Transient receptor potential vanilloid 1 and 4 double knockout leads to increased bone mass in mice

Haruki Nishimura; Makoto Kawasaki; Manabu Tsukamoto; Kunitaka Menuki; Hitoshi Suzuki; Takanori Matsuura; Kazuhiko Baba; Yasuhito Motojima; Teruaki Fujitani; Hideo Ohnishi; Yoshiaki Yamanaka; Kenji Kosugi; Yasuaki Okada; Kotaro Tokuda; Takafumi Tajima; Toru Yoshioka; Nobukazu Okimoto; Yoichi Ueta; Akinori Sakai

doi:10.1016/j.bonr.2020.100268

Transient receptor potential vanilloid 1 and 4 double knockout leads to increased bone mass in mice

Bone Rep. 2020 Apr 23:12:100268. doi: 10.1016/j.bonr.2020.100268. eCollection 2020 Jun.

Authors

Haruki Nishimura¹, Makoto Kawasaki¹, Manabu Tsukamoto¹, Kunitaka Menuki¹, Hitoshi Suzuki¹, Takanori Matsuura¹, Kazuhiko Baba¹, Yasuhito Motojima¹, Teruaki Fujitani¹, Hideo Ohnishi¹, Yoshiaki Yamanaka¹, Kenji Kosugi¹, Yasuaki Okada¹, Kotaro Tokuda¹, Takafumi Tajima¹, Toru Yoshioka², Nobukazu Okimoto³, Yoichi Ueta⁴, Akinori Sakai¹

Affiliations

¹ Department of Orthopaedic Surgery, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan.
² Department of Orthopaedics, Shimura Hospital, 3-13 Funairimachi Naka-ku, Hiroshima 730-0841, Japan.
³ Okimoto Clinic, 185-4 Yutakamachikubi, Kure, Hiroshima 734-0304, Japan.
⁴ Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan.

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.

Keywords: ALP, alkaline phosphatase; BMD, bone mineral density; BMSCs, bone marrow mesenchymal stem cells; Bone histomorphometry; CB, cannabinoid; CT, computed tomography; Cell culture; DKO, double knock out; DXA, dual-energy X-ray absorption; MNCs, multinucleated cells; Micro-CT; Osteogenesis; PCR, polymerase chain reaction; POc, preosteoclast; Preosteoclast; RANK, receptor activator of nuclear factor-kappa B; RANKL, receptor activator of nuclear factor-kappa B ligand; TRACP, tartrate-resistant acid phosphatase; TRPV, transient receptor potential vanilloid; Transient receptor potential vanilloid; V1KO, TRPV1 knock out; V4KO, TRPV4 knock out; WT, wild type.