일주기 리듬(circadian rhythm)은 생명체가 나타내는 생체주기 중 약 24시간의 주기로 나타나는 생물학적 리듬을 말한다. 인체는 외부 환경의 변화에 따른 자극에 반응하여 체내 생체주기와 이를 동기화시킴으로써 일주기 리듬을 조절한다. 인간을 포함한 포유류의 일주기 리듬은 뇌시상하부의 시신경교차상핵(suprachiasmatic nucleus, SCN)에 존재하는 중추 생체시계에 의해 주관되며, 중추시계는 말초시계에 신호를 전달하여 일주기 리듬을 동조화한다. 일주기 리듬을 구성하는 일주기 시계 유전자들이 말초조직에서 다양한 역할을 담당하고 있음이 계속해서 보고되고 있으며 뼈와 혈관 조직에서도 이들 일주기 시계가 중요한 역할을 한다는 증거가 축적되고 있다. 뼈는 조골세포와 파골세포의 상호 균형 속에서 조절되며 뼈의 석회화를 담당하는 것은 조골세포이다. 한편, 조골세포에서 발생하는 석회화 과정은 혈관 평활근세포에서도 비슷한 기전으로 발생한다고 알려져 있지만 조골세포와 다른 것은 혈관에서 발생하는 석회화는 이소성 석회화라는 점이다. 본 연구에서는 일주기 시계 유전자가 조골세포와 혈관 평활근 세포의 석회화에 어떠한 영향을 미치는지 조사하였다. Brain and muscle Arnt-like protein-1 (BMAL1) 유전자는 조골세포에서 인슐린에 의해 mRNA 및 단백질 발현이 증가하였으며 BMAL1 유전자의 과발현은 Bone morphogenetic protein 2 (BMP2)를 비롯한 inhibitor of differentiation 1 (Id1), Runt-related transcription factor 2 (Runx2), osteocalcin (OC)과 같은 조골세포 분화 표지 유전자의 발현을 증가시켰다. 인슐린에 의해 유도되는 조골세포 분화에서 BMAL1 유전자의 역할을 확인하기 위하여 wild type 세포와 BMAL1 knockout 세포에 인슐린을 처리하여 분석한 결과에서 BMAL1이 손실된 조골세포는 인슐린에 의한 분화 유도가 나타나지 않았다. Per1 유전자는 강력한 조골세포 분화 유도 인자인 BMP2에 의해서 발현이 증가되었다. Per1 유전자의 과발현 실험은 Runx2, OC와 같은 분화 표지 유전자의 발현을 증가시켰으며 ALP 염색 및 alizarin red S 염색의 정도를 증가시켰다. 에스트라디올은 estrogen receptor α (ERα), retinoic acid-related orphan receptor α (RORα) 및 조골세포 분화 표지 유전자들의 발현을 유도하였다. 이후 에스트라디올에 의한 조골세포 분화에 있어 RORα의 역할을 확인하기 위한 과발현과 knockdown을 실시하였으며 각각 조골세포 분화 표지 유전자의 발현을 증가 혹은 감소시키는 것을 확인하였다. 또한 luciferase 분석을 통하여 BMP2 프로모터의 -1910/+284 부위가 RORα의 작용에 중요함을 확인하였다. OVO homolog-like 1 (OVOL1) 유전자는 ascorbic acid와 β-glycerophosphate로 유도한 조골세포 분화에서 발현이 증가하였으며 OVOL1 유전자의 과발현은 조골세포 분화 표지 유전자의 발현을 증가시켰다. 또한 OVOL1을 매개한 조골세포의 분화는 레스베라트롤에 의해 유도될 수 있음을 확인하였다. 혈관 평활근세포 BMAL1 유전자는 무기인산에 의해 유도되는 석회화 과정에서 발현이 증가하였으며 이는 멜라토닌에 의해 억제되었다. 본 연구의 결과를 요약하면 BMAL1, Per1, RORα, OVOL1 유전자가 각각 조골세포의 분화를 유도할 수 있으며, 특히 BMAL1 유전자는 조골세포와 혈관 평활근세포 모두에서 석회화를 유도함을 밝힌 것이다.

Bone is a metabolically and undergoes continuous remodeling throughout the life of an organism. Osteoblast is differentiated from mesenchymal stem cells (MSCs) which can differentiate into adipocytes, osteoblasts, chondrocytes, and myocytes. Osteoblast differentiation is regulated by complex signaling pathways. Bone morphogenetic proteins (BMPs), the transforming growth factor-β (TGF-β) superfamily members, play important roles in various cellular pathways, including development, cell growth, and bone formation. BMP2 is the most dominant positive regulator of osteoblast differentiation, and stimulates bone formation by activating the Smad signaling cascade and Runt-related transcription factor 2 (Runx2), which regulates expression of osteogenic genes such as collagen type Ι, osteocalcin (OC), bone sialoprotein, and osteopontin.
Vascular calcification is an active and multifactorial process regulated by osteo/chondrogenic reprogramming of vascular smooth muscle cells (VSMCs). This process shares many similarities with bone mineralization. Transdifferentiation of VSMCs stimulated by various pathological factors, particularly phosphate, is important for vascular calcification. During calcification of VSMCs, various stimuli induce expression of osteogenic markers such as distal-less homeobox 5 (Dlx5), Runx2, and alkaline phosphatase (ALP).
