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Ubiquitin E3 Ligase LNX2 is Critical for Osteoclastogenesis In Vitro by Regulating M-CSF/RANKL Signaling and Notch2

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Abstract

The Notch signaling pathway plays a crucial role in skeletal development and homeostasis by regulating the proliferation and differentiation of osteoblasts and osteoclasts. However, the molecular mechanisms modulating the level and activity of Notch receptors in bone cells remain unknown. In this study, we uncovered that LNX2, an E3 ubiquitin ligase and Notch inhibitor Numb binding protein, was up-regulated during osteoclast differentiation. Knocking-down LNX2 expression in bone marrow macrophages by lentivirus-mediated short hairpin RNAs markedly inhibited osteoclast formation. Decreased LNX2 expression attenuated macrophage colony-stimulating factor (M-CSF)-induced ERK and AKT activation and RANKL-stimulated activation of NF-κB and JNK pathways; therefore, accelerated osteoclast apoptosis. Additionally, loss of LNX2 led to an increased accumulation of Numb, which promoted the degradation of Notch and caused a reduction of the expression of the Notch downstream target gene, Hes1. We conclude that LNX2 regulates M-CSF/RANKL and the Notch signaling pathways during osteoclastogenesis.

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Acknowledgments

The authors would like to thank Drs. Stavros C Manolagas, Charles A Obrien, and Ms. GibAnn Berryhill for the critics of the manuscript prior submission. Erin Hogan is thanked for her support in microscopes. The work was supported by NIH Grants AR062012 and P01 AG13918.

Conflict of interest

Jian Zhou, Toshifumi Fujiwara, Shiqiao Ye, Xiaolin Li, Haibo Zhao state that they have no conflicts of interest.

Human and Animal Rights and Informed Consent

All animal procedures were approved by Institutional Animal Care and Use Committee at University of Arkansas for Medical Sciences.

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    Authors and Affiliations

    1. Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Xuhui District, Shanghai, 200233, People’s Republic of China

      Jian Zhou & Xiaolin Li

    2. Division of Endocrinology and Metabolism, Department of Internal Medicine, Center for Osteoporosis and Bone Metabolic Diseases, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 587, Little Rock, AR, 72205, USA

      Jian Zhou, Toshifumi Fujiwara, Shiqiao Ye & Haibo Zhao

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    Jian Zhou and Toshifumi Fujiwara have contributed equally to this work.

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    Zhou, J., Fujiwara, T., Ye, S. et al. Ubiquitin E3 Ligase LNX2 is Critical for Osteoclastogenesis In Vitro by Regulating M-CSF/RANKL Signaling and Notch2. Calcif Tissue Int 96, 465–475 (2015). https://doi.org/10.1007/s00223-015-9967-7

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