Undercarboxylated, But Not Carboxylated, Osteocalcin Suppresses TNF-α-Induced Inflammatory Signaling Pathway in Myoblasts

Danbi Park; Do-Yeon Kim; Mi Ran Byun; Hyorin Hwang; Seong-Hee Ko; Jeong-Hwa Baek; Kyunghwa Baek

doi:10.1210/jendso/bvac084

Undercarboxylated, But Not Carboxylated, Osteocalcin Suppresses TNF-α-Induced Inflammatory Signaling Pathway in Myoblasts

J Endocr Soc. 2022 May 29;6(8):bvac084. doi: 10.1210/jendso/bvac084. eCollection 2022 Aug 1.

Authors

Danbi Park¹, Do-Yeon Kim², Mi Ran Byun³, Hyorin Hwang¹, Seong-Hee Ko¹, Jeong-Hwa Baek⁴, Kyunghwa Baek¹

Affiliations

¹ Department of Pharmacology, College of Dentistry and Research Institute of Oral Science, Gangneung-Wonju National University, Gangwondo 25457, Republic of Korea.
² Department of Pharmacology, School of Dentistry, Kyungpook National University, Daegu 41940, Republic of Korea.
³ Department of Pharmacology, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea.
⁴ Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 08826, Republic of Korea.

Abstract

Undercarboxylated osteocalcin (ucOCN) has been considered to be an important endocrine factor, especially to regulate bone and energy metabolism. Even with the mounting evidence showing the consistent inverse correlation of ucOCN levels in chronic inflammatory diseases, however, the mechanism underlying the involvement of ucOCN in the muscular inflammation has not been fully understood. In the present study, we explored 1) the endocrine role of ucOCN in the regulation of inflammation in C2C12 myoblasts and primary myoblasts and the underlying intracellular signaling mechanisms, and 2) whether G protein-coupled receptor family C group 6 member A (GPRC6A) is the ucOCN-sensing receptor associated with the ucOCN-mediated anti-inflammatory signaling pathway in myoblasts. ucOCN suppressed the tumor necrosis factor-α (TNF-α)-induced expressions of major inflammatory cytokines, including interleukin-1β (IL-1β) and inhibited the TNF-α-stimulated activities of transcription factors, including NF-κB, in C2C12 and primary myoblasts. Both knockdown and knockout of GPRC6A, by using siRNA or a CRISPR/CAS9 system, respectively, did not reverse the effect of ucOCN on IL-1β expression in myoblasts. Interestingly, TNF-α-induced IL-1β expression was inhibited by knockdown or deletion of GPRC6A itself, regardless of the ucOCN treatment. ucOCN was rapidly internalized into the cytoplasmic region via caveolae-mediated endocytosis, suggesting the presence of new target proteins in the cell membrane and/or in the cytoplasm for interaction with ucOCN in myoblasts. Taken together, these findings indicate that ucOCN suppresses the TNF-α-induced inflammatory signaling pathway in myoblasts. GPRC6A is not a sensing receptor associated with the ucOCN-mediated anti-inflammatory signaling pathway in myoblasts.

Keywords: C2C12; GPRC6A; IL-1β; anti-inflammation; sarcopenia; undercarboxylated osteocalcin.