Activation of Key Profibrotic Mechanisms in Transgenic Fibroblasts Expressing Kinase-deficient Type II Transforming Growth Factor-β Receptor (TβRIIΔk)

• Published in: The Journal of biological chemistry 2005-04, Vol.280 (16), p.16053
• Main Authors: Christopher P. Denton, Gisela E. Lindahl, Korsa Khan, Xu Shiwen, Voon H. Ong, Nicholas J. Gaspar, Konstantinos Lazaridis, Dylan R. Edwards, Andrew Leask, Mark Eastwood, Patricia Leoni, Elisabetta A. Renzoni, George Bou Gharios, David J. Abraham, Carol M. Black
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M413134200

We have generated transgenic mice expressing a kinase-deficient type II transforming growth factor-β (TGFβ) receptor selectively on fibroblasts (TβRIIΔk-fib). These mice develop dermal and pulmonary fibrosis. In the present study we explore activation of TGFβ signaling pathways in this strain and examine the profibrotic properties of explanted transgenic fibroblasts including myofibroblast differentiation and abnormal metalloproteinase production. Gene expression profiles of littermate wild type or transgenic fibroblasts were compared using high-density gene arrays and validated by Taqman reverse transcriptase-PCR, Northern and Western blotting. Using a specific inhibitor (SD-208) we demonstrate that the abnormal phenotype of these cells is dependent upon TβRI kinase (ALK5) activity, and that transgenic fibroblasts show enhanced expression and activation of TGFβ together with increased levels of wild type TβRII. Moreover, we confirm that transgene expression is itself regulated by TGFβ and that expression at low levels facilitates signaling, whereas high level expression is inhibitory. For a subset of TGFβ responsive genes basal up-regulation is normalized or suppressed by exogenous recombinant TGFβ1 at time points coincident with increased transgene expression. These findings explain the profound refractoriness of TβRIIΔk-fib fibroblasts to exogenous TGFβ1, despite their activated phenotype. Thus, transgenic fibroblasts recapitulate many hallmark biochemical properties of fibrotic cells, including high level CTGF (CCN2) expression and type I collagen overproduction, altered MMP production, and myofibroblast differentiation. These cells also show an enhanced ability to contract collagen gel matrices. Our study demonstrates that altered high affinity TGFβ receptor function may lead to ligand-dependent activation of downstream signaling, and provides further evidence of a pivotal role for sustained TGFβ overactivity in fibrosis.