72 MAST CELLS AND EOSINOPHILS STIMULATE MYOFIBROBLAST DIFFERENTIATION BY SIGNALING THROUGH THE TRANSFORMING GROWTH FACTOR b TYPE I (ALK5) RECEPTOR.

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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Faculty Opinions recommendation of Lactic acid is elevated in idiopathic pulmonary fibrosis and induces myofibroblast differentiation via pH-dependent activation of transforming growth factor-β.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717967484.793467143 ◽

2012 ◽

Author(s):

Moisés Selman ◽

Carolina Garcia de Alba

Keyword(s):

Lactic Acid ◽

Growth Factor ◽

Idiopathic Pulmonary Fibrosis ◽

Pulmonary Fibrosis ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Myofibroblast Differentiation ◽

Ph Dependent

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Cellular distribution of type I and type II receptors for transforming growth factor-beta.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)67611-2 ◽

1986 ◽

Vol 261 (21) ◽

pp. 9972-9978 ◽

Cited By ~ 7

Author(s):

S Cheifetz ◽

B Like ◽

J Massagué

Keyword(s):

Growth Factor ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Cellular Distribution ◽

Type I ◽

Type Ii ◽

Growth Factor Beta

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Utilizing the ABC Transporter for Growth Factor Production by fleQ Deletion Mutant of Pseudomonas fluorescens

Biomedicines ◽

10.3390/biomedicines9060679 ◽

2021 ◽

Vol 9 (6) ◽

pp. 679

Author(s):

Benedict-Uy Fabia ◽

Joshua Bingwa ◽

Jiyeon Park ◽

Nguyen-Mihn Hieu ◽

Jung-Hoon Ahn

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Pseudomonas Fluorescens ◽

Abc Transporter ◽

Transforming Growth Factor Beta ◽

Deletion Mutant ◽

Transforming Growth Factor ◽

Gene Knockout ◽

Type I ◽

Double Deletion

Pseudomonas fluorescens, a gram-negative bacterium, has been proven to be a capable protein manufacturing factory (PMF). Utilizing its ATP-binding cassette (ABC) transporter, a type I secretion system, P. fluorescens has successfully produced recombinant proteins. However, besides the target proteins, P. fluorescens also secretes unnecessary background proteins that complicate protein purification and other downstream processes. One of the background proteins produced in large amounts is FliC, a flagellin protein. In this study, the master regulator of flagella gene expression, fleQ, was deleted from P. fluorescens Δtp, a lipase and protease double-deletion mutant, via targeted gene knockout. FleQ directs flagella synthesis, so the new strain, P. fluorescens ΔfleQ, does not produce flagella-related proteins. This not only simplifies purification but also makes P. fluorescens ΔfleQ an eco-friendly expression host because it will not survive outside a controlled environment. Six recombinant growth factors, namely, insulin-like growth factors I and II, beta-nerve growth factor, fibroblast growth factor 1, transforming growth factor beta, and tumor necrosis factor beta, prepared using our supercharging method, were successfully secreted by P. fluorescens ΔfleQ. Our findings demonstrate the potential of P. fluorescens ΔfleQ, combined with our supercharging process, as a PMF.

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Mechanisms of Transforming Growth Factor-β Receptor Endocytosis and Intracellular Sorting Differ between Fibroblasts and Epithelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.12.3.675 ◽

2001 ◽

Vol 12 (3) ◽

pp. 675-684 ◽

Cited By ~ 38

Author(s):

Jules J.E. Doré ◽

Diying Yao ◽

Maryanne Edens ◽

Nandor Garamszegi ◽

Elizabeth L. Sholl ◽

...

Keyword(s):

Growth Factor ◽

Epithelial Cells ◽

Ligand Binding ◽

Transforming Growth Factor ◽

Point Mutations ◽

Type I ◽

Receptor Complex ◽

Type Ii ◽

Down Regulation ◽

Β Receptor

Transforming growth factor-βs (TGF-β) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-β type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-β receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling.

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