The circadian master clock, located in the suprachiasmatic nucleus (SCN), is synchronized by light coming through the eye, and the output signal from the SCN synchronizes with peripheral tissues through the sympathetic nervous system and humoral routes. In mammals, the circadian system is regulated by transcriptional-translational autoregulatory loops consisting of circadian locomotor output cycles kaput (CLOCK), brain and muscle Arnt-like protein 1 (BMAL1), period proteins, and cryptochromes. CLOCK/BMAL1 controls circadian insulin sensitivity changes in mouse hepatocytes. Further, BMAL1 increases de novo lipogenesis by regulating insulin signaling in the liver. In adipocytes, BMAL1 regulates brown fat adipogenesis through TGF-β/BMP signaling. Sympathetic signaling can induce expression of clock genes in human osteoblasts. Moreover, BMP4 is affected by circadian clocks in osteoblasts. However, exactly how the circadian oscillator regulates osteoblast differentiation remains unclear.
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 Id1, Runx2, and OC. Interestingly, expression of BMP2, an important upstream factor of Id1, Runx2, and OC, was markedly increased by Bmal1. Finally, 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. These results demonstrate that Bmal1 promotes osteoblast differentiation by regulating BMP2 expression in MC3T3-E1 cells.
MC3T3-E1 cells were treated with BMP2 for induction of osteoblastic differentiation. Osteogenic maker gene and Per1 mRNA expression was measured using real-time PCR. Interestingly, BMP2 treatment induces Per1 mRNA expression in MC3T3-E1 cells. To further investigate the function of Per1 on osteoblast differentiation, MC3T3-E1 cells were transiently transfected with pCMV-Per1. Per1 overexpression increased Runx2 mRNA and protein levels. Also, mRNA expression and promoter activity of osteocalcin were upregulated by Per1 overexpression. To investigate the effect of interaction between Per1 and osteogenic condition, MC3T3-E1 cells were cultured in osteogenic medium containing ascorbic acid and β-glycerophosphate. Osteogenic medium-induced ALP staining level and mineralization were synergistically increased by overexpression of Per1. Taken together, these results demonstrate that Per1 is positive regulator of osteoblast differentiation.
The retinoic acid receptor-related orphan receptor alpha (RORα) is a member of the nuclear hormone receptor superfamily. Several studies show that estradiol is related to RORα expression. However, the link between estradiol and RORα in osteoblast differentiation remains unknown. This study showed that estradiol induces RORα expression in C3H10T1/2 and MC3T3-E1 cells. RORα overexpression increased the expression of osteogenic genes including BMP2, Dlx5, Id1, Runx2, and OC. In addition, RORα increased phosphorylation of smad1/5/9. Furthermore, RORα knockdown suppressed estradiol-induced BMP2 and Runx2 protein level. Also, estradiol-induced ALP staining and matrix mineralization was attenuated in RORα knockdown. Summarily, these results suggest that estradiol-induced RORα promotes osteoblast differentiation.
OVO homologue-like 1 (OVOL1) encodes a C2H2 zinc finger protein and is an evolutionarily conserved gene in mammals. OVOL1 expression is required for development. However, the function of OVOL1 on bone metabolism remains unreported. This study showed for the first time the role of OVOL1 in osteoblast differentiation. To determine the role of OVOL1 on osteogenic differentiation, OVOL1 expression was analyzed in pre-osteoblastic cell line. OVOL1 mRNA expression was induced during osteoblast differentiation. In addition, OVOL1 overexpression enhanced the expression of osteogenic genes including BMP2, Id1, Dlx5, Runx2, OC, and ALP. Moreover, mineralization of the extracellular matrix was increased by OVOL1 overexpression in MC3T3-E1 cells. Furthermore, knockdown of OVOL1 experiment demonstrated that OVOL1 is required for osteoblast differentiation. Collectively, these results suggest that OVOL1 function as an important regulator of osteoblast differentiation by inducing BMP2 expression in MC3T3-E1 cells.
There is growing evidence that the circadian rhythm plays an important role in cardiovascular disease. BMAL1 is regulated by melatonin and has been reported to function in blood vessels. However, it is unknown whether BMAL1 is involved in calcification of VSMCs. First, the effect of melatonin on calcification of VSMCs was investigated. Aortic VSMCs were harvested from rats, cultured, and incubated with inorganic phosphate to induce calcification. Melatonin inhibited phosphate-induced calcification of VSMCs. Next, the expression of BMAL1 was measured in calcified VSMCs and it was investigated whether it mediated the protective effect of melatonin against phosphate-induced calcification. Western blot and real-time PCR analyses showed that BMAL1 expression was increased by phosphate and that this effect was abolished by melatonin. Moreover, melatonin induced TIMP4 expression, which is repressed by BMAL1. Knockdown of BMAL1 attenuated phosphate-induced calcification of VSMCs. Finally, the effects of TIMP4 were investigated in VSMCs. Overexpression of TIMP4 suppressed phosphate-induced calcification. TIMP4 knockdown induced calcification of VSMCs. These results indicate that melatonin protects against phosphate-induced calcification by inhibiting BMAL1.