Elsevier

Bone

Volume 49, Issue 6, December 2011, Pages 1131-1140
Bone

Original Full Length Article
Sclerostin expression is induced by BMPs in human Saos-2 osteosarcoma cells but not via direct effects on the sclerostin gene promoter or ECR5 element

https://doi.org/10.1016/j.bone.2011.08.016Get rights and content

Abstract

Sclerostin is a secreted inhibitor of Wnt signaling and plays an essential role in the regulation of bone mass. The expression of sclerostin is largely restricted to osteocytes although its mode of transcriptional regulation is not well understood. We observed regulated expression of sclerostin mRNA and protein that was directly correlated with the mineralization response in cultured human Saos-2 osteosarcoma cells and rat primary calvarial cells. Sclerostin mRNA and protein levels were increased following treatment of cells with BMP2, BMP4 and BMP7. Analysis of deletion mutants from the − 7.4 kb upstream region of the human sclerostin promoter did not reveal any specific regions that were responsive to BMPs, Wnt3a, PTH, TGFβ1 or Activin A in Saos-2 cells. The downstream ECR5 element did not show enhancer activity in Saos-2 cells and also was not affected when Saos-2 cells were treated with BMPs or PTH. Genome-wide microarray analysis of Saos-2 cells treated with BMP2 showed significant changes in expression of several transcription factors with putative consensus DNA binding sites in the region of the sclerostin promoter. However, whereas most factors tested showed either a range of inhibitory activity (DLX family, MSX2, HEY1, SMAD6/7) or lack of activity on the sclerostin promoter including SMAD9, only MEF2B showed a positive effect on both the promoter and ECR5 element. These results suggest that the dramatic induction of sclerostin gene expression by BMPs in Saos-2 cells occurs indirectly and is associated with late stage differentiation of osteoblasts and the mineralization process.

Highlights

► Sclerostin expression enhanced by BMPs and coupled to osteoblast mineralization. ► BMPs, Wnt3a and PTH do not directly activate sclerostin promoter. ► BMP2 increases expression of several sclerostin inhibitors. ► MEF2B increased activity of the promoter and ECR5 element. ► No ECR5 enhancer activity in human osteosarcoma cells.

Introduction

Bone mass is regulated by several signaling pathways and the critical role of BMPs, PTH and β-Catenin-dependent Wnt signaling is well established. The soluble Wnt inhibitor sclerostin appears to be linked to each of these pathways and is known to profoundly affect the biology of the skeleton [1], [2].

Loss of function mutations in the sclerostin gene results in a high bone mass condition known as sclerosteosis [3], [4] whereas a 52 kb deletion downstream of the gene leads to van Buchem's disease also associated with high bone mass [5], [6]. Deletion of the gene in mice also leads to a profound high bone mass phenotype [7] and transgenic overexpression severely decreases bone mass [8], [9], thus clearly demonstrating a fundamental role for this gene in bone biology. Furthermore SNP polymorphisms in the human sclerostin gene appear to be associated with changes in BMD [10], [11], [12], [13].

In intact bone sclerostin appears to be secreted predominantly by osteocytes but in vitro studies have also shown sclerostin to be expressed by several osteoblast cell lines grown in media to stimulate osteogenic differentiation. Sclerostin inhibits Wnt signaling in osteoblasts via binding to the propeller 1 region of LRP5/6 Wnt co-receptor [14], [15]. Activating mutations in LRP5/6 genes that impact propeller 1 appears to prevent the binding of sclerostin thus allowing for enhancement of Wnt signaling [16], [17]. Treatment of animals with anti-sclerostin antibodies increases bone mass [18], [19], [20] and has led to initiation of clinical trials with an anti-sclerostin antibody for the treatment of osteoporosis and fracture repair [21].

Despite a growing understanding of the role of sclerostin in Wnt signaling, little is known about the regulation of sclerostin gene expression. Previous studies have shown stimulatory effects of BMPs or BMPs in combination with retinoic acid or 1,25-dihydroyvitamin D3 [22] and induction by glucocorticoids [23], the cytokine TWEAK [24] and calcitonin [25]. Other studies have shown inhibition of sclerostin expression by PTH [26], [27], [28], [29] and the cytokines oncostatin M, leukemia inhibitory factor and cardiotrophin-1 [30]. In addition it has been shown that alterations in mechanical loading of bone lead to changes in sclerostin expression, where increased loading is inversely related to sclerostin expression [31], [32], [33]. However, despite these observations it is not known how loading signals might impact transcriptional activity of the sclerostin gene.

The purpose of the present study was to determine if transcriptional activity of the sclerostin promoter was directly affected by BMPs in human Saos-2 osteosarcoma cells. Despite induction of sclerostin expression by BMPs which was associated with upregulation of SMAD9 expression, neither the promoter nor the ECR5 element showed direct responsiveness to BMP treatment. Of the factors tested, only MEF2B increased the activity of the gene ECR5 element. These results suggest that sclerostin expression is not directly regulated by BMPs in Saos-2 cells but correlates with conditions that favor enhancement of mineralization.

