Research ArticlePhysiology

Protein kinase N3 promotes bone resorption by osteoclasts in response to Wnt5a-Ror2 signaling

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Science Signaling  29 Aug 2017:
Vol. 10, Issue 494, eaan0023
DOI: 10.1126/scisignal.aan0023
  • Fig. 1 Wnt5a secreted from osteoclasts regulates osteoclast bone-resorbing activities.

    (A) RT-PCR analysis of Ctsk and Wnt expression in BMM cultures treated with or without Rankl and Csf1. n = 5 dishes for each time point. BMMs were prepared from five mice. ND, not detected. (B) Immunoblotting analysis of Wnt5a abundance in BMM cultures treated with glutathione S-transferase (GST)–Rankl plus Csf1. n = 3 biological replicates. (C and D) Effects of recombinant Wnt5a on the formation of resorption pits (hematoxylin staining) (C) and actin rings (D) by osteoclasts derived from wild-type (WT) and Wnt5a−/− liver macrophages on dentin slices. n = 5 slices for each genotype. Osteoclasts were prepared from three mice for each genotype. Scale bars, 100 μm. In (A), (C), and (D), error bars represent SD. *P < 0.05, **P < 0.01. n.s., not significant. For statistical analyses, Kruskal-Wallis and Steel-Dwass test (A) or analysis of variance (ANOVA) and Scheffé test (C and D) were used.

  • Fig. 2 Ror2-mediated signals are required for bone-resorbing activity of osteoclasts.

    (A) Reverse transcription PCR analysis of Ror1 and Ror2 in osteoclasts. n = 3 biological replicates. (B) RT-PCR analysis of Ror2 mRNA in BMMs and osteoclasts from Ror2fl/fl (Control) and Ror2fl/fl: CtskCre/+ (Ror2ΔOcl/ΔOcl) mice. n = 5 cultures of BMMs and osteoclasts for each genotype. (C) Immunoblotting of Ror2 in osteoclasts. n = 3 biological replicates. (D) Micro-CT of distal femurs from male control (Ror2fl/fl) and Ror2ΔOcl/ΔOcl mice. n = 8 mice for each genotype. Scale bar, 1 mm. (E) TRAP and hematoxylin staining of the distal femurs. Scale bar, 50 μm. Osteoclast number per bone perimeter from control and Ror2ΔOcl/ΔOcl mice. n = 8 mice for each genotype. (F) Erosion depth and eroded surface per bone surface. Erosion depth: 120 resorption lacunae assessed from seven control and nine Ror2ΔOcl/ΔOcl mice. Eroded surface per bone surface: n = 8 mice for each genotype. (G) Serum collagen type I cross-linked CTX in control and Ror2ΔOcl/ΔOcl mice. n = 8 mice for each genotype. (H) Alkaline phosphatase activity in serum. n = 8 mice for each genotype. In (B) and (D) to (H), error bars represent SD. ***P < 0.001, **P < 0.01, *P < 0.05. For statistical analyses, Kruskal-Wallis and Steel-Dwass test (B) or two-tailed Student’s t test (D to H) was used.

  • Fig. 3 Daam2 is a critical scaffold molecule linking Ror2 and Rho.

    (A) Wnt5a-induced Rac and Rho activity in osteoclasts. n = 5 dishes for each genotype. a.u, arbitrary units. (B) Immunoblotting of RhoA and Rac1 in osteoclasts expressing CA-RhoA or CA-Rac1. n = 3 biological replicates. (C and D) Effects of CA-RhoA and CA-Rac1 on actin ring formation (C) and resorbing pits (D). n = 5 dentine slices for each genotype. Scale bars, 100 μm. (E) RT-PCR of Daam1 and Daam2 expression. n = 5 dishes for each genotype. (F) RT-PCR of Daam2 expression in osteoclasts transfected with shDaam2. n = 5 dishes for each condition. (G) Effects of shRNA-mediated knockdown of Daam2 on Wnt5a-induced Rho activity in osteoclasts. n = 5 dishes for each group. (H and I) Effects of the knockdown of Daam2 and overexpression of CA-RhoA on actin ring formation (H) and resorbing pits (I) in osteoclasts. n = 5 dentine slices for each group. Scale bars, 100 μm. In (A) and (C) to (I), error bars represent SD. **P < 0.01, *P < 0.05. For statistical analyses, Kruskal-Wallis and Steel-Dwass test (A and G), ANOVA and Scheffé test (C, D, H, and I), two-tailed Student’s t test (F), or two-tailed Welch’s t test (E) was used.

