Abstract
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.
Highlights
A growing body of evidence implicates overactivity of transforming growth factor  (TGF)1 in fibrosis [1, 2] leading to sustained extracellular matrix overproduction and promoting myofibroblast differentiation [3,4,5]
The present study extends our analysis of a novel genetically determined mouse model of fibrosis, the TRII⌬k-fib strain
In our initial description we reported that a kinase-deficient human type II TGF receptor leads to fibrosis in transgenic mice [21]
Summary
A growing body of evidence implicates overactivity of transforming growth factor  (TGF) in fibrosis [1, 2] leading to sustained extracellular matrix overproduction and promoting myofibroblast differentiation [3,4,5]. The prototypic multisystem fibrotic disease systemic sclerosis (SSc) has been associated with increased activity of TGF signaling pathways [6, 7], altered expression of high and low affinity TGF receptors (8 –11), and autocrine overproduction of several TGFregulated genes [12, 13]. We achieved our goal of sustained activation of TGF signaling by expressing a kinase-deficient type II TGF receptor (TRII⌬k) in fibroblasts. In the present study the fibrotic phenotype of explanted dermal fibroblasts from TRII⌬k-fib mice is delineated. Fibrotic Mechanisms in TRII⌬k-fib Transgenic Mice activation is dependent upon endogenous TRI receptor kinase activity. We confirm that the fibroblast-specific expression cassette is itself regulated by TGF and that low level expression of the kinase-deficient TRII activates TGF signaling, whereas higher expression levels are inhibitory. Consequences of blocking TGF ligand or receptor activity are unpredictable, and animal models such as the TRII⌬k-fib strain are likely to be valuable tools in the development and evaluation of targeted antifibrotic therapies
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