Abstract
Accumulating evidence indicates that cryptochrome circadian regulatory (CRY) proteins have emerged as crucial regulators of osteogenic differentiation. However, the associated mechanisms are quite elusive. In this study, we show that knockdown of CRY2 downregulated the expression of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN) to facilitate osteoblast differentiation. Further study identified that CRY2 was directly targeted by microRNA (miR)-7-5p, which was highly induced during osteoblast differentiation. The expression of Runx2, ALP, collagen type I alpha 1 (Col1a1), and OCN was upregulated by overexpression of miR-7-5p and induction of osteoblast differentiation. Moreover, signal transducer and activator of transcription 3 (STAT3) transcriptionally activated miR-7-5p to significantly enhance the expression of above osteogenic marker genes and mineral formation. However, overexpression of CRY2 abolished the osteogenic differentiation induced by miR-7-5p overexpression. Silencing of CRY2 unraveled the binding of CRY2 with the circadian locomotor output cycles kaput (CLOCK)/brain and muscle ARNT-like 1 (BMAL1) complex to release CLOCK/BMAL1, which facilitated the binding of CLOCK/BMAL1 to the promoter region of the P300 E-box to stimulate the transcription of P300. P300 subsequently promoted the acetylation of histone 3 and the formation of a transcriptional complex with Runx2 to enhance osteogenesis. Taken together, our study revealed that CRY2 is repressed by STAT3/miR-7-5p to promote osteogenic differentiation through CLOCK/BMAL1/P300 signaling. The involved molecules may be potentially targeted for treatment of osteoporosis.
Highlights
Osteoblast differentiation and bone remodeling are regulated by a concerted communication between osteoblasts and osteoclasts.[1,2] Osteoblast differentiation requires numerous transcription factors to regulate both the formation and maintenance of bone.[3]
We identified that the expression of CRY2 was directly inhibited by signal transducer and activator of transcription 3 (STAT3)-transactivated miR-7-5p overexpression, which upregulated the expression of circadian locomotor output cycles kaput (CLOCK)/brain and muscle ARNT-like 1 (BMAL1) and the transcription of P300 to stimulate osteogenesis through the enhanced histone 3 acetylation and the formation of a transcriptional complex with runt-related transcription factor 2 (Runx2)
Knockdown of CRY2 Enhances Osteogenic Differentiation To identify the role of CRY2, a transcriptional repressor and a core component of the circadian clock that may play a crucial role in osteogenic differentiation, we first silenced the endogenous expression of CRY2 in C3H10 and C2C12 cell lines by using a couple of short hairpins RNAs
Summary
Osteoblast differentiation and bone remodeling are regulated by a concerted communication between osteoblasts and osteoclasts.[1,2] Osteoblast differentiation requires numerous transcription factors to regulate both the formation and maintenance of bone.[3]. CRY2, the cryptochrome circadian regulator 2, is a core component of the circadian clock necessary for the generation and maintenance of circadian rhythms.[5] CRY2 acts as a transcriptional repressor to negatively regulate the gene transcription and is associated with diverse physiological processes.[6] More and more reports suggest that the circadian rhythm may regulate specific osteogenic differentiation. Animals with Cry2À/À displayed a significant increase of the bone volume at the age of 12 weeks.[7] the expression of peroxisome proliferator-activated receptor d (PPARd) genes was elevated in myotubes and muscles of the Cry2À/À animal,[8] which facilitates osteogenic differentiation through the sirtuin 1-dependent signaling pathway.[9] the specific mechanism associated with CRY2 is unknown
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