J Bone Miner Res. 2012 Aug;27(8):1708-21. doi: 10.1002/jbmr.1629.

Calcium/calmodulin-signaling supports TRPV4 activation in osteoclasts and regulates bone mass.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Masuyama, R., Mizuno, A., Komori, H., Kajiya, H., Uekawa, A., Kitaura, H., Okabe, K., Ohyama, K., Komori, T.,
["Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. ritsuko@nagasaki-u.ac.jp"]
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.
PMID: 22492541External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Screening Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPV4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Myosin-10 Affinity purification-mass spectrometry Mouse Full-length Human HEK293 Cell lystaes 22492541
TRPV4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Myosin-HIIA Affinity purification-mass spectrometry Mouse Full-length Human HEK293 Cell lystaes 22492541
TRPV4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RPS27A Affinity purification-mass spectrometry Mouse Full-length Human HEK293 Cell lystaes 22492541
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vitro validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with recombinant proteins Reference
TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Expression system Species Region Expression system
TRPV4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Calmodulin Fusion protein-pull down assay Mouse Full-length HEK293 lysates Not specified Not specified In vitro translation 22492541
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vivo validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with endogenous proteins Assay with overexpressed proteins Reference
Cell or tissue Cell or tissue TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Species Region
TRPV4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Myosin-10 Co-immunofluorescence staining HEK293 Mouse Full-length Not used 22492541
TRPV4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Myosin-HIIA Co-immunofluorescence staining HEK293 Mouse Full-length Not used 22492541
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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