Skip to main content

Advertisement

SpringerLink
Log in

Inhibitor of protein kinase N3 suppresses excessive bone resorption in ovariectomized mice

  • Original Article
  • Published:
Journal of Bone and Mineral Metabolism Aims and scope Submit manuscript

Abstract

Introduction

The long-term inhibition of bone resorption suppresses new bone formation because these processes are coupled during physiological bone remodeling. The development of anti-bone-resorbing agents that do not suppress bone formation is urgently needed. We previously demonstrated that Wnt5a-Ror2 signaling in mature osteoclasts promoted bone-resorbing activity through protein kinase N3 (Pkn3). The p38 MAPK inhibitor SB202190 reportedly inhibited Pkn3 with a low Ki value (0.004 μM). We herein examined the effects of SB202190 on osteoclast differentiation and function in vitro and in vivo.

Materials and methods

Bone marrow cells were cultured in the presence of M-csf and GST-Rankl to differentiate into multinucleated osteoclasts. Osteoclasts were treated with increasing concentrations of SB202190. For in vivo study, 10-week-old female mice were subjected to ovariectomy (OVX). OVX mice were intraperitoneally administered with a Pkn3 inhibitor at 2 mg/kg or vehicle for 4 weeks, and bone mass was analyzed by micro-CT.

Results

SB202190 suppressed the auto-phosphorylation of Pkn3 in osteoclast cultures. SB202190 significantly inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation. SB202190 reduced c-Src activity in osteoclast cultures without affecting the interaction between Pkn3 and c-Src. A treatment with SB202190 attenuated OVX-induced bone loss without affecting the number of osteoclasts or bone formation by osteoblasts.

Conclusions

Our results showed that Pkn3 has potential as a therapeutic target for bone loss due to increased bone resorption. SB202190 is promising as a lead compound for the development of novel anti-bone-resorbing agents.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Takeda S, Elefteriou F, Karsenty G (2003) Common endocrine control of body weight, reproduction, and bone mass. Annu Rev Nutr 23:403–411

    Article  CAS  Google Scholar 

  2. Zaidi M (2007) Skeletal remodeling in health and disease. Nat Med 13:791–801

    Article  CAS  Google Scholar 

  3. Eastell R, O’Neill TW, Hofbauer LC, Langdahl B, Reid IR, Gold DT, Cummings SR (2016) Postmenopausal osteoporosis. Nat Rev Dis Primers 2:16069

    Article  Google Scholar 

  4. Okamoto K, Nakashima T, Shinohara M, Negishi-Koga T, Komatsu N, Terashima A, Sawa S, Nitta T, Takayanagi H (2017) Osteoimmunology: The conceptual framework unifying the immune and skeletal systems. Physiol Rev 97:1295–1349

    Article  CAS  Google Scholar 

  5. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ (1999) Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20:345–357

    Article  CAS  Google Scholar 

  6. Yoshida H, Hayashi S, Kunisada T, Ogawa M, Nishikawa S, Okamura H, Sudo T, Shultz LD, Nishikawa S (1990) The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345:442–444

    Article  CAS  Google Scholar 

  7. Nakashima T, Hayashi M, Fukunaga T, Kurata K, Oh-Hora M, Feng JQ, Bonewald LF, Kodama T, Wutz A, Wagner EF, Penninger JM, Takayanagi H (2011) Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nat Med 17:1231–1234

    Article  CAS  Google Scholar 

  8. Xiong J, Onal M, Jilka RL, Weinstein RS, Manolagas SC, O’Brien CA (2011) Matrix-embedded cells control osteoclast formation. Nat Med 17:1235–1241

    Article  CAS  Google Scholar 

  9. Anastasilakis AD, Polyzos SA, Makras P (2018) THERAPY OF ENDOCRINE DISEASE: Denosumab vs bisphosphonates for the treatment of postmenopausal osteoporosis. Eur J Endocrinol 179:R31-45

    Article  CAS  Google Scholar 

  10. Crane JL, Cao X (2014) Bone marrow mesenchymal stem cells and TGF-β signaling in bone remodeling. J Clin Invest 124:466–472

    Article  CAS  Google Scholar 

  11. Tang SY, Alliston T (2013) Regulation of postnatal bone homeostasis by TGFβ. Bonekey Rep 2:255

    Article  Google Scholar 

  12. Crane JL, Cao X (2014) Function of matrix IGF-1 in coupling bone resorption and formation. J Mol Med (Berl) 92:107–115

