Tohoku J. Exp. Med., 2010, 222(2)

Investigating Bone Morphogenetic Protein (BMP) Signaling in a Newly Established Human Cell Line Expressing BMP Receptor Type II

TADA-AKI KUDO,1 HIROYASU KANETAKA,2,3 AKIRA WATANABE,4 AYAKO OKUMOTO,2 MASANOBU ASANO,1 YE ZHANG,2 FEI ZHAO,1 MITSUHIRO KANO,5 YOSHINAKA SHIMIZU,5 SHINRI TAMURA6 and HARUHIDE HAYASHI1

1Division of Oral Physiology, Tohoku University Graduate School of Dentistry, Sendai, Japan
2Department of Physical Medicine and Rehabilitation, Tohoku University Graduate School of Biomedical Engineering, Sendai, Japan
3Division of Advanced Prosthetic Dentistry, Tohoku University Graduate School of Dentistry, Sendai, Japan
4Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan
5Division of Oral and Craniofacial Anatomy, Tohoku University Graduate School of Dentistry, Sendai, Japan
6Department of Biochemistry, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan

Bone morphogenetic proteins (BMPs), members of the transforming growth factor β cytokine superfamily, elicit various biological effects in different tissues. BMP receptor type II (BMPRII) contains a unique carboxyl-terminal region that interacts with multiple signaling molecules. However, expression of endogenous BMPRII is low in various mammalian cell lines, which hampers the analysis of BMP signaling. Therefore, we established a human cell line expressing BMPRII tagged with a Flag epitope (BMPRII-Flag) using the tetracycline-controlled Flp-In T-REx gene expression system. The BMPRII-Flag gene was introduced into the Flp-In T-REx 293 (FT293) cell line, a derivative of human 293 embryonic kidney fibroblasts. Then we analyzed the expression of key BMP target genes, inhibitors of DNA binding (Id) family members (Id1, Id2, and Id3) and the inhibitory Smads Smad6 and Smad7, in parental FT293 cells and an established cell line, FT293-BMPRII, by quantitative real-time PCR. Tetracycline treatment significantly increased the expression of BMPRII-Flag mRNA and protein in FT293-BMPRII cells, but induced no significant changes in expression of Id1, Id2, Id3, Smad6, or Smad7 mRNA. In contrast, treatment with a BMPRII ligand BMP2 induced the expression of Id1, Id2, Id3, and Smad6 in parental FT293 cells and FT293-BMPRII cells. Tetracycline-induced BMPRII-Flag expression significantly enhanced the induction of Id1, Id3, and Smad6 mRNA expression in FT293-BMPRII cells treated with BMP2. These findings provide evidence that although BMPRII has no obvious effect on the expression of representative BMP target genes, it differentially modulates the responsiveness of target genes to BMP2.

keywords —— bone morphogenetic protein; BMP receptor type II; stable cell line; tetracycline regulation; Flag-epitope tagging

2010 Tohoku University Medical Press

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Tohoku J. Exp. Med., 2010, 222, 121-129

Correspondence: Tada-aki Kudo, Division of Oral Physiology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.

e-mail: tkudo@m.tohoku.ac.jp