title: Calcium/calmodulin-signaling supports TRPV4 activation in osteoclasts and regulates bone mass.
abstract: Osteoclast differentiation is critically dependent on calcium (Ca(2+)) signaling. Transient receptor potential vanilloid 4 (TRPV4), mediates Ca(2+) influx in the late stage of osteoclast differentiation and thereby regulates Ca(2+) signaling. However, the system-modifying effect of TRPV4 activity remains to be determined. To elucidate the mechanisms underlying TRPV4 activation based on osteoclast differentiation, TRPV4 gain-of-function mutants were generated by the amino acid substitutions R616Q and V620I in TRPV4 and were introduced into osteoclast lineage in Trpv4 null mice to generate Trpv4(R616Q/V620I) transgenic mice. As expected, TRPV4 activation in osteoclasts increased the number of osteoclasts and their resorption activity, thereby resulting in bone loss. During in vitro analysis, Trpv4(R616Q/V620I) osteoclasts showed activated Ca(2+)/calmodulin signaling compared with osteoclasts lacking Trpv4. In addition, studies of Trpv4(R616Q/V620I) mice that lacked the calmodulin-binding domain indicated that bone loss due to TRPV4 activation was abrogated by loss of interactions between Ca(2+)/calmodulin signaling and TRPV4. Finally, modulators of TRPV4 interactions with the calmodulin-binding domain were investigated by proteomic analysis. Interestingly, nonmuscle myosin IIa was identified by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis, which was confirmed by immunoblotting following coimmunoprecipitation with TRPV4. Furthermore, myosin IIa gene silencing significantly reduced TRPV4 activation concomitant with impaired osteoclast maturation. These results indicate that TRPV4 activation reciprocally regulates Ca(2+)/calmodulin signaling, which involves an association of TRPV4 with myosin IIa, and promotes sufficient osteoclast function.
authors.affiliation: Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. ritsuko@nagasaki-u.ac.jp
authors.forename: Ritsuko
authors.initials: R
authors.lastname: Masuyama
authors.forename: Atsuko
authors.initials: A
authors.lastname: Mizuno
authors.forename: Hisato
authors.initials: H
authors.lastname: Komori
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kajiya
authors.forename: Atsushi
authors.initials: A
authors.lastname: Uekawa
authors.forename: Hideki
authors.initials: H
authors.lastname: Kitaura
authors.forename: Koji
authors.initials: K
authors.lastname: Okabe
authors.forename: Kaname
authors.initials: K
authors.lastname: Ohyama
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Komori
mesh_terms: D000818:Animals; D001862:Bone Resorption / Q000097:blood / Q000473:pathology; D001842:Bone and Bones / Q000378:metabolism* / Q000473:pathology*; D020013:Calcium Signaling; D002147:Calmodulin / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D051379:Mice; D009218:Myosins / Q000378:metabolism; D009929:Organ Size; D010010:Osteoclasts / Q000378:metabolism* / Q000473:pathology*; D017434:Protein Structure, Tertiary; D050916:TRPV Cation Channels / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D050916:TRPV Cation Channels; C486576:Trpv4 protein, mouse; D009218:Myosins