USP10 Promotes Fibronectin Recycling, Secretion, and Organization.
USP10 Promotes Fibronectin Recycling, Secretion, and Organization.
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DOI：
10.1167/iovs.62.13.15
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发表时间：
2021-10-04
影响因子：
4.4
通讯作者：
Bernstein AM
中科院分区：
文献类型：
--
作者：
Phillips AT;Boumil EF;Castro N;Venkatesan A;Gallo E;Adams JJ;Sidhu SS;Bernstein AM
整合素在肌成纤维细胞病理性粘附、过度收缩和TGFβ活化中起核心作用。以前，我们证明了角膜创伤后，αv整合素通过上调去泛素化酶USP 10而免受细胞内降解，导致细胞表面整合素积累。由于整合素结合并内化细胞外基质（ECM），我们测试了细胞外纤连蛋白（FN）的积累是否可以导致在原代人角膜成纤维细胞（HCF）的整合素和基质再循环的增加。从尸体眼中分离原发性HCF。通过核转染用USP 10 cDNA或对照cDNA转染HCF。用FN ELISA定量内化的FN。通过活细胞共聚焦显微镜（Zeiss LSM 780），用链霉亲和素-488检测包被的细胞外整联蛋白和FN。免疫细胞化学法检测内源性FN胞外区A。通过流式细胞术测定细胞大小和从细胞表面去除FN。USP 10过表达增加α5β1（1.9倍; P < 0.001）和αv（1.7倍; P < 0.05）整联蛋白再循环，同时增加生物素化FN内化（2.1倍; P < 0.05）和4天内再循环（1.7-2.2倍; P < 0.05）。通过α5β1和αv整联蛋白阻断抗体证实了FN再循环对整联蛋白的依赖性，与对照IgG相比，其降低了生物素化FN再循环（分别为62%和84%; P < 0.05）。总之，我们确定细胞外FN由约1/3的再循环生物素化FN和2/3的内源性分泌FN组成。我们的数据表明，减少整合素降解与随后增加的整合素/FN回收伤后可能是一个新的识别机制ECM的特征性积累在角膜瘢痕组织。
Integrins play a central role in myofibroblast pathological adhesion, over-contraction, and TGFβ activation. Previously, we demonstrated that after corneal wounding, αv integrins are protected from intracellular degradation by upregulation of the deubiquitinase USP10, leading to cell-surface integrin accumulation. Because integrins bind to and internalize extracellular matrix (ECM), we tested whether extracellular fibronectin (FN) accumulation can result from an increase in integrin and matrix recycling in primary human corneal fibroblasts (HCFs). Primary HCFs were isolated from cadaver eyes. HCFs were transfected with either USP10 cDNA or control cDNA by nucleofection. Internalized FN was quantified with a FN ELISA. Recycled extracellular integrin and FN were detected with streptavidin-488 by live cell confocal microscopy (Zeiss LSM 780). Endogenous FN extra domain A was detected by immunocytochemistry. Cell size and removal of FN from the cell surface was determined by flow cytometry. USP10 overexpression increased α5β1 (1.9-fold; P < 0.001) and αv (1.7-fold; P < 0.05) integrin recycling, with a concomitant increase in biotinylated FN internalization (2.1-fold; P < 0.05) and recycling over 4 days (1.7–2.2-fold; P < 0.05). The dependence of FN recycling on integrins was demonstrated by α5β1 and αv integrin blocking antibodies, which, compared with control IgG, decreased biotinylated FN recycling (62% and 84%, respectively; P < 0.05). Overall, we established that extracellular FN was composed of approximately 1/3 recycled biotinylated FN and 2/3 endogenously secreted FN. Our data suggest that reduced integrin degradation with a subsequent increase in integrin/FN recycling after wounding may be a newly identified mechanism for the characteristic accumulation of ECM in corneal scar tissue.
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