; POc, preosteoclast; RANKL, receptor activator of nuclear factor-kappa B ligand; RANK, receptor activator of nuclear factor-kappa B; BMD, bone mineral density; TRACP, tartrate-resistant acid phosphatase; ALP, alkaline phosphatase; MNCs, multinucleated cells; CB, cannabinoid; BMSCs, bone marrow mesenchymal stem cells
The results of DXA, micro-CT, and bone morphometry clearly showed that DKO mice had higher bone mass and had higher BMD, BV/ TV, Tb N, and OS/BS
Summary
The skeletal system is affected by both intracellular Ca2+ signaling and external Ca2+ balance in bone cells. A previous study reported that TRPV4 knockout (V4KO) osteoclasts had lower intracellular Ca2+ levels, NFATc1 activity, osteoclast differentiation, and resorptive capacity than did wild type osteoclasts (Masuyama et al, 2012). V4KO mice had higher bone mass, but lower osteoclast abundance and bone resorption than did wild type mice (Masuyama et al, 2008) Both TRPV1 and TRPV4 modulate osteoclast differentiation, and impairing these channels increases bone mass. Our results show that DKO mice have a higher bone mass than wild type mice due to the suppression of osteoclast differentiation and the exaggeration of osteoblast differentiation Taking this result into consideration, the concurrent deficiency of TRPV1 and TRPV4 is probably more effective for an increase in bone mass than individual deficiency. Our findings may contribute to the further investigation of the mechanism and a new treatment of osteoporosis associated with TRPV1 and TRPV4
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