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

Abstract

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

Contribution of activin receptor–like kinase 5 (transforming growth factor β receptor type I) signaling to the fibrotic phenotype of scleroderma fibroblasts

“…Dermal dcSSc fibroblasts overexpress an array of proadhesive and procontractile proteins (23), including CCN2, which is also known as connective tissue growth factor (4,21,24,25). To assess whether endogenous TGF␤ signaling through the type I TGF␤ receptor is involved in the overexpression of profibrotic genes by dcSSc fibroblasts, we incubated dermal fibroblasts derived from healthy controls and from lesional areas of the skin of dcSSc patients for 24 hours with SD208, an ALK-5/TGF␤RI kinase inhibitor (19,20). Cell extracts were then prepared, and subjected to SDS-PAGE and (25 g) from dermal fibroblasts derived from normal subjects and from lesional areas of the skin of patients with dcSSc were subjected to Western blot analysis with antimoesin, antivinculin, anti-CCN2, antiezrin, anti-␣-smooth muscle actin (anti-␣-SMA), anti-␣4 integrin, anti-␤1 integrin, and anti-GAPDH antibodies.…”

“…Fibroblasts from patients with dcSSc overexpress a series of profibrotic proteins. In this study, we investigated the ability of SD208, a TGF␤RI kinase (ALK-5) inhibitor (19,20), to prevent the overexpression of profibrotic proteins by dermal fibroblasts from patients with earlyonset dcSSc, as well as the ability of dermal fibroblasts from patients with early-onset dcSSc to excessively adhere to and contract ECM.…”

“…SD208 has an 50% inhibition concentration of 49 nmoles/liter, based on direct, cell-free enzymatic assay of TGF␤RI kinase (ALK-5) activity with a specificity of Ͼ100-fold against TGF␤RII and at least 17-fold over members of a panel of related protein kinases, including p38␣, p38␤, p38␦, JNK1, epidermal growth factor receptor, MAPKAPK-2, MKK-6, ERK-2, protein kinases C, A, and D, Cdc2, and Ca 2ϩ /calmodulin-regulated kinase II. Based upon data from our earlier studies of the effect of SD208 on murine dermal fibroblast ECM biosynthesis (19), a concentration of 1 M of SD208 was used for experiments examining control or SSc human dermal fibroblasts.…”

“…Cells were cultured until confluence in DMEM/10% FBS and incubated in DMEM/0.5% bovine serum albumin (BSA; Sigma, St. Louis, MO) for 18 hours, and then incubated with 1 M SD208 (19,20) for an additional 24 hours. Cells were used at passage 3.…”

Recombinant human anti–transforming growth factor β1 antibody therapy in systemic sclerosis: A multicenter, randomized, placebo‐controlled phase I/II trial of CAT‐192

“…There is a large body of indirect evidence implicating the overexpression of TGF␤ ligand or receptor in the pathogenesis of SSc (23), and several recent studies have demonstrated that blocking TGF␤ ligand may abrogate fibrosis in a number of mouse models (24). In addition, activation of TGF␤ signaling pathways constitutively in fibroblasts replicates many of the key histologic and biochemical features of established SSc (25). In this context, it is disappointing that our study did not show any evidence of efficacy for .…”