Original Full Length ArticleSclerostin expression is induced by BMPs in human Saos-2 osteosarcoma cells but not via direct effects on the sclerostin gene promoter or ECR5 element
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.
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2017, BoneCitation 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.
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2017, BoneCitation 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].
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2015, Journal of Biological ChemistryCitation 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).