    Article  CAS  Google Scholar 

  13. Pederson L, Ruan M, Westendorf JJ, Khosla S, Oursler MJ (2008) Regulation of bone formation by osteoclasts involves Wnt/BMP signaling and the chemokine sphingosine-1-phosphate. Proc Natl Acad Sci U S A 105:20764–20769

    Article  CAS  Google Scholar 

  14. Takeshita S, Fumoto T, Matsuoka K, Park KA, Aburatani H, Kato S, Ito M, Ikeda K (2013) Osteoclast-secreted CTHRC1 in the coupling of bone resorption to formation. J Clin Invest 123:3914–3924

    Article  CAS  Google Scholar 

  15. Walker EC, McGregor NE, Poulton IJ, Pompolo S, Allan EH, Quinn JM, Gillespie MT, Martin TJ, Sims NA (2008) Cardiotrophin-1 is an osteoclast-derived stimulus of bone formation required for normal bone remodeling. J Bone Miner Res 23:2025–2032

    Article  CAS  Google Scholar 

  16. Koide M, Kobayashi Y, Yamashita T, Uehara S, Nakamura M, Hiraoka BY, Ozaki Y, Iimura T, Yasuda H, Takahashi N, Udagawa N (2017) Bone formation is coupled to resorption via suppression of sclerostin expression by osteoclasts. J Bone Miner Res 32:2074–2086

    Article  CAS  Google Scholar 

  17. Gauthier JY, Chauret N, Cromlish W et al (2008) The discovery of odanacatib (MK-0822), a selective inhibitor of cathepsin K. Bioorg Med Chem Lett 18:923–928

    Article  CAS  Google Scholar 

  18. Bone HG, McClung MR, Roux C, Recker RR, Eisman JA, Verbruggen N, Hustad CM, DaSilva C, Santora AC, Ince BA (2010) Odanacatib, a cathepsin-K inhibitor for osteoporosis: a two-year study in postmenopausal women with low bone density. J Bone Miner Res 25:937–947

    PubMed  Google Scholar 

  19. Mullard A (2016) Merck & Co. drops osteoporosis drug odanacatib. Nat Rev Drug Discov 15:669

    PubMed  PubMed Central  Google Scholar 

  20. Boyle WJ, Simonet WS, Lacey DL (2003) Osteoclast differentiation and activation. Nature 423:337–342

    Article  CAS  Google Scholar 

  21. Uehara S, Udagawa N, Kobayashi Y (2018) Non-canonical Wnt signals regulate cytoskeletal remodeling in osteoclasts. Cell Mol Life Sci 75:3683–3692

    Article  CAS  Google Scholar 

  22. Uehara S, Udagawa N, Mukai H, Ishihara A, Maeda K, Yamashita T, Murakami K, Nishita M, Nakamura T, Kato S, Minami Y, Takahashi N, Kobayashi Y (2017) Protein kinase N3 promotes bone resorption by osteoclasts in response to Wnt5a-Ror2 signaling. Sci Signal. https://doi.org/10.1126/scisignal.aan0023

    Article  PubMed  Google Scholar 

  23. Falk MD, Liu W, Bolaños B, Unsal-Kacmaz K, Klippel A, Grant S, Brooun A, Timofeevski S (2014) Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors. Biosci Rep. https://doi.org/10.1042/BSR20140010

  24. Lee SE, Woo KM, Kim SY, Kim HM, Kwack K, Lee ZH, Kim HH (2002) The phosphatidylinositol 3-kinase, p38, and extracellular signal-regulated kinase pathways are involved in osteoclast differentiation. Bone 30:71–77

    Article  CAS  Google Scholar 

  25. Wilson KP, McCaffrey PG, Hsiao K, Pazhanisamy S, Galullo V, Bemis GW, Fitzgibbon MJ, Caron PR, Murcko MA, Su MS (1997) The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAP kinase. Chem Biol 4:423–431

    Article  CAS  Google Scholar 

  26. Mukai H, Muramatsu A, Mashud R, Kubouchi K, Tsujimoto S, Hongu T, Kanaho Y, Tsubaki M, Nishida S, Shioi G, Danno S, Mehruba M, Satoh R, Sugiura R (2016) PKN3 is the major regulator of angiogenesis and tumor metastasis in mice. Sci Rep 6:18979