  • Fig. 4 Pkn3 acts as a Rho effector for the bone-resorbing activity of osteoclasts.

    (A) RT-PCR analysis of the expression of mRNAs encoding Rho effectors in BMMs and osteoclasts. n = 5 dishes of BMM and osteoclast cultures. (B) Effect of shRNA-mediated knockdown of Pkn family members on the bone-resorbing activity of osteoclasts. n = 5 slices for each group. Osteoclasts were prepared from five mice. Scale bar, 100 μm. (C) Effects of the shRNA-mediated knockdown of mDia2 on the bone-resorbing activity of osteoclasts. n = 5 slices for each group. Osteoclasts were prepared from three mice. Scale bar, 100 μm. (D) Effects of Y27632 on stress fiber formation in bone marrow stromal cells. n = 5 wells for each treatment. Bone marrow stromal cells were prepared from two mice. Scale bar, 100 μm. (E) Effects of Y27632 on the formation of actin rings (the left two images and the left bar graph) and resorbing pits (the right two images and the right bar graph) in osteoclasts cultured on dentin slices. n = 5 slices for each treatment. Osteoclasts were prepared from two mice. Scale bars, 100 μm. In (A) to (E), error bars represent SD. **P < 0.01. For statistical analyses, Mann-Whitney U test (A), Kruskal-Wallis and Steel-Dwass test (B), or two-tailed Student’s t test (C to E) was used.

  • Fig. 5 Impaired bone-resorbing activity of osteoclasts in Pkn3−/− mice.

    (A) Immunoblotting of Pkn3 in osteoclasts formed from Pkn3−/− mice. n = 3 biological replicates. (B) Micro-CT analysis of femurs. n = 7 mice for each genotype. Scale bar, 1 mm. (C) TRAP and hematoxylin staining images and bone histomorphometric analysis of femurs. n = 7 mice for each genotype. Scale bar, 50 μm. (D) Erosion depth and the frequency distribution of the erosion depth in femurs. n = 7 mice for each genotype. Erosion depth: 200 resorption lacunae were assessed. (E) Serum CTX in WT and Pkn3−/− mice. n = 7 mice for each genotype. (F) Bone histomorphometric analysis of bone formation parameters in distal femurs. n = 7 mice for each genotype. (G) Ex vivo analysis of actin ring formations in osteoclasts. (H) Ex vivo analysis of resorbing pits in WT and Pkn3−/− mice–derived osteoclasts. In (G) and (H), n = 5 slices for each genotype. Osteoclasts were prepared from three mice for each genotype. Scale bars, 100 μm. In (B) to (H), error bars represent SD. ***P < 0.001, **P < 0.01, *P < 0.05. For statistical analyses, two-tailed Student’s t test (B to F) or two-tailed Welch’s t test (G and H) was used.

  • Fig. 6 Pkn3 forms complexes with c-Src and Pyk2 to promote bone-resorbing activity.

