Activation of key profibrotic mechanisms in transgenic fibroblasts expressing kinase-deficient type II transforming growth factor-β receptor (TβRIIΔk)

We have generated transgenic mice expressing a kinase-deficient type II transforming growth factor-beta (TGF beta) receptor selectively on fibroblasts (T beta RII Delta k-fib). These mice develop dermal and pulmonary fibrosis. In the present study we explore activation of TGF beta 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 T beta RI kinase (ALK5) activity, and that transgenic fibroblasts show enhanced expression and activation of TGF beta together with increased levels of wild type T beta RII. Moreover, we confirm that transgene expression is itself regulated by TGF beta and that expression at low levels facilitates signaling, whereas high level expression is inhibitory. For a subset of TGF beta responsive genes basal up-regulation is normalized or suppressed by exogenous recombinant TGF beta 1 at time points coincident with increased transgene expression. These findings explain the profound refractoriness of T beta RII Delta k-fib fibroblasts to exogenous TGF beta 1, 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 TGF beta receptor function may lead to ligand-dependent activation of downstream signaling, and provides further evidence of a pivotal role for sustained TGF beta overactivity in fibrosis.