Cell-penetrating peptides selectively targeting SMAD3 inhibit profibrotic TGF-β signaling
Jeong-Han Kang, … , Danielle M. Hernandez, Edward B. Leof
Jeong-Han Kang, … , Danielle M. Hernandez, Edward B. Leof
Published May 22, 2017
Citation Information: J Clin Invest. 2017;127(7):2541-2554. https://doi.org/10.1172/JCI88696.
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Research Article Cell biology

Cell-penetrating peptides selectively targeting SMAD3 inhibit profibrotic TGF-β signaling

Abstract

TGF-β is considered a master switch in the pathogenesis of organ fibrosis. The primary mediators of this activity are the SMAD proteins, particularly SMAD3. In the current study, we have developed a cell-penetrating peptide (CPP) conjugate of the HIV TAT protein that is fused to an aminoterminal sequence of sorting nexin 9 (SNX9), which was previously shown to bind phosphorylated SMAD3 (pSMAD3). We determined that specifically preventing the nuclear import of pSMAD3 using the TAT-SNX9 peptide inhibited profibrotic TGF-β activity in murine cells and human lung fibroblasts as well as in vivo with no demonstrable toxicity. TGF-β signaling mediated by pSMAD2, bone morphogenetic protein 4 (BMP4), EGF, or PDGF was unaffected by the TAT-SNX9 peptide. Furthermore, while the TAT-SNX9 peptide prevented TGF-β’s profibrotic activity in vitro as well as in 2 murine treatment models of pulmonary fibrosis, a 3–amino acid point mutant that was unable to bind pSMAD3 proved ineffective. These findings indicate that specifically targeting pSMAD3 can ameliorate both the direct and indirect fibroproliferative actions of TGF-β.

Authors

Jeong-Han Kang, Mi-Yeon Jung, Xueqian Yin, Mahefatiana Andrianifahanana, Danielle M. Hernandez, Edward B. Leof

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Figure 5

TAT–SH3-2 inhibits profibrotic responses in human lung fibroblasts.

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TAT–SH3-2 inhibits profibrotic responses in human lung fibroblasts. (A) ...
(A) NHLF or lung fibroblasts from IPF patients were transduced with the indicated TAT peptide (1.5 μM) as in Figure 1. TGF-β (5 ng/ml) or SB431542 (10 μM) was then added for an additional 24 hours. Images were obtained on an LSM510 confocal microscope following F-actin labeling with phalloidin-TRITC and DAPI nuclei staining. Photographs are representative of 3 separate experiments. Scale bars: 10 μm. (B) Western blot analysis (representative of 3 separate experiments) for the indicated proteins subsequent to TAT peptide transduction (1.5 μM) and 24-hour treatment in the absence (–) or presence (+) of TGF-β (5 ng/ml) or SB431542 (10 μM). (C) NHLF or IPF fibroblasts were treated as in A and qPCR performed as described in Methods and Figure 3D. Results represent mean ± SEM from 3 independent experiments. ***P < 0.0005, 1-way ANOVA followed by Dunnett’s multiple comparisons test.