TY - JOUR
T1 - Investigating bone morphogenetic protein (BMP) signaling in a newly established human cell line expressing BMP receptor type II
AU - Kudo, Tada Aki
AU - Kanetaka, Hiroyasu
AU - Watanabe, Akira
AU - Okumoto, Ayako
AU - Asano, Masanobu
AU - Zhang, Ye
AU - Zhao, Fei
AU - Kano, Mitsuhiro
AU - Shimizu, Yoshinaka
AU - Tamura, Shinri
AU - Hayashi, Haruhide
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
KW - BMP receptor type II
KW - Bone morphogenetic protein
KW - Flag-epitope tagging
KW - Stable cell line
KW - Tetracycline regulation
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U2 - 10.1620/tjem.222.121
DO - 10.1620/tjem.222.121
M3 - Article
C2 - 20919447
AN - SCOPUS:77958118529
VL - 222
SP - 121
EP - 129
JO - Tohoku Journal of Experimental Medicine
JF - Tohoku Journal of Experimental Medicine
SN - 0040-8727
IS - 2
ER -