Spontaneous Ca2+ fluctuations mediated by TRPM7 channels in growth plate chondrocytes

[Speaker] Atsuhiko Ichimura:1
[Co-author] Qian Nianchao:1, Daisuke Takei:1, Reiko Sakaguchi:2, Miyuki Nishi:1, Yasuo Mori:2, Hiroshi Takeshima:1
1:Department of Biological Chemistry, Graduate School of Pharmaceutical Science, Kyoto University, Japan, 2:Department of Synthetic and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan

During embryonic bone development, condensations of mesenchymal progenitor cells give rise to round chondrocytes. Subsequently, round chondrocytes proliferate, differentiate into columnar chondrocytes, and then further differentiate into hypertrophic chondrocytes. Ca2+ signaling may play essential roles in this process. However, Ca2+ handling is largely unknown in growth plate chondrocytes. In this study, we developed a new fluorometric imaging method to survey Ca2+ signaling in growth plate chondrocytes using femoral bone slice preparations from E17.5 mouse embryos. Weak Ca2+ rises and falls, named spontaneous Ca2+ fluctuations, were observed in appreciable populations of round and columnar chondrocytes. Ca2+ removal and non-specific Ca2+ channel inhibitor Gd3+ addition abolished the Ca2+ fluctuations. On the other hand, sarco/endoplasmic reticulum Ca2+-pump inhibitors, thapsigargin and cyclopiazonic acid activated the Ca2+ fluctuations. Therefore, we confirmed that Ca2+ entry is essential for generating the Ca2+ fluctuations. Microarray analysis was conducted to identify the Ca2+ channel responsible for the Ca2+ fluctuations. We found that transient receptor potential melastatin subfamily 7 (TRPM7) channel, a non-selective cation channel that can conduct Ca2+ and Mg2+, is expressed in growth plate chondrocytes. Reported TRPM7 inhibitors, FTY720 and NS8593, attenuated the Ca2+ fluctuations while TRPM7 activators, naltriben and NNC550396, facilitated the Ca2+ fluctuations. Furthermore, the Ca2+ fluctuations were depressed by phospholipase C (PLC) inhibitor U73122, while they were stimulated by the large-conductance calcium-activated-K+ (BK) channel activator NS1619. Therefore, we conclude that TRPM7 channels mediated spontaneous Ca2+ fluctuations and were functionally coupled by PLC and BK channels in growth plate chondrocytes. Currently, we are trying to clarify the physiological function of the spontaneous Ca2+ fluctuations mediated TRPM7 channels in bone development.
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