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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.

In vertebrates, BMP signaling has been demonstrated to be sufficient for bone formation in several tissue contexts. This suggests that genes necessary for bone formation are expressed in a BMP signaling dependent manner. However, till date no gene has been reported to be expressed in a BMP signaling dependent manner in bone. Our aim was to identify such genes. On searching the literature we found that several microarray experiments have been conducted where the transcriptome of osteogenic cells in absence and presence of BMP signaling activation have been compared. However, till date, there is no evidence to suggest that any of the genes found to be upregulated in presence of BMP signaling in these microarray analyses is indeed a target of BMP signaling in bone. We wanted to utilize this publicly available information to identify candidate BMP signaling target genes in vivo. We performed a meta-analysis of six such comparable microarray datasets. This analysis and subsequent experiments led to the identification of five targets of BMP signaling in bone that are conserved both in mouse and chick. Of these Lox, Klf10 and Gpr97 are likely to be direct transcriptional targets of BMP signaling pathway. Dpysl3, is a novel BMP signaling target identified in our study. Our data demonstrate that Dpysl3 is important for osteogenic differentiation of mesenchymal cells and is involved in cell secretion. We have demonstrated that the expression of Dpysl3 is co-operatively regulated by BMP signaling and Runx2. Based on our experimental data, in silico analysis of the putative promoter-enhancer regions of Bmp target genes and existing literature, we hypothesize that BMP signaling collaborates with multiple signaling pathways to regulate the expression of a unique set of genes involved in endochondral ossification.

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