Hyeon-Young Min1, Kyeong-Min Kim2, Gabbine Wee3, Eun-Jung Kim4, Won-Gu Jang5. 1. Department of Biotechnology, College of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea. Electronic address: jminhy@gmail.com. 2. Department of Biotechnology, College of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea. Electronic address: kkm97655@naver.com. 3. Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), 80 Cheombok-ro, Dong-gu, Daegu 41061, Republic of Korea. Electronic address: gabbine@dgmif.re.kr. 4. Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea. Electronic address: ejkim4164@gamil.com. 5. Department of Biotechnology, College of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea. Electronic address: jangwg@daegu.ac.kr.
Abstract
AIMS: Mammalian circadian rhythms regulate many metabolic processes. Recent studies suggest that brain and muscle Arnt-like 1 (BMAL1), an important component of mammalian circadian rhythm, is associated with insulin signaling. Several studies have shown that insulin is associated with bone metabolism; however, the relationship between BMAL1 and osteoblasts remains unclear. MAIN METHODS: Expression of osteogenic markers and Bmal1 in MC3T3-E1 cells was measured by RT-PCR and Western blotting. Alizarin red S staining was performed to assess matrix mineralization in MC3T3-E1 cells. KEY FINDINGS: mRNA levels of osteogenic genes and Bmal1 were up-regulated in MC3T3-E1 cells upon insulin treatment. In addition, Bmal1 overexpression increased the expression of osteogenic genes including inhibitor of DNA binding (Id1), Runt-related transcription factor 2 (Runx2), and osteocalcin (OC). Interestingly, expression of Bone morphogenetic protein-2 (BMP2), an important upstream factor of Id1, Runx2, and OC, was markedly increased by Bmal1. Finally, we confirmed that insulin-induced BMP2 expression was attenuated in Bmal1 knockout (KO) cells. PCR analysis and alizarin red S staining showed that insulin-mediated increases gene expression and calcium deposition were reduced in Bmal1 KO cells compared to wild-type cells. SIGNIFICANCE: Taken together, these results demonstrate that Bmal1 promotes osteoblast differentiation by regulating BMP2 expression in MC3T3-E1 cells.
AIMS: Mammalian circadian rhythms regulate many metabolic processes. Recent studies suggest that brain and muscle Arnt-like 1 (BMAL1), an important component of mammalian circadian rhythm, is associated with insulin signaling. Several studies have shown that insulin is associated with bone metabolism; however, the relationship between BMAL1 and osteoblasts remains unclear. MAIN METHODS: Expression of osteogenic markers and Bmal1 in MC3T3-E1 cells was measured by RT-PCR and Western blotting. Alizarin red S staining was performed to assess matrix mineralization in MC3T3-E1 cells. KEY FINDINGS: mRNA levels of osteogenic genes and Bmal1 were up-regulated in MC3T3-E1 cells upon insulin treatment. In addition, Bmal1 overexpression increased the expression of osteogenic genes including inhibitor of DNA binding (Id1), Runt-related transcription factor 2 (Runx2), and osteocalcin (OC). Interestingly, expression of Bone morphogenetic protein-2 (BMP2), an important upstream factor of Id1, Runx2, and OC, was markedly increased by Bmal1. Finally, we confirmed that insulin-induced BMP2 expression was attenuated in Bmal1 knockout (KO) cells. PCR analysis and alizarin red S staining showed that insulin-mediated increases gene expression and calcium deposition were reduced in Bmal1 KO cells compared to wild-type cells. SIGNIFICANCE: Taken together, these results demonstrate that Bmal1 promotes osteoblast differentiation by regulating BMP2 expression in MC3T3-E1 cells.