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.
View: Text | PDF
Research Article Article has an altmetric score of 22

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

×

Figure 7

DLAM derivatives suppress TGF-β–dependent transcription by inhibiting the DNA binding of SMAD3.

Options: View larger image (or click on image) Download as PowerPoint
DLAM derivatives suppress TGF-β–dependent transcription by inhibiting th...
(A) Analysis of the interaction between the DLAM-bound VDR and SRC-1 or SMAD3. Flag-tagged SRC-1 (aa 593–730) was expressed in HEK293 cells, and the cell extracts or purified recombinant His-tagged SMAD3-MH1 (aa 1–132) were incubated with GST-VDR-LBD in the presence or absence of 1,25(OH)2D3. Mixtures were then analyzed by in vitro pulldown assay. (B) DLAM-iPr and DLAM-4P interfere with binding of SMAD3 to the SMAD3-binding element. Purified recombinant SMAD3-MH1 and VDR-LBD were incubated with a DNA probe containing the SMAD3-binding element in the presence or absence of the indicated ligands, and binding of SMAD3-MH1 to the probe was analyzed by EMSA (top). Protein levels were aslo assessed by Western blotting (middle and bottom). (C) Inhibition of TGF-β–dependent SMAD3 recruitment to the Serpine1 and Acta2 promoters by DLAM-iPr and DLAM-4P. TCMK-1 cells were cultured in the presence or absence of 5 ng/ml TGF-β, 10 nM 1,25(OH)2D3, 1 μM DLAM-iPr, or 1 μM DLAM-4P. ChIP assays were performed with control IgG or anti-SMAD3 antibodies. Immunoprecipitated DNA was examined by qPCR with primers specific for the Serpine1 or Acta2 promoter. Samples were normalized to input DNA level. **P < 0.01; ***P < 0.001.