Chemokine ligand 28 (CCL28) negatively regulates trabecular bone mass by suppressing osteoblast and osteoclast activities

Journal of Bone and Mineral Metabolism (2021)Cite this article

Abstract

Introduction

Bone metabolism imbalances cause bone metabolism diseases, like osteoporosis, through aging. Although some chemokines are known to be involved in bone mass regulation, many have not been investigated. Thus, the present study aimed to investigate the role of chemokine ligand 28 (CCL28) on bone metabolism.

Materials and methods

To investigate the role of CCL28 on bone metabolism, 10-week-old male wild-type and Ccl28 knockout (Ccl28 KO) mice were analyzed. Microcomputed tomography analysis and bone tissue morphometry were used to investigate the effect of Ccl28 deficiency on the bone. CCL28 localization in bone tissue was assumed by immunohistochemistry. Osteoblast and osteoclast markers were evaluated by enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction. Finally, in vitro experiments using MC3T3-E1 and bone marrow macrophages revealed the direct effect of CCL28 on osteoblast and osteoclast.

Results

This study showed that Ccl28 deficiency significantly increased bone mass and the number of mature osteoblasts. Immunoreactivity for CCL28 was observed in osteoblasts and osteoclasts on bone tissue. Additionally, Ccl28 deficiency promoted osteoblast and osteoclast maturation. Moreover, CCL28 treatment decreased osteoblast and osteoclast activities but did not affect differentiation.

Conclusion

In summary, this study indicated that CCL28 is one of the negative regulators of bone mass by suppressing osteoblast and osteoclast activities. These results provide important insights into bone immunology and the selection of new osteoporosis treatments.

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Acknowledgements

The authors thank Dr. Keiichiro Yogo (Shizuoka University) for providing the NIH3T3 cells that stably expressed the human M-CSF vector and Dr. Tomohisa Ishikawa and Dr. Momoka Yamaguchi (University of Shizuoka) for providing the antibodies. Moreover, Mr. Fumiya Kamiya, Kurumi Maeda, Hirofumi Fukazawa, Ayano Hashimoto, and Sano Takayuki (Shizuoka University) are also thanked for their technical assistance. This work was supported, in part, by a grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology (16K12720).

Author information

Affiliations

  1. Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan

    Rina Iwamoto, Kazutoshi Sayama & Akira Yukita

  2. Department of Agriculture, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan

    Takumi Takahashi & Kazutoshi Sayama

  3. Laboratory Animal Research Center, Division of Animal Genetics, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Kazuto Yoshimi

  4. Mouse Genomics Resource Laboratory, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka, 411-8540, Japan

    Yuji Imai & Tsuyoshi Koide

  5. Department of Oral Diagnostics and Comprehensive Dentistry, Matsumoto Dental University Hospital, 1780 Hirooka-gobara, Shiojiri, Nagano, 399-0781, Japan

    Miroku Hara

  6. Department of Anatomy, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan

    Tadashi Ninomiya

  7. Department of Oral Histology, Matsumoto Dental University, 1780 Hirooka-gobara, Shiojiri, Nagano, 399-0781, Japan

    Hiroaki Nakamura

  8. Department of Education (Sciences), Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan

    Akira Yukita

  9. Department of Science, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan

    Akira Yukita

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  1. Rina Iwamoto
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  2. Takumi Takahashi
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  3. Kazuto Yoshimi
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Contributions

RI, TT, KS, and AY contributed to the conception and design of the research. RI, TT, KY, YI, TK, MH, and TN performed the experiments and analyzed the data. RI, TT, NH, KS, and AY interpreted the results. RI and TT prepared the figures. RI drafted the manuscript, and HN, KS, and AY edited and revised the manuscript. All authors approved the final version of the manuscript.

Corresponding author

Correspondence to Akira Yukita.

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Supplementary Information

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Supplementary file1 Fig. S1 genotyping in a conventional PCR with specific primers and sequence. a Ccl28 gene knockout target region. b Sequence results confirmed that the Ccl28 gene was mutated by a 13-base deletion. c Primer and amplification regions used for genotyping of mice used in the experiment. d Primer1 and primer2 can amplify DNA with WT and Ccl28 KO, respectively. Ccl28 KO reduces the amplification size by 13 bases because primer3 can amplify the region containing the defective site. PCR results confirmed that the mice used were Ccl28 KO. (TIFF 185 KB)

Supplementary file2 Fig. S2. Effect of genotype and treatment on organ weights in male and female animals. The animals were sacrificed and the a body, b kidney, c liver, d colon, e ileum, f caecal, g spleen, and h thymus weights were measured. Data are presented as mean ± SEM; **P < 0.01, *****P < 0.00001 vs WT. (TIFF 114 KB)

Supplementary file3 Fig. S3. The expressions of Ccl28 and Ccr3 in MC3T3-E1. a, b The relative expression levels of the indicated genes were measured by qRT-PCR in MC3T3-E1 treated with αMEM or OMM (a Ccl28, b Ccr3). These markers were normalized to Gapdh expression. Data are presented as mean ± SE, n = 3; *P < 0.05 vs αMEM. (TIFF 65 KB)

Supplementary file4 Fig. S4. The expressions of Ccr1 and Il-6 in the bones of 10-week-old WT and Ccl28 KO mice. a The relative expression levels of Ccr1 was measured by qRT-PCR in the bones of 10-week-old WT and Ccl28 KO mice. b The relative expression levels of Il-6 were measured by QRT-PCR in the livers of 10-week-old WT and Ccl28 KO mice. These markers were normalized to Gapdh expression. Data are presented as mean ± SE, n = 3; *P < 0.05 vs WT mice. (TIFF 64 KB)

Supplementary file5 Fig. S5. Changes in CCL28, CCR3, and CCR10 expressions with aging. a–c The relative expression levels of the indicated genes were measured by qRT-PCR in 10-week-old (young) and 84-week-old (aged) mice bone (a Ccl28, b Ccr3, and c Ccr10). These markers were normalized to Gapdh expression. Data are presented as mean ± SE, n = 3; *P < 0.05, ***P < 0.001, *****P < 0.00001 vs young group (TIFF 26370 KB)

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Iwamoto, R., Takahashi, T., Yoshimi, K. et al. Chemokine ligand 28 (CCL28) negatively regulates trabecular bone mass by suppressing osteoblast and osteoclast activities. J Bone Miner Metab (2021). https://doi.org/10.1007/s00774-021-01210-9

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Keywords

  • Chemokine ligand 28
  • Bone metabolism
  • Osteoblast
  • Osteoclast