    Article  CAS  Google Scholar 

  27. Yamamoto M, Fisher JE, Gentile M, Seedor JG, Leu CT, Rodan SB, Rodan GA (1998) The integrin ligand echistatin prevents bone loss in ovariectomized mice and rats. Endocrinology 139:1411–1419

    Article  CAS  Google Scholar 

  28. Maeda K, Kobayashi Y, Udagawa N, Uehara S, Ishihara A, Mizoguchi T, Kikuchi Y, Takada I, Kato S, Kani S, Nishita M, Marumo K, Martin TJ, Minami Y, Takahashi N (2012) Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis. Nat Med 18:405–412

    Article  CAS  Google Scholar 

  29. Takahashi N, Udagawa N, Kobayashi Y, Suda T (2007) Generation of osteoclasts in vitro, and assay of osteoclast activity. Methods Mol Med 135:285–301

    Article  CAS  Google Scholar 

  30. Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20:87–90

    Article  CAS  Google Scholar 

  31. Okamoto M, Udagawa N, Uehara S, Maeda K, Yamashita T, Nakamichi Y, Kato H, Saito N, Minami Y, Takahashi N, Kobayashi Y (2014) Noncanonical Wnt5a enhances Wnt/β-catenin signaling during osteoblastogenesis. Sci Rep 4:4493

    Article  Google Scholar 

  32. Matsumoto M, Sudo T, Saito T, Osada H, Tsujimoto M (2000) Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL). J Biol Chem 275:31155–31161

    Article  CAS  Google Scholar 

  33. Li X, Udagawa N, Itoh K, Suda K, Murase Y, Nishihara T, Suda T, Takahashi N (2002) p38 MAPK-mediated signals are required for inducing osteoclast differentiation but not for osteoclast function. Endocrinology 143:3105–3113

    Article  CAS  Google Scholar 

  34. Leenders F, Möpert K, Schmiedeknecht A, Santel A, Czauderna F, Aleku M, Penschuck S, Dames S, Sternberger M, Röhl T, Wellmann A, Arnold W, Giese K, Kaufmann J, Klippel A (2004) PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase. EMBO J 23:3303–3313

    Article  CAS  Google Scholar 

  35. Soriano P, Montgomery C, Geske R, Bradley A (1991) Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64:693–702

    Article  CAS  Google Scholar 

  36. Duong LT, Lakkakorpi PT, Nakamura I, Machwate M, Nagy RM, Rodan GA (1998) PYK2 in osteoclasts is an adhesion kinase, localized in the sealing zone, activated by ligation of alpha(v)beta3 integrin, and phosphorylated by src kinase. J Clin Invest 102:881–892

    Article  CAS  Google Scholar 

  37. Greenblatt MB, Shim JH, Zou W, Sitara D, Schweitzer M, Hu D, Lotinun S, Sano Y, Baron R, Park JM, Arthur S, Xie M, Schneider MD, Zhai B, Gygi S, Davis R, Glimcher LH (2010) The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice. J Clin Invest 120:2457–2473

    Article  CAS  Google Scholar 

  38. Song L, Bi YN, Zhang PY, Yuan XM, Liu Y, Zhang Y, Huang JY, Zhou K (2017) Optimization of the Time Window of Interest in Ovariectomized Imprinting Control Region Mice for Antiosteoporosis Research. Biomed Res Int. https://doi.org/10.1155/2017/8417814

    Article  PubMed  PubMed Central  Google Scholar 

  39. Chen D, Li Y, Guo F, Lu Z, Hei C, Li P, Jin Q (2015) Protective effect of p38 MAPK inhibitor on wear debris-induced inflammatory osteolysis through downregulating RANK/RANKL in a mouse model. Genet Mol Res 14:40–52

    Article  CAS  Google Scholar 

  40. Badger AM, Bradbeer JN, Votta B, Lee JC, Adams JL, Griswold DE (1996) Pharmacological profile of SB203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function. J Pharmacol Exp Ther 279:1453–1461

    CAS  PubMed  Google Scholar 

  41. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW, Strickler JE, McLaughlin MM, Siemens IR, Fisher SM, Livi GP, White JR, Adams JL, Young PR (1994) A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:739–746

