BRITER: A BMP Responsive Osteoblast Reporter Cell Line

Prem Swaroop Yadav,Paritosh Prashar,Amitabha Bandyopadhyay,Austin John Cooney

doi:10.1371/journal.pone.0037134

Prem Swaroop Yadav, Paritosh Prashar + Show 2 more

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https://doi.org/10.1371/journal.pone.0037134

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Journal: PloS one	Publication Date: May 14, 2012
Citations: 29	License type: CC BY 4.0

Affiliation: Indian Institute of Technology Kanpur

Abstract

BackgroundBMP signaling pathway is critical for vertebrate development and tissue homeostasis. High-throughput molecular genetic screening may reveal novel players regulating BMP signaling response while chemical genetic screening of BMP signaling modifiers may have clinical significance. It is therefore important to generate a cell-based tool to execute such screens.Methodology/Principal FindingsWe have established a BMP responsive reporter cell line by stably integrating a BMP responsive dual luciferase reporter construct in the immortalized calvarial osteoblast cells isolated from tamoxifen inducible Bmp2; Bmp4 double conditional knockout mouse strain. This cell line, named BRITER (BMP Responsive Immortalized Reporter cell line), responds robustly, promptly and specifically to exogenously added BMP2 protein. The sensitivity to added BMP may be further increased by depleting the endogenous BMP2 and BMP4 proteins.ConclusionAs the dynamic range of the assay (for BMP responsiveness) is very high for BRITER and as it responds specifically and promptly to exogenously added BMP2 protein, BRITER may be used effectively for chemical or molecular genetic screening for BMP signaling modifiers. Identification of novel molecular players capable of influencing BMP signaling pathway may have clinical significance.

Highlights

Bone Morphogenetic Protein (BMP) activity is the molecular basis of the intrinsic regenerative ability of bone [1]
20 different BMP molecules have been identified in the humans [3,4] of which BMP2, BMP4, BMP5, BMP6 and BMP7 are characterized as osteogenic BMPs due to their ability to induce ectopic bone formation [5]
Osteoblast cells themselves are known to produce a variety of BMPs [37,38] and production of endogenous BMP by the reporter cell line is expected to reduce its sensitivity towards exogenously added BMP as well as reduce the dynamic range of the assay

Summary

Introduction

Bone Morphogenetic Protein (BMP) activity is the molecular basis of the intrinsic regenerative ability of bone [1]. BMPs are members of the transforming Growth Factor-b (TGF-b) superfamily of signaling molecules [2]. It is known that Bone Morphogenetic Proteins (BMPs) are necessary and sufficient for bone formation [1,6]. BMP ligands bind to typeI and typeII serine/threonine kinase cell surface receptors. The typeII receptors phosphorylate type I receptors which in turn phosphorylate a group of transcription factors known as receptor-regulated SMADs (R-SMADs) i.e. SMAD1, SMAD5 or SMAD8. Phosphorylated R-SMADs form a heterodimer with SMAD4 and translocate into the nucleus and regulate transcription of a host of BMP downstream target genes [7,8,9]. It is important to generate a cell-based tool to execute such screens

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