Biochemical and Biophysical Research Communications
The transcriptional modulator Ifrd1 is a negative regulator of BMP-2-dependent osteoblastogenesis
Introduction
Bone homeostasis is maintained by the collaborative function of bone-forming osteoblasts, derived from mesenchymal cells, and bone-resorbing osteoclasts, derived from hematopoietic cells [1]. An imbalance between bone formation and bone resorption leads to various metabolic bone diseases, including osteoporosis and osteopetrosis [2]. A complex network of various systems, including endocrine, paracrine/autocrine, and neuronal systems, work synergistically to orchestrate osteoblastogenesis [3]. These systems generate intracellular signals and control cellular functions for proliferation, survivability, differentiation, and maturation in osteoblasts by modulating the expression and activity of various transcription factors, including runt-related transcription factor 2 (Runx2), Osterix, activator protein 1 (AP-1), and nuclear factor-κB (NF-κB), controlled by transcriptional modulators (activators or repressors) [4], [5], [6], [7].
Interferon-related developmental regulator 1 (Ifrd1) is a transcriptional coactivator/repressor that controls gene expression patterns by interacting with transcription factors or histone deacetylase complexes [8]. We demonstrated that osteoblastic Ifrd1 represses osteoblastogenesis and activates osteoclastogenesis by modulating the NF-κB/osterix and β-catenin/osteoprotegerin pathways, respectively [9]. Moreover, we revealed that Ifrd1 regulates bone homeostasis through its expression in the osteoclast lineage under pathologic conditions and the modulation of osteoclastogenesis via NF-κB/NFATc1 signaling in a cell-autonomous fashion [10].
Bone morphogenetic proteins (BMPs) were originally identified as proteins that induced ectopic bone formation when implanted into muscular tissue, and they are strong inducers of osteoblast differentiation and bone formation [11], [12]. Here, we showed that BMP-2, one of the potent osteoinductive cytokines, directly induces Ifrd1 expression at the transcriptional level in osteoblasts via the Smad pathway. Moreover, we showed that Ifrd1 negatively regulates the BMP-2-dependent differentiation and maturation of osteoblasts. Our findings provide a novel molecular framework that identifies potential future targets for the discovery and development of treatments for a variety of metabolic bone diseases.
Section snippets
Materials
Recombinant human BMP-2 was purchased from Peprotech (Rocky Hill, NJ, USA). Both LDN193189 and SB431542 were purchased from Wako Pure Chemical Industries (Osaka, Japan). The pCMV5 Flag-Smad1 (#14044) and pCMV5 Flag-DPC4 (#14039) were obtained from Addgene (Cambridge, MA, USA). THUNDERBIRD SYBR qPCR Mix was supplied by TOYOBO (Osaka, Japan). Other chemicals used were all of the highest purity commercially available.
Cell culture and luciferase assay
Mouse primary osteoblasts were prepared from calvaria of newborn mice by the
BMP-2 transcriptionally activates Ifrd1 expression in osteoblasts
We tested if Ifrd1 expression was responsive to BMP-2 stimulation in primary osteoblasts. Primary osteoblasts were exposed to 100 ng/mL BMP-2 for periods of 1–4 days and then Ifrd1 protein expression was determined. Ifrd1 expression was markedly upregulated by BMP-2 stimulation over time, and the protein expression of Runx2, a master regulator of osteoblastogenesis, was markedly induced in osteoblasts (Fig. 1A). Moreover, BMP-2 stimulation led to a significant induction of Ifrd1 mRNA expression
Discussion
In this study, we determined that BMP-2 induced Ifrd1 expression via activation of the canonical Smad pathway in osteoblasts. Although the transcription factors ATF6 and AP-1 directly upregulate Ifrd1 expression in adipocytes and osteoclasts, respectively, in vitro [10], [14], the upstream mechanisms for Ifrd1 expression are not well elucidated. In contrast, the downstream signaling pathways inducted by Ifrd1 expression are well characterized. The BMP signaling pathway involves the
Conflict of interest
All authors have no conflict of interest.
Acknowledgements
This work was supported in part by Grants-in-Aids for Scientific Research to E.H. (No. 24659113) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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These authors equally contributed to this work.