Fibroblast-specific Expression of a Kinase-deficient Type II Transforming Growth Factor β (TGFβ) Receptor Leads to Paradoxical Activation of TGFβ Signaling Pathways with Fibrosis in Transgenic Mice

Abstract

To better understand the role of disrupted transforming growth factor ␤ (TGF␤) signaling in fibrosis, we have selectively expressed a kinase-deficient human type II TGF␤ receptor (T␤RII⌬k) in fibroblasts of transgenic mice, using a lineage-specific expression cassette subcloned from the pro-␣2(I) collagen gene. Surprisingly, despite previous studies that characterized T␤RII⌬k as a dominant negative inhibitor of TGF␤ signaling, adult mice expressing this construct demonstrated TGF␤ overactivity and developed dermal and pulmonary fibrosis. Compared with wild type cells, transgenic fibroblasts proliferated more rapidly, produced more extracellular matrix, and showed increased expression of key markers of TGF␤ activation, including plasminogen activator inhibitor-1, connective tissue growth factor, Smad3, Smad4, and Smad7. Smad2/3 phosphorylation was increased in transgenic fibroblasts. Overall, the gene expression profile of explanted transgenic fibroblasts using cDNA microarrays was very similar to that of littermate wild type cells treated with recombinant TGF␤1. Despite basal up-regulation of TGF␤ signaling pathways, transgenic fibroblasts were relatively refractory to further stimulation with TGF␤1. Thus, responsiveness of endogenous genes to TGF␤ was reduced, and TGF␤-regulated promoter-reporter constructs transiently transfected into transgenic fibroblasts showed little activation by recombinant TGF␤1. Responsiveness was partially restored by overexpression of wild type type II TGF␤ receptors. Activation of MAPK pathways by recombinant TGF␤1 appeared to be less perturbed than Smad-dependent signaling. Our results show that expression of T␤RII⌬k selectively in fibroblasts leads to paradoxical ligand-dependent activation of downstream signaling pathways and causes skin and lung fibrosis. As well as confirming the potential for nonsignaling receptors to regulate TGF␤ activity, these findings support a direct role for perturbed TGF␤ signaling in fibrosis and provide a novel genetically determined animal model of fibrotic disease.Transforming growth factor ␤ (TGF␤) 1 isoforms ␤1-␤3 are important regulators of embryonic and postnatal cell differentiation and proliferation. They are also potent profibrotic factors in vitro. Transgenic and gene-targeted mutant mice have proven valuable for investigating the important roles of the TGF␤ family in growth and development. For example, mice lacking TGF␤1 show substantial embryonic lethality, and those that are born develop a fatal disseminated inflammatory illness within the first postnatal month. Conversely, mice lacking TGF␤2 all die neonatally with defective epithelial-mesenchymal interaction, whereas mice lacking TGF␤3 show a perinatal lethal phenotype with cleft palate (1). These different knock-out phenotypes suggest somewhat distinct biological functions in vivo, although all three TGF␤ isoforms signal though the same specific receptor complex, so differential effects are likely to reflect patterns of ligand expression or the influence of accessory receptors at the...

TGFβ pathobiology in the eye

“…57 However, blocking TGFb type II receptor function by dominant negative expression in collagen I-expressing fibroblasts in a transgenic mouse results instead in a paradoxical systemic tissue fibrosis in association with an uncontrolled Smad signaling activation. 58 The mechanism of this phenomenon could be explained the fact that Smad3 is phosphorylated by various MAPK at their middle linker region, which might promote nuclear translocation of Smad and might stimulate fibrosis-related gene expression.…”

Recent progress in genetics of Marfan syndrome and Marfan-associated disorders

“…The heterozygous state (with one normal allele and the other mutant) of TGFBR2 abnormality in affected individuals might not simply reflect a loss-of-function nature of the mutation, probably because of the complex regulation of the TGFb signaling pathway (Loeys et al 2005). Paradoxically, increased TGFb signaling was also shown in the kinase-deficient TbRII transgenic mice (Denton et al 2003).…”

A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2

“…Such events might culminate in excess, rather than decreased, TGFb superfamily signaling. This hypothesis was bolstered by the observation that transgenic mice expressing a dominant-negative (kinase domain-deleted) TbRII showed tissue fibrosis, upregulation of TGFb ligands, increased expression of TGFb-responsive genes and increased responsiveness to exogenous ligand 24,25 . We now present evidence that a similar net effect can be …”