TGF-β triggers rapid fibrillogenesis via a novel TβRII-dependent fibronectin-trafficking mechanism

Varadaraj, Archana; Jenkins, Laura M.; Singh, Priyanka; Chanda, Anindya; Snider, J. Caleb; Lee, N. Y.; Amsalem-Zafran, Ayelet R.; Ehrlich, Marcelo; Henis, Yoav I.; Mythreye, Karthikeyan

doi:10.1091/mbc.e16-08-0601

Cited by 28 publications

(36 citation statements)

References 91 publications

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“…Alternatively, if the total FN protein increases with treatment, then the increase in pellet FN may be due to an increase in the total protein and not specifically a difference in pellet FN fraction between treatments. Our study (refer Figure 1C in Varadaraj et al , 2017) showed that TGFβ1 and TGFβ2 increases FN fibril fraction, we measured total FN levels in parallel experiments to fractionation experiments to confirm that FN fibril fraction was increasing with treatments.…”

Section: Discussionmentioning

confidence: 61%

“…To represent the analyzed data as a figure in a publication, convert the ratios to fold difference/relative difference, create a bar graph of the fold difference between UN and treated samples, the UN sample being ‘1’, calculate SEM and perform appropriate statistics (Refer Figure 4E in Varadaraj et al , 2017). …”

Section: Discussionmentioning

confidence: 99%

“…Since we were interested to investigate whether TGFβ1 and TGFβ2 increases FN fibrillogenesis via recycling, we had a control condition with only growth medium, growth medium containing 10 ng/ml TGFβ1 and growth medium containing 10 ng/ml TGFβ2 (Varadaraj et al , 2017). …”

Section: Methodsmentioning

confidence: 99%

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Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells

2018

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Fibronectin (FN) is an extracellular matrix protein that is secreted by many cell types and binds predominantly to the cell surface receptor Integrin α5β1. Integrin α5β1 binding initiates the step-wise assembly of FN into fibrils, a process called fibrillogenesis. We and several others have demonstrated critical effects of fibrillogenesis on cell migration and metastasis. While immunostaining and microscopy methods help visualize FN incorporation into fibrils, with each fibril being at least 3 μm in length, the first study that developed a method to biochemically fractionate FN to quantify fibril incorporated FN was published by Jean Schwarzbauer’s group in 1996. Our protocol was adapted from the original publication, and has been tested on multiple cell types including as shown here in MCF10A mammary epithelial and Caki-1 renal cancer epithelial cells. Using two detergent extractions, cellular FN is separated into detergent insoluble or fibril incorporated FN and soluble FN or unincorporated fractions. To determine whether fibrillogenesis utilizes a recycled pool of FN, we have used a Biotin labeled FN (FN-Biotin) recycling assay, that has been modified from a previous study. Using a combination of the recycling assay and deoxycholate fractionation methods, one can quantitatively demonstrate the extent of fibrillogenesis in cells under different experimental conditions and determine the source of FN for fibrillogenesis.

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Section: Discussionmentioning

confidence: 61%

Section: Discussionmentioning

confidence: 99%

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Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells

2018

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“…Emerging evidence suggests that TGFβ, through increased fibronectin trafficking, is a direct inducer of the fibrillogenesis required for TGFβ-induced cell migration. In response to TGFβ, internalized fibronectin is not degraded by lysosome but instead is recycled for fibrillogenesis, a process dependent of the TGFR2 [34,81].…”

