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Fibroblast-specific TGF-β–Smad2/3 signaling underlies cardiac fibrosis
Hadi Khalil, … , Jason Karch, Jeffery D. Molkentin
Hadi Khalil, … , Jason Karch, Jeffery D. Molkentin
Published September 11, 2017
Citation Information: J Clin Invest. 2017;127(10):3770-3783. https://doi.org/10.1172/JCI94753.
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Research Article Cardiology Cell biology

Fibroblast-specific TGF-β–Smad2/3 signaling underlies cardiac fibrosis

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Abstract

The master cytokine TGF-β mediates tissue fibrosis associated with inflammation and tissue injury. TGF-β induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins. Canonical TGF-β signaling mobilizes Smad2 and Smad3 transcription factors that control fibrosis by promoting gene expression. However, the importance of TGF-β–Smad2/3 signaling in fibroblast-mediated cardiac fibrosis has not been directly evaluated in vivo. Here, we examined pressure overload–induced cardiac fibrosis in fibroblast- and myofibroblast-specific inducible Cre-expressing mouse lines with selective deletion of the TGF-β receptors Tgfbr1/2, Smad2, or Smad3. Fibroblast-specific deletion of Tgfbr1/2 or Smad3, but not Smad2, markedly reduced the pressure overload–induced fibrotic response as well as fibrosis mediated by a heart-specific, latency-resistant TGF-β mutant transgene. Interestingly, cardiac fibroblast–specific deletion of Tgfbr1/2, but not Smad2/3, attenuated the cardiac hypertrophic response to pressure overload stimulation. Mechanistically, loss of Smad2/3 from tissue-resident fibroblasts attenuated injury-induced cellular expansion within the heart and the expression of fibrosis-mediating genes. Deletion of Smad2/3 or Tgfbr1/2 from cardiac fibroblasts similarly inhibited the gene program for fibrosis and extracellular matrix remodeling, although deletion of Tgfbr1/2 uniquely altered expression of an array of regulatory genes involved in cardiomyocyte homeostasis and disease compensation. These findings implicate TGF-β–Smad2/3 signaling in activated tissue-resident cardiac fibroblasts as principal mediators of the fibrotic response.

Authors

Hadi Khalil, Onur Kanisicak, Vikram Prasad, Robert N. Correll, Xing Fu, Tobias Schips, Ronald J. Vagnozzi, Ruijie Liu, Thanh Huynh, Se-Jin Lee, Jason Karch, Jeffery D. Molkentin

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Figure 2

Generation of fibroblast-specific canonical TGF-β–deleted mice.

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Generation of fibroblast-specific canonical TGF-β–deleted mice.
(A) Sche...
(A) Schematic representation of different mouse lines used, including the Postn genetic locus containing a tamoxifen-regulated MCM cDNA cassette inserted into exon 1 (E1), which was crossed with Smad2- and/or Smad3-loxP–containing gene-targeted lines, along with the Rosa26 reporter allele(R26EGFP). The mouse chromosome associated with each allele is shown. (B) Experimental scheme whereby mice were subjected to TAC injury or sham procedure for 4 and 12 weeks. Mice were fed tamoxifen diet 48 hours before surgery and then maintained on tamoxifen until harvesting. (C) Representative histological section showing EGFP-labeled interstitial cells in hearts of PostnMCM/+ R26EGFP/+ mice after 4 weeks of TAC injury (n = 4). Scale bar: 10 μm. (D) Western blot analysis for Smad2, Smad3, and EGFP from 500,000 cells isolated from hearts of the 2 genotypes of mice shown. (E) Western blot for Tgfbr2 in total heart mesenchymal cells lacking myocytes, CD31, and CD45 cells and conditionally targeted for Tgfbr1/2-loxP with PostnMCM in cardiac-activated fibroblasts after TAC stimulation.

Copyright © 2022 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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