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-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.

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Distinct Endocytic Responses of Heteromeric and Homomeric Transforming Growth Factor β Receptors

Molecular Biology of the Cell ◽

10.1091/mbc.8.11.2133 ◽

1997 ◽

Vol 8 (11) ◽

pp. 2133-2143 ◽

Cited By ~ 49

Author(s):

Robert A. Anders ◽

Sandra L. Arline ◽

Jules J.E. Doré ◽

Edward B. Leof

Keyword(s):

Growth Factor ◽

Ligand Binding ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Cell Surface Receptor ◽

Type I ◽

Type Ii ◽

Down Regulation ◽

Tgfβ Receptors ◽

Tgfβ Receptor

Transforming growth factor β (TGFβ) family ligands initiate a cascade of events capable of modulating cellular growth and differentiation. The receptors responsible for transducing these cellular signals are referred to as the type I and type II TGFβ receptors. Ligand binding to the type II receptor results in the transphosphorylation and activation of the type I receptor. This heteromeric complex then propagates the signal(s) to downstream effectors. There is presently little data concerning the fate of TGFβ receptors after ligand binding, with conflicting reports indicating no change or decreasing cell surface receptor numbers. To address the fate of ligand-activated receptors, we have used our previously characterized chimeric receptors consisting of the ligand binding domain from the granulocyte/macrophage colony-stimulating factor α or β receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGFβ receptor. This system not only provides the necessary sensitivity and specificity to address these types of questions but also permits the differentiation of endocytic responses to either homomeric or heteromeric intracellular TGFβ receptor oligomerization. Data are presented that show, within minutes of ligand binding, chimeric TGFβ receptors are internalized. However, although all the chimeric receptor combinations show similar internalization rates, receptor down-regulation occurs only after activation of heteromeric TGFβ receptors. These results indicate that effective receptor down-regulation requires cross-talk between the type I and type II TGFβ receptors and that TGFβ receptor heteromers and homomers show distinct trafficking behavior.

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Integrin β1Signaling Is Necessary for Transforming Growth Factor-β Activation of p38MAPK and Epithelial Plasticity

Journal of Biological Chemistry ◽

10.1074/jbc.m106176200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 46707-46713 ◽

Cited By ~ 264

Author(s):

Neil A. Bhowmick ◽

Roy Zent ◽

Mayshan Ghiassi ◽

Maureen McDonnell ◽

Harold L. Moses

Keyword(s):

Extracellular Matrix ◽

Growth Factor ◽

Transcriptional Activation ◽

Intracellular Signaling ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Dominant Negative ◽

Receptor Expression ◽

Type I ◽

Type Ii

Transforming growth factor-β (TGF-β) can induce epithelial to mesenchymal transdifferentiation (EMT) in mammary epithelial cells. TGF-β-meditated EMT involves the stimulation of a number of signaling pathways by the sequential binding of the type II and type I serine/threonine kinase receptors, respectively. Integrins comprise a family of heterodimeric extracellular matrix receptors that mediate cell adhesion and intracellular signaling, hence making them crucial for EMT progression. In light of substantial evidence indicating TGF-β regulation of various β1integrins and their extracellular matrix ligands, we examined the cross-talk between the TGF-β and integrin signal transduction pathways. Using an inducible system for the expression of a cytoplasmically truncated dominant negative TGF-β type II receptor, we blocked TGF-β-mediated growth inhibition, transcriptional activation, and EMT progression. Dominant negative TGF-β type II receptor expression inhibited TGF-β signaling to the SMAD and AKT pathways, but did not block TGF-β-mediated p38MAPK activation. Interestingly, blocking integrin β1function inhibited TGF-β-mediated p38MAPK activation and EMT progression. Limiting p38MAPK activity through the expression of a dominant negative-p38MAPK also blocked TGF-β-mediated EMT. In summary, TGF-β-mediated p38MAPK activation is dependent on functional integrin β1, and p38MAPK activity is required but is not sufficient to induce EMT.

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Expression of Transforming Growth Factor-β Type I and Type II Receptors Is Altered in Rat Lungs Undergoing Bleomycin-Induced Pulmonary Fibrosis

Experimental and Molecular Pathology ◽

10.1006/exmp.2000.2319 ◽

2000 ◽

Vol 69 (2) ◽

pp. 67-78 ◽

Cited By ~ 16

Author(s):

Yun Zhao ◽

Deepti U. Shah

Keyword(s):

Growth Factor ◽

Pulmonary Fibrosis ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Type I ◽

Type Ii ◽

Rat Lungs

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Protein tyrosine phosphatase-α amplifies transforming growth factor-β-dependent profibrotic signaling in lung fibroblasts

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00235.2019 ◽

2020 ◽

Vol 319 (2) ◽

pp. L294-L311 ◽

Cited By ~ 1

Author(s):

Yael Aschner ◽

Meghan Nelson ◽

Matthew Brenner ◽

Helen Roybal ◽

Keriann Beke ◽

...

Keyword(s):

Growth Factor ◽

Epithelial Cells ◽

Pulmonary Fibrosis ◽

Protein Tyrosine Phosphatase ◽

Transforming Growth Factor ◽

Tyrosine Phosphatase ◽

Transforming Growth Factor Β ◽

Lung Fibroblasts ◽

Type Ii ◽

Wild Type

Idiopathic pulmonary fibrosis (IPF) is a progressive, often fatal, fibrosing lung disease for which treatment remains suboptimal. Fibrogenic cytokines, including transforming growth factor-β (TGF-β), are central to its pathogenesis. Protein tyrosine phosphatase-α (PTPα) has emerged as a key regulator of fibrogenic signaling in fibroblasts. We have reported that mice globally deficient in PTPα ( Ptpra−/−) were protected from experimental pulmonary fibrosis, in part via alterations in TGF-β signaling. The goal of this study was to determine the lung cell types and mechanisms by which PTPα controls fibrogenic pathways and whether these pathways are relevant to human disease. Immunohistochemical analysis of lungs from patients with IPF revealed that PTPα was highly expressed by mesenchymal cells in fibroblastic foci and by airway and alveolar epithelial cells. To determine whether PTPα promotes profibrotic signaling pathways in lung fibroblasts and/or epithelial cells, we generated mice with conditional (floxed) Ptpra alleles ( Ptpraf/f). These mice were crossed with Dermo1-Cre or with Sftpc-CreERT2 mice to delete Ptpra in mesenchymal cells and alveolar type II cells, respectively. Dermo1-Cre/ Ptpraf/f mice were protected from bleomycin-induced pulmonary fibrosis, whereas Sftpc-CreERT2 /Ptpraf/f mice developed pulmonary fibrosis equivalent to controls. Both canonical and noncanonical TGF-β signaling and downstream TGF-β-induced fibrogenic responses were attenuated in isolated Ptpra−/− compared with wild-type fibroblasts. Furthermore, TGF-β-induced tyrosine phosphorylation of TGF-β type II receptor and of PTPα were attenuated in Ptpra−/− compared with wild-type fibroblasts. The phenotype of cells genetically deficient in PTPα was recapitulated with the use of a Src inhibitor. These findings suggest that PTPα amplifies profibrotic TGF-β-dependent pathway signaling in lung fibroblasts.

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