    Article  CAS  Google Scholar 

  42. Beyaert R, Cuenda A, Vanden Berghe W, Plaisance S, Lee JC, Haegeman G, Cohen P, Fiers W (1996) The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor. EMBO J 15:1914–1923

    Article  CAS  Google Scholar 

  43. Kassis S, Kumar S, Badger A, Adams JL (1999) p38 mitogen-activated protein kinase inhibitors–mechanisms and therapeutic potentials. Pharmacol Ther 82:389–397

    Article  Google Scholar 

  44. Asquith RMC, Temme L, Laitinen T, Pickett J, Kwarcinski EF, Sinha P, Wells IC, Tizzard GJ, Zutshi R, Drewry HD (2020) Identification and Optimization of cell active 4-anilino-quin(az)oline Inhibitors for Protein Kinase Novel 3 (PKN3). Biorxiv. https://doi.org/10.1101/2020.03.02.972943

    Article  Google Scholar 

Download references

Acknowledgements

The present study was supported by the Japan Society for the Promotion of Science KAKENHI Grant Numbers JP19K10050 (SU), JP17K19776 (NU), and JP16H02691 (YK).

Author information

Authors and Affiliations

  1. Department of Biochemistry, Matsumoto Dental University, Nagano, 399-0781, Japan

    Shunsuke Uehara & Nobuyuki Udagawa

  2. Biosignal Research Center, Kobe University, Hyogo, 657-8501, Japan

    Hideyuki Mukai

  3. Department of Clinical Laboratory, Kitano Hospital, Osaka, 530-8480, Japan

    Hideyuki Mukai

  4. Institute for Oral Science, Matsumoto Dental University, 1780 Gobara, Hiro-oka , Shiojiri-shi, Nagano, 399-0781, Japan

    Teruhito Yamashita, Masanori Koide & Yasuhiro Kobayashi

  5. Laboratory of Immunology, Faculty of Veterinary Medicine, Okayama University of Science, Ehime, 794-8555, Japan

    Kohei Murakami

Authors
  1. Shunsuke Uehara
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hideyuki Mukai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Teruhito Yamashita
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Masanori Koide
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kohei Murakami
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nobuyuki Udagawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yasuhiro Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yasuhiro Kobayashi.

Ethics declarations

Conflict of interests

The authors declare no conflict of financial interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

774_2021_1296_MOESM1_ESM.docx

Supplementary file1 (DOCX 85 KB) The administration of SB202190 did not affect body or uterus weights. A: Schematic representation of the study design. B: Increases in body weight during the experimental period. C: The wet weight of the uterus was measured. B and C: n = 9 mice for each group. Error bars, s.d. *: p < 0.05 significantly different from sham. In statistical analyses, an ANOVA and Scheffé’s test were used

774_2021_1296_MOESM2_ESM.docx

Supplementary file2 (DOCX 47 KB) SB202190 did not affect bone nodule formation in osteoblasts in vitro. A, B: Alizarin Red S staining. Representative photographs were shown in A. Alizarin Red S-positive areas were calculated with ImageJ software (B). n = 5 wells for each group. Error bars, s.d. In statistical analyses, an ANOVA and Scheffé’s test were used

774_2021_1296_MOESM3_ESM.docx

Supplementary file3 (DOCX 43 KB) Comparison of amino acid sequences in ATP pocket of mouse Pkn3 and p38α. Matched amino acid residues and similar amino acid residues (+) were shown in middle rows between the sequence of Pkn3 and that of p38α. Matched amino acid residues were depicted on a yellow background. The seven amino acid residues (K53, L75, L86, L104, V105, T106, M109) required for binding SB203580 to p38α were shown in blue characters. Asterisks indicate 5 amino acid residues conserved in Pkn3 among the above 7 amino acid residues. The two amino acid residues (E629 and L631) required for binding Pkn3 to the novel inhibitors were shown in red characters

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Uehara, S., Mukai, H., Yamashita, T. et al. Inhibitor of protein kinase N3 suppresses excessive bone resorption in ovariectomized mice. J Bone Miner Metab 40, 251–261 (2022). https://doi.org/10.1007/s00774-021-01296-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00774-021-01296-1

Keywords

Search

Navigation