Abstract
New bone anabolic agents for the effective treatment of bone metabolic diseases like osteoporosis are of high clinical demand. In the present study, we reveal the function of salt-inducible kinase 1 (SIK1) in regulating osteoblast differentiation. Gene knockdown of SIK1 but not of SIK2 or SIK3 expression in primary preosteoblasts increased osteoblast differentiation and bone matrix mineralization. SIK1 also regulated the proliferation of osteoblastic precursor cells in osteogenesis. This negative control of osteoblasts required the catalytic activity of SIK1. SIK1 phosphorylated CREB regulated transcription coactivator 1 (CRTC1), preventing CRTC1 from enhancing CREB transcriptional activity for the expression of osteogenic genes like Id1. Furthermore, SIK1 knockout (KO) mice had higher bone mass, osteoblast number, and bone formation rate versus littermate wild-type (WT) mice. Preosteoblasts from SIK1 KO mice showed more osteoblastogenic potential than did WT cells, whereas osteoclast generation among KO and WT precursors was indifferent. In addition, bone morphogenic protein 2 (BMP2) suppressed both SIK1 expression as well as SIK1 activity by protein kinase A (PKA)–dependent mechanisms to stimulate osteogenesis. Taken together, our results indicate that SIK1 is a key negative regulator of preosteoblast proliferation and osteoblast differentiation and that the repression of SIK1 is crucial for BMP2 signaling for osteogenesis. Therefore, we propose SIK1 to be a useful therapeutic target for the development of bone anabolic strategies.
Highlights
An imbalance in the amount of bone-formation by osteoblasts and bone resorption by osteoclasts in bone remodeling leads to bone metabolic diseases like osteoporosis
Mouse RANKL and OPG ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). pcDNA3SIK1-HA, pcDNA3-salt-inducible kinase 1 (SIK1)-FLAG, and pcDNA3-SIK1T182A-FLAG plasmids were as previously described39. pGL-Id1 (−1231/+88) plasmid was provided by Dr Korchynskyi (National Academy of Sciences in Ukraine)40. siRNA oligonucleotides for control, SIK1, SIK2, SIK3, or CREB regulated transcription coactivator 1 (CRTC1) were purchased from Thermo Fisher Scientific (Waltham, MA, USA)
In osteogenic culture with medium containing β-glycerophosphate and ascorbic acid of primary mouse precursor cells, the SIK1 mRNA levels were dramatically decreased within two days, whereas the mRNA levels of SIK2 and SIK3 were almost constant until the late stage (Fig. 1a)
Summary
An imbalance in the amount of bone-formation by osteoblasts and bone resorption by osteoclasts in bone remodeling leads to bone metabolic diseases like osteoporosis. Osteoblast-targeted anabolic agents were developed for osteoporosis and fracture treatments[2,3]. Osteoblast differentiation is achieved through a series of ill-demarcated steps including the commitment of mesenchymal stem cells to osteoprogenitors, proliferation of progenitor cells, early differentiation to immature osteoblasts, and differentiation to mature osteoblasts[6,7]. These multiple steps are regulated by diverse groups of extracellular signals like bone morphogenic protein (BMP), Wnt, hedgehog, and parathyroid hormone-related peptide (PTHrP) via distinct or overlapping intracellular signaling pathways[6]
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