The transcriptional modulator Ifrd1 is a negative regulator of BMP-2-dependent osteoblastogenesis

https://doi.org/10.1016/j.bbrc.2016.11.063Get rights and content

Highlights

  • Ifrd1 expression is upregulated by BMP-2 in osteoblasts at the transcriptional level.

  • Smad4/Smad1 stimulates Ifrd1 promoter activity in osteoblasts.

  • BMP-2 accelerates recruitment of Smad1 to the Ifrd1 promoter in osteoblasts.

  • Ifrd1 knockdown enhances BMP-2-dependent osteoblastogenesis.

Abstract

We previously demonstrated that the transcriptional coactivator/repressor interferon-related developmental regulator 1 (Ifrd1) was expressed in osteoblasts and participated in the regulation of bone homeostasis. However, it remains unclear how Ifrd1 expression itself is regulated in osteoblasts. In the present study, we investigated the upstream regulatory mechanisms of Ifrd1 in osteoblasts during osteoblastogenesis. Ifrd1 protein expression and runt-related transcription factor 2, the master regulator of osteoblastogenesis, were markedly upregulated by bone morphogenetic protein 2 (BMP-2) stimulation in primary osteoblasts. Moreover, BMP-2 stimulation significantly induced Ifrd1 mRNA expression and promoter activity in osteoblasts. LDN193189, an inhibitor of activin-like kinase 2/3, almost completely inhibited the BMP-2-induced increase in Ifrd1 protein expression. There were at least two putative Smad-binding elements in the 5′-flanking region, which was highly conserved between mouse and human Ifrd1 genes. Co-introduction of both Smad4 and Smad1 significantly increased Ifrd1 promoter activity in osteoblasts. In addition, BMP-2 induced the recruitment of Smad1 to the Ifrd1 promoter in osteoblasts. Moreover, BMP-2-dependent osteoblastogenesis was further enhanced in Ifrd1 knocked-down osteoblasts, as determined by the intensity of Alizarin red stain and marker gene expression. These results suggest that BMP-2 directly induces Ifrd1 expression at the transcriptional level in osteoblasts via the Smad pathway, and Ifrd1 negatively regulates 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.

References (25)

  • S. Harada et al.

    Control of osteoblast function and regulation of bone mass

    Nature

    (2003)
  • J. Chang et al.

    Inhibition of osteoblastic bone formation by nuclear factor-kappaB

    Nat. Med.

    (2009)
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