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 8

TAT–SH3-2 has no demonstrable effect on murine liver enzymes or inflammatory cell recruitment.

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TAT–SH3-2 has no demonstrable effect on murine liver enzymes or inflamma...
C57BL/6 mice were treated as described in Methods and Figure 7. On day 14, all animals began daily treatment with either vehicle (METHOCEL/saline) or 1.0 mg/kg of TAT–SH3-2. Blood samples were obtained at days 0, 14, and 28 from the facial vein of unanesthetized animals and assessed for effect on the indicated liver enzymes and inflammatory cells. Serum levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), and albumin were determined using a Piccolo Xpress Chemistry Analyzer. Quantification of lymphocytes, monocytes, and neutrophils was measured using a VetScan HM5 Analyzer. The Toxicology and Pharmacology Laboratory in the Department of Molecular Medicine at the Mayo Clinic performed all analyses. Data are presented as mean ± SEM of n = 5.