Section: Tgfβ1 and Agap2 In Hsc Proliferation And Migrationmentioning

confidence: 99%

AGAP2: Modulating TGFβ1-Signaling in the Regulation of Liver Fibrosis

Navarro‐Corcuera

Ansorena

Montiel-Duarte

et al. 2020

IJMS

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AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat and PH domain 2) isoform 2 is a protein that belongs to the Arf GAP (GTPase activating protein) protein family. These proteins act as GTPase switches for Arfs, which are Ras superfamily members, being therefore involved in signaling regulation. Arf GAP proteins have been shown to participate in several cellular functions including membrane trafficking and actin cytoskeleton remodeling. AGAP2 is a multi-tasking Arf GAP that also presents GTPase activity and is involved in several signaling pathways related with apoptosis, cell survival, migration, and receptor trafficking. The increase of AGAP2 levels is associated with pathologies as cancer and fibrosis. Transforming growth factor beta-1 (TGF-β1) is the most potent pro-fibrotic cytokine identified to date, currently accepted as the principal mediator of the fibrotic response in liver, lung, and kidney. Recent literature has described that the expression of AGAP2 modulates some of the pro-fibrotic effects described for TGF-β1 in the liver. The present review is focused on the interrelated molecular effects between AGAP2 and TGFβ1 expression, presenting AGAP2 as a new player in the signaling of this pro-fibrotic cytokine, thereby contributing to the progression of hepatic fibrosis.

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“…Accumulating evidence indicates the presence of crosstalk between growth factors and adhesive signaling pathways. Firstly, TGF-β may regulate integrin signaling through physical interaction between TGF-β receptors (TGF-βR) and integrins (6), stable interactions between the Type II TGF-β receptor (TβRII) and α5β1 integrin have also been described in rapid fibrillogenesis (7), and the association of integrin αvβ3 with TGF-βR has been suggested to enhance TGF-β-induced invasion of breast cancer cells and contribute to abnormal wound healing in lung fibroblasts (8,9). Secondly, TGF-β may upregulate integrin expression: TGF-β signaling increased α5β1 integrin expression in keratinocytes during wound healing and promoted carcinoma cell migration (10).…”

Section: The Role Of Focal Adhesion Kinase In Transforming Growth Facmentioning

confidence: 99%

The role of focal adhesion kinase in transforming growth factor-β2 induced migration of human lens epithelial cells

Liu

et al. 2018

Int J Mol Med

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The migration of lens epithelial cells towards the posterior capsule is a key event in the development of posterior capsule opacification (PCO). Accumulating evidence has described crosstalk between growth factors and adhesive signaling pathways in wound healing and cell migration. The aim of the present study was to elucidate an aberrant transforming growth factor (TGF)-β2 signaling pathway that regulated the migration of lens epithelial cells in the pathological context of PCO. The expression of fibronectin, focal adhesion kinase (FAK) and phosphorylated (p)-FAK in HLE-B3 cells following TGF-β2 treatment was determined by western blot analysis and the expression of integrin α5β1 was detected by flow cytometry. Cell migration capacity was measured by wound healing and Transwell assays in the presence of 1,2,4,5-tetraaminobenzene tetrahydrochloride, a selective FAK inhibitor, fibronectin small interfering RNA interference, arginylglycylaspartic acid peptides or α5β1-integrin neutralizing antibodies. The 1,2,4,5-tetraaminobenzene tetrahydrochloride was administered daily to 16 rabbits following cataract surgery. Fibronectin and TGF-β expression were increased in the PCO group, demonstrated by immunofluorescence assays. PCO grading was conducted by slit-lamp biomicroscopy and evaluation of posterior capsule opacification software. It was observed that TGF-β2 promoted HLE-B3 cell migration and upregulated fibronectin expression, which was followed by an increased phosphorylation of FAK. In addition, TGF-β2 treatment and fibronectin surface coating significantly increased cell migration and FAK activation, which was inhibited by disrupting fibronectin-integrin α5β1 interaction with the arginylglycylaspartic acid peptide, α5β1-integrin neutralizing antibody or fibronectin depletion. Finally, suppression of FAK signaling by its inhibitor significantly decreased cell migration in vitro and attenuated PCO development in vivo. In summary, TGF-β2 was indicated to promote the migration of lens epithelial cells through the TGF-β2/fibronectin/integrin/FAK axis. Inhibition of FAK activity decreased TGF-β2-mediated cell migration in vitro and improved the symptoms of PCO in a rabbit model.

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TGF-β triggers rapid fibrillogenesis via a novel TβRII-dependent fibronectin-trafficking mechanism

Cited by 28 publications

References 91 publications

Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells

Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells

AGAP2: Modulating TGFβ1-Signaling in the Regulation of Liver Fibrosis

The role of focal adhesion kinase in transforming growth factor-β2 induced migration of human lens epithelial cells

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