Indian Hedgehog Gene Is a Target of the Bone Morphogenetic Protein Signaling Pathway

Kenji Seki,Akiko Hata

doi:10.1074/jbc.m311592200

Abstract

Bone morphogenetic proteins (BMPs), a large subgroup of the transforming growth factor-beta family of growth factors, are key mediators of many fundamental processes in embryonic development. The BMP target genes mediating these effects and the mechanism of their selective regulation are poorly characterized. In this study, we used a chromatin immunoprecipitation-based gene cloning method to identify BMP target genes in a mouse embryonic carcinoma cell line. We identified the Indian hedgehog (Ihh) gene as a target of BMP signaling. Both reporter and reverses transcription-PCR assays revealed that Ihh is up-regulated in embryonic cells upon BMP treatment. The BMP response element of the Ihh promoter contains multiple GC-rich motifs known as the "Mad/Medea binding box" found in the Drosophila tinman gene and in the mouse Id1 gene. DNA binding studies revealed that Smad4 binds to these GC-rich motifs. These findings indicate that BMP stimulates Ihh expression. We also suggest expression of the Ihh gene, which contains multiple Smad binding sites, might be finely regulated by a gradient of BMP concentrations.

Highlights

The bone morphogenetic proteins (BMPs)1 were originally identified as peptides that induce ectopic bone and cartilage production [1]
The mouse teratocarcinoma cell line P19 is well characterized as a cell line that responds to Bone morphogenetic proteins (BMPs) [10] and expresses some of the BMP target genes, such as Smad6 [13]
Using a chromatin immunoprecipitation (ChIP)-based cloning method, we found that the Indian hedgehog (Ihh) gene is a direct target of the BMP-Smad signaling pathway in the mouse embryonic carcinoma P19 cell line

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—COS-1 and P19 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone). The nuclei were resuspended in chip buffer (20 mM Tris-HCl, pH 7.4, 1 M sodium chloride, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% bovine serum albumin, proteinase inhibitors) and incubated on ice for 10 min. Deletion mutants of the ZHC8 genomic fragment were generated by PCR and products and were ligated to XhoI and HindIII sites of pE1bTATA-luc vector. Biotinylated Oligonucleotide Precipitation Assays—48 h after transfection with Smad, Smad, and constitutive active BMPRIB vectors, COS-1 cells were lysed by sonication in T-NET buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.05% of Nonidet P-40) containing protease and phosphatase inhibitors. DNA-bound proteins were collected with streptavidin-agarose beads for 1 h, washed with T-NET buffer, separated on a SDS-PAGE gel, and identified by Western blot. Western Blot—Smad, Smad, and p21/Waf1/Cip proteins were detected with anti-Smad rabbit polyclonal antibodies (H-552, Santa Cruz Biotechnology), anti-Smad rabbit polyclonal antibodies

RESULTS

DISCUSSION

Kenji Seki and Akiko Hata