Dynamic visualization of RANKL and Th17-mediated osteoclast function
Junichi Kikuta, Yoh Wada, Toshiyuki Kowada, Ze Wang, Ge-Hong Sun-Wada, Issei Nishiyama, Shin Mizukami, Nobuhiko Maiya, Hisataka Yasuda, Atsushi Kumanogoh, Kazuya Kikuchi, Ronald N. Germain, Masaru Ishii
Junichi Kikuta, Yoh Wada, Toshiyuki Kowada, Ze Wang, Ge-Hong Sun-Wada, Issei Nishiyama, Shin Mizukami, Nobuhiko Maiya, Hisataka Yasuda, Atsushi Kumanogoh, Kazuya Kikuchi, Ronald N. Germain, Masaru Ishii
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Technical Advance Bone biology

Dynamic visualization of RANKL and Th17-mediated osteoclast function

Abstract

Osteoclasts are bone resorbing, multinucleate cells that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursor cells. Although previous studies have revealed important molecular signals, how the bone resorptive functions of such cells are controlled in vivo remains less well characterized. Here, we visualized fluorescently labeled mature osteoclasts in intact mouse bone tissues using intravital multiphoton microscopy. Within this mature population, we observed cells with distinct motility behaviors and function, with the relative proportion of static – bone resorptive (R) to moving – nonresorptive (N) varying in accordance with the pathophysiological conditions of the bone. We also found that rapid application of the osteoclast-activation factor RANKL converted many N osteoclasts to R, suggesting a novel point of action in RANKL-mediated control of mature osteoclast function. Furthermore, we showed that Th17 cells, a subset of RANKL-expressing CD4+ T cells, could induce rapid N-to-R conversion of mature osteoclasts via cell-cell contact. These findings provide new insights into the activities of mature osteoclasts in situ and identify actions of RANKL-expressing Th17 cells in inflammatory bone destruction.

Authors

Junichi Kikuta, Yoh Wada, Toshiyuki Kowada, Ze Wang, Ge-Hong Sun-Wada, Issei Nishiyama, Shin Mizukami, Nobuhiko Maiya, Hisataka Yasuda, Atsushi Kumanogoh, Kazuya Kikuchi, Ronald N. Germain, Masaru Ishii

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Figure 4

RANKL-mediated rapid control of mature osteoclast function.

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RANKL-mediated rapid control of mature osteoclast function. (A) Intravit...
(A) Intravital multiphoton imaging of osteoclasts in mouse bone tissues of a3-GFP mice under control conditions (Supplemental Video 6). Mature osteoclasts expressing GFP-fused V-type H+ ATPase a3 subunit are in green. Blue, bone surface. Cell borders are marked in white lines. Scale bar: 40 μm. (B) Representative computer-processed images of mature osteoclasts and their RANKL-mediated rapid shape changes. Images 1–4 were computer extracted from images under the initial condition from A (left panels) and again 10 minutes after i.v. injection of 1 mg/kg of GST-RANKL (right panels) (as in Figure 1, E and F). Scale bars: 5 μm. (C) Representative time courses of the CDI for the 4 individual cells shown in B. Moving (high CDI) osteoclasts underwent transition to the static (low CDI) state less than 10 minutes after i.v. administration of RANKL. (D) The summary of CDIs under control conditions (control) and 40 minutes after i.v. injection of 0.1 mg/kg of GST-RANKL (RANKL 0.1), 1 mg/kg of GST-RANKL (RANKL 1), or GST alone (n = 13 for control, n = 5 for 0.1 mg/kg of GST-RANKL, n = 13 for 1 mg/kg of GST-RANKL, and n = 21 for GST alone, compiled from 3 independent experiments).