A nonclassical vitamin D receptor pathway suppresses renal fibrosis
Ichiaki Ito, … , Kazuo Nagasawa, Junn Yanagisawa
Ichiaki Ito, … , Kazuo Nagasawa, Junn Yanagisawa
Published October 25, 2013
Citation Information: J Clin Invest. 2013;123(11):4579-4594. https://doi.org/10.1172/JCI67804.
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Research Article

A nonclassical vitamin D receptor pathway suppresses renal fibrosis

Abstract

The TGF-β superfamily comprises pleiotropic cytokines that regulate SMAD and non-SMAD signaling. TGF-β–SMAD signal transduction is known to be involved in tissue fibrosis, including renal fibrosis. Here, we found that 1,25-dihydroxyvitamin D3–bound [1,25(OH)2D3-bound] vitamin D receptor (VDR) specifically inhibits TGF-β–SMAD signal transduction through direct interaction with SMAD3. In mouse models of tissue fibrosis, 1,25(OH)2D3 treatment prevented renal fibrosis through the suppression of TGF-β–SMAD signal transduction. Based on the structure of the VDR-ligand complex, we generated 2 synthetic ligands. These ligands selectively inhibited TGF-β–SMAD signal transduction without activating VDR-mediated transcription and significantly attenuated renal fibrosis in mice. These results indicate that 1,25(OH)2D3-dependent suppression of TGF-β–SMAD signal transduction is independent of VDR-mediated transcriptional activity. In addition, these ligands did not cause hypercalcemia resulting from stimulation of the transcriptional activity of the VDR. Thus, our study provides a new strategy for generating chemical compounds that specifically inhibit TGF-β–SMAD signal transduction. Since TGF-β–SMAD signal transduction is reportedly involved in several disorders, our results will aid in the development of new drugs that do not cause detectable adverse effects, such as hypercalcemia.

Authors

Ichiaki Ito, Tsuyoshi Waku, Masato Aoki, Rumi Abe, Yu Nagai, Tatsuya Watanabe, Yuka Nakajima, Ichiro Ohkido, Keitaro Yokoyama, Hiroyuki Miyachi, Toshiyuki Shimizu, Akiko Murayama, Hiroyuki Kishimoto, Kazuo Nagasawa, Junn Yanagisawa

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Figure 3

1,25(OH)2D3 disrupts binding of SMAD3 to DNA via VDR.

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1,25(OH)2D3 disrupts binding of SMAD3 to DNA via VDR. (A) VDR mutants ...
(A) VDR mutants lacking DNA binding activity due to mutation or deletion of the C domain are shown at top. HEK293 cells were transfected with plasmids encoding ALK5 TD, VDR, VDR-mC, or VDR-LBD and the reporter plasmids VDRE-Luc or CAGA-Luc. After culturing transfected cells with or without 1,25(OH)2D3 for 24 hours, cell extracts were analyzed using luciferase assay. DBD, DNA-binding domain. (B) Purified recombinant His-tagged SMAD2, SMAD3, and SMAD4 were incubated with GST-VDR-LBD in the presence or absence of 1,25(OH)2D3. (C) Purified recombinant His-tagged SMAD3-MH1 (aa 1–132), SMAD3-MH1L (aa 1–225), or SMAD3-MH2 (aa 226–425) were incubated with GST-VDR-LBD in the presence or absence of 1,25(OH)2D3, after which the mixtures were examined using in vitro pulldown assay. (D) Recombinant SMAD3-MH1 and/or VDR-LBD were mixed with a DNA probe containing the SMAD3-binding element in the presence or absence of 1,25(OH)2D3, after which binding between SMAD3-MH1 and the probe was analyzed by EMSA. Protein levels were determined by Western blotting. The quantified shifted band (bound fraction) is shown at right (n = 3). + and ++ indicate the ratios of sample volumes applied to the assay. *P < 0.05; **P < 0.01; ***P < 0.001.