Section snippets

Plasmid constructs and DNA sequencing

The human sclerostin gene − 7.4 kb promoter was cloned by PCR using human genomic DNA (Beckton Dickenson Clontech #6550-1) as template. Four oligonucleotide primers were used to generate two PCR products that were ligated to produce the 7.4 kb fragment. The sequences of the primers were as follows: forward primer #1: 5′CGCTAGCTGAACCACCTTGTTTTTTTGTCCTTCTTATCACAGTTGG3′; reverse primer #1: 5′GAAAAAAGGGAAAATTCGAAC3′; forward primer #2: 5′GAAAATTCGAACACAGAGAGAAC3′ and reverse primer #2: 5′

Sclerostin protein and mRNA in osteoblast cells

Using a new ELISA assay, secreted sclerostin protein was detected at very high levels up to ~ 40,000 pg/ml in several rat osteosarcoma lines including UMR106 and ROS (data for UMR108 not shown). High sclerostin mRNA levels were previously reported in rat UMR106 cells [27]. In the current study, however, protein and mRNA levels did not appear to be strongly correlated (Figs. 1A and B). Furthermore, protein levels were remarkably similar when cells were grown either in GM or DM suggesting that

Discussion

The present results showed that whereas BMPs stimulated sclerostin expression in a dose and time-dependent manner in Saos-2 cells, the effects were not dependent on direct activation of the sclerostin gene promoter. Although several transcription factors with putative sclerostin promoter binding sites were shown to be up-regulated by BMP2 treatment, none of the ones tested in this study stimulated promoter activity. Of the additional factors explored only MEF2B showed a modest positive effect

Disclosures

This research was funded by Amgen, Inc. All authors are employees of Amgen, Inc. and have received stock and stock options from Amgen, Inc.

Acknowledgments

This research was funded by Amgen, Inc. We acknowledge Chris Paszty for comments on the manuscript.

References (50)

  • C.Y. Han et al.

    Small molecules with potent osteogenic-inducing activity in osteoblast cells

    Bioorg Med Chem Lett

    (2009)
  • I. Kramer et al.

    Does osteocytic SOST suppression mediate PTH bone anabolism?

    Trends Endocrinol Metab

    (2010)
  • B. Sevetson et al.

    Cbfa1/RUNX2 directs specific expression of the sclerosteosis gene (SOST)

    J Biol Chem

    (2004)
  • S.L. Cheng et al.

    Msx2 exerts bone anabolism via canonical Wnt signaling

    J Biol Chem

    (2008)
  • H. Li et al.

    Expression and function of Dlx genes in the osteoblast lineage

    Dev Biol

    (2008)
  • F. Yang et al.

    Sclerostin is a direct target of osteoblast-specific transcription factor osterix 1

    Biochem Biophys Res Commun

    (2010)
  • M.J. Moester et al.

    Sclerostin: current knowledge and future perspectives

    Calcif Tissue Int

    (2010)
  • C. Paszty et al.

    Sclerostin: a gem from the genome leads to bone-building antibodies

    J Bone Miner Res

    (2010)
  • W. Balemans et al.

    Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST)

    Hum Mol Genet

    (2001)
  • W. Balemans et al.

    Identification of a 52 kb deletion downstream of the SOST gene in patients with van Buchem disease

    J Med Genet

    (2002)
  • K. Staehling-Hampton et al.

    A 52-kb deletion in the SOST-MEOX1 intergenic region on 17q12–q21 is associated with van Buchem disease in the Dutch population

    Am J Med Genet

    (2002)
  • X. Li et al.

    Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength

    J Bone Miner Res

    (2008)
  • D.G. Winkler et al.

    Osteocyte control of bone formation via sclerostin, a novel BMP antagonist

    EMBO J

    (2003)
  • G.G. Loots et al.

    Genomic deletion of a long-range bone enhancer misregulates sclerostin in Van Buchem disease

    Genome Res

    (2005)
  • A.M. Sims et al.

    Genetic analyses in a sample of individuals with high or low BMD shows association with multiple Wnt pathway genes

    J Bone Miner Res

    (2008)
  • Cited by (28)

    • Growth factors, cytokines, and pediatric malignant primary bones tumors

      2021, Bone Cancer: Bone Sarcomas and Bone Metastases - From Bench to Bedside
    • Transcriptional control of Sost in bone

      2017, Bone
      Citation Excerpt :

      Osteoblast-targeted conditional deletion of BMP receptor type IA (Bmpr1a) in mice or blocking BMP signaling by adenovirus-mediated transfection of BMP inhibitor noggin resulted in significant reduction of Sost expression whereas mice expressing constitutively active Bmpr1a showed a significant increase in Sost levels [61,63,64]. It has been suggested that BMPs induce SOST expression mainly by targeting the SOST promoter [58]; however, BMPs may also regulate SOST expression independent of the SOST promoter [65]. The distal enhancer ECR5 is a 255-bp evolutionarily conserved sequence within the VBD deletion region.

    • Sclerostin's role in bone's adaptive response to mechanical loading

      2017, Bone
      Citation Excerpt :

      Primary osteoblasts do not express readily detectable levels of Sost until they form mineralised matrix, which precludes their use for in vitro strain studies. Mouse osteocytic MLO cell lines do not reliably produce readily detectable levels of Sost [38] and their expression of the constitutively active SV40 antigen [39] impacts PI3K/AKT signalling, which is a stain-responsive pathway [40]. The more recently developed IDG-SW3 cell line promises to circumvent this limitation, but these cells only express Sost after prolonged periods of differentiation [41].

    • Heavy metal ion regulation of gene expression: Mechanisms by which lead inhibits osteoblastic bone-forming activity through modulation of the Wnt/β-catenin signaling pathway

      2015, Journal of Biological Chemistry
      Citation Excerpt :

      Genes of interest were normalized to β-actin expression. Initially, MC3T3-E1 cells were plated and transfected with TOPFLASH (LEF/TCF basal c-Fos-luciferase), human 7-kb sclerostin upstream promoter (SOST-Luc) (15), Smad3 reporter (p3TP-lux), or constructively active Smad3 construct (CA-Smad3) (16). Then, transfections were performed using FuGENE HD (Qiagen) according to the manufacturer's instructions and as described previously (17).

    View all citing articles on Scopus
    View full text