    (A) Immunoblotting of phosphorylated Pkns in osteoclasts. n = 3 biological replicates. (B) RT-PCR of Pkn3 mRNA in osteoclasts. n = 5 dishes for each genotype. (C) Confocal microscopic images in osteoclasts expressing Venus-Pkn3 and DsRed-actin proteins. n = 3 biological replicates. Scale bar, 50 μm. (D) Interactions between Venus-Pkn3 and c-Src in Ror2ΔOcl/ΔOcl osteoclasts. n = 3 biological replicates. (E) c-Src kinase activity in Ror2ΔOcl/ΔOcl osteoclasts. n = 5 dishes of BMM and osteoclast cultures for each genotype. (F) Effects of Daam2 knockdown on interactions between Pkn3, c-Src, and Pyk2. n = 3 biological replicates. (G) Immunoprecipitation (IP) analysis of osteoclasts expressing full-length Pkn3-Venus (full), Pkn3-Venus lacking the PRR domain (ΔPRR), and Pkn3-Venus lacking the kinase domain (Δkinase). n = 3 biological replicates. (H and I) Effects of the enforced expression of Pkn3 full, Pkn3-ΔPRR, and Pkn3-Δkinase on the actin ring formation (H) and resorbing pits (I) of Pkn3−/− osteoclasts. n = 5 slices for each group. Osteoclasts were prepared from three mice. Scale bars, 100 μm. In (B), (E), (G), and (H), error bars represent SD. **P < 0.01. (J) Proposed pathway in our current study. For statistical analyses, Mann-Whitney U test (B), Kruskal-Wallis and Steel-Dwass (E), or ANOVA and Scheffé test (H and I) were used.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/494/eaan0023/DC1

    Fig. S1. Rankl-induced formation of osteoclasts from Wnt5a−/− liver macrophages on dentin slices.

    Fig. S2. Micro-CT analysis of femurs and lumbar vertebrae in Ror2ΔOcl/ΔOcl mice and bone-resorbing activity of osteoclasts formed from Ror2ΔOcl/ΔOcl mice.

    Fig. S3. Effects of suppression of Daam2 on osteoclasts.

    Fig. S4. Effects of shRNA-mediated knockdown of Pkns and mDia2 on osteoclasts.

    Fig. S5. Micro-CT analysis of femurs and lumbar vertebrae of Pkn3−/− mice.

    Fig. S6. Osteoclast and osteoblast differentiation in cultures prepared from Pkn3−/− mice.

    Fig. S7. The expression of c-Src, phosphorylation of Pkn3, and schematic of Pkn3.

  • Supplementary Materials for:

    Protein kinase N3 promotes bone resorption by osteoclasts in response to Wnt5a-Ror2 signaling

    Shunsuke Uehara, Nobuyuki Udagawa, Hideyuki Mukai, Akihiro Ishihara, Kazuhiro Maeda, Teruhito Yamashita, Kohei Murakami, Michiru Nishita, Takashi Nakamura, Shigeaki Kato, Yasuhiro Minami, Naoyuki Takahashi, Yasuhiro Kobayashi*

    *Corresponding author. Email: ykoba{at}po.mdu.ac.jp

    This PDF file includes:

    • Fig. S1. Rankl-induced formation of osteoclasts from Wnt5a−/− liver macrophages on dentin slices.
    • Fig. S2. Micro-CT analysis of femurs and lumbar vertebrae in Ror2ΔOcl/ΔOcl mice and bone-resorbing activity of osteoclasts formed from Ror2ΔOcl/ΔOclmice.
    • Fig. S3. Effects of suppression of Daam2 on osteoclasts.
    • Fig. S4. Effects of shRNA-mediated knockdown of Pkns and mDia2 on osteoclasts.
    • Fig. S5. Micro-CT analysis of femurs and lumbar vertebrae of Pkn3−/− mice.
    • Fig. S6. Osteoclast and osteoblast differentiation in cultures prepared from Pkn3−/− mice.
    • Fig. S7. The expression of c-Src, phosphorylation of Pkn3, and schematic of Pkn3.

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    Citation: S. Uehara, N. Udagawa, H. Mukai, A. Ishihara, K. Maeda, T. Yamashita, K. Murakami, M. Nishita, T. Nakamura, S. Kato, Y. Minami, N. Takahashi, Y. Kobayashi, Protein kinase N3 promotes bone resorption by osteoclasts in response to Wnt5a-Ror2 signaling. Sci. Signal.10, eaan0023 (2017).

    © 2017 American Association for the Advancement of Science

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