Quantification of Extracellular Matrix Fiber Systems Related to ADAMTS Proteins

Zhang, Rong-Mo; Kumra, Heena; Reinhardt, Dieter P.

doi:10.1007/978-1-4939-9698-8_19

Cited by 12 publications

(15 citation statements)

References 21 publications

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“…S5 a , the area of extracellular FN stained was quantified and normalized to the total area of each image and cell numbers. The amount of FN branches was quantified as described in Zhang et al (2020) . Briefly, pictures were set to binary 8 bits, the same threshold was set for all of them, noise was removed with a despeckle function and Skeleton plugin, and analysis was applied by Fiji ImageJ.…”

Section: Methodsmentioning

confidence: 99%

αv-Class integrin binding to fibronectin is solely mediated by RGD and unaffected by an RGE mutation

Benito-Jardón

Strohmeyer

Ortega-Sanchís

et al. 2020

Journal of Cell Biology

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Fibronectin (FN) is an essential glycoprotein of the extracellular matrix; binds integrins, syndecans, collagens, and growth factors; and is assembled by cells into complex fibrillar networks. The RGD motif in FN facilitates cell binding and fibrillogenesis through binding to α5β1 and αv-class integrins. However, whether RGD is the sole binding site for αv-class integrins is unclear. Most notably, substituting aspartate with glutamate (RGE) was shown to eliminate integrin binding in vitro, while mouse genetics revealed that FNRGE preserves αv-class integrin binding and fibrillogenesis. To address this conflict, we employed single-cell force spectroscopy, engineered cells, and RGD motif–deficient mice (Fn1ΔRGD/ΔRGD) to search for additional αv-class integrin–binding sites. Our results demonstrate that α5β1 and αv-class integrins solely recognize the FN-RGD motif and that αv-class, but not α5β1, integrins retain FN-RGE binding. Furthermore, Fn1ΔRGD/ΔRGD tissues and cells assemble abnormal and dysfunctional FNΔRGD fibrils in a syndecan-dependent manner. Our data highlight the central role of FN-RGD and the functionality of FN-RGE for αv-class integrins.

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Section: Methodsmentioning

confidence: 99%

αv-Class integrin binding to fibronectin is solely mediated by RGD and unaffected by an RGE mutation

Benito-Jardón

Strohmeyer

Ortega-Sanchís

et al. 2020

Journal of Cell Biology

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“…10 μM of SCH772984 or DMSO control (1:1000 v/v) was added at cell seeding. Nuclei were counterstained with DAPI (blue) (B) Quantification of Ki67‐positive cells shown in (A) using ImageJ, as described in 45 . Y ‐axis values indicate the percentage of Ki67‐positive cells ± standard deviation.…”

Section: Resultsmentioning

confidence: 99%

“…B and D, Quantification of Ki67-positive cells using ImageJ. 45 Y-axis values show the percentage of Ki67-positive cells ± standard deviation. Twosample t test was performed to analyze statistical significance (*P value < .05)…”

Section: Mir-1208 Negatively Regulates Fibroblast Proliferationmentioning

confidence: 99%

“…Original images in the TIFF format were exported for cell counting using ImageJ, 44 as previously described. 45…”

Section: Indirect Immunofluorescencementioning

confidence: 99%

“…Nuclei were counterstained with DAPI (blue) (B) Quantification of Ki67-positive cells shown in (A) using ImageJ, as described in. 45 Y-axis values indicate the percentage of Ki67-positive cells ± standard deviation. Statistical significance was analyzed with the two-sample t test (*P value < .05).…”

Section: Inhibitors Of Jnk and Src Kinase Stimulate Mir-1208 Expressionmentioning

confidence: 99%

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The fibrillin‐1 RGD motif posttranscriptionally regulates ERK1/2 signaling and fibroblast proliferation via miR‐1208

et al. 2021

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Fibrillin-1 is an extracellular matrix protein which contains one conserved RGD integrin-binding motif. It constitutes the backbone of microfibrils in many tissues, and mutations in fibrillin-1 cause various connective tissue disorders. Although it is well established that fibrillin-1 interacts with several RGD-dependent integrins, very little is known about the associated intracellular signaling pathways. Recent published evidence identified a subset of miRNAs regulated by fibrillin-1 RGD-cell adhesion, with miR-1208 among the most downregulated. The present study shows that the downregulated miR-1208 controls fibroblast proliferation. Inhibitor experiments revealed that fibrillin-1 RGD suppressed miR-1208 expression via c-Src kinase and the downstream JNK signaling. Bioinformatic prediction and experimental target sequence validation demonstrated four miR-1208 binding sites on the ERK2 mRNA and one on the MEK1 mRNA. ERK2 and MEK1 are critical proliferationpromoting kinases. Decreased miR-1208 levels elevated the total and phosphorylated ERK1/2 and MEK1/2 protein levels and the phosphorylated to total ERK1/2 ratio.Together, the data demonstrate a novel outside-in signaling mechanism explaining how fibrillin-1 RGD-cell binding regulates fibroblast proliferation.

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Fibrillin-1-regulated miR-122 has a critical role in thoracic aortic aneurysm formation

Zhang

Tiedemann

Muthu

et al. 2022

Cell. Mol. Life Sci.

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Quantification of Extracellular Matrix Fiber Systems Related to ADAMTS Proteins

Cited by 12 publications

References 21 publications

αv-Class integrin binding to fibronectin is solely mediated by RGD and unaffected by an RGE mutation

αv-Class integrin binding to fibronectin is solely mediated by RGD and unaffected by an RGE mutation

The fibrillin‐1 RGD motif posttranscriptionally regulates ERK1/2 signaling and fibroblast proliferation via miR‐1208

Fibrillin-1-regulated miR-122 has a critical role in thoracic aortic aneurysm formation

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