Decorin antagonizes corneal fibroblast migration via caveolae-mediated endocytosis of epidermal growth factor receptor.
Decorin antagonizes corneal fibroblast migration via caveolae-mediated endocytosis of epidermal growth factor receptor.
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DOI：
10.1016/j.exer.2019.01.001
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发表时间：
2019-03
影响因子：
3.4
通讯作者：
Chaurasia, Shyam S.
中科院分区：
文献类型：
--
作者：
Mohan, Rajiv R.;Tripathi, Ratnakar;Sharma, Ajay;Sinha, Prashant R.;Giuliano, Elizabeth A.;Hesemann, Nathan P.;Chaurasia, Shyam S.
核心蛋白聚糖（Dcn）是一种富含亮氨酸的小分子蛋白多糖，参与角膜创伤愈合的调节。表皮生长因子受体（EGFR）在角膜成纤维细胞增殖、迁移和细胞外基质（ECM）调节中起着关键作用。本研究旨在探讨Dcn在EGFR内化中调节角膜基质成纤维细胞（CSFs）迁移的机制，这是角膜伤口愈合的关键步骤。人角膜基质成纤维细胞（hCSF）培养物由供体角膜产生。在70%汇合时，将细胞转换到无血清条件48小时，然后在EGF（100 ng/ml）存在或不存在下用核心蛋白聚糖（250 nM）处理不同的时间点（10-60分钟）。对细胞裂解物进行蛋白质组阵列分析筛选42种不同的磷酸化人受体酪氨酸激酶（RTK）、免疫细胞化学和蛋白质印迹以分析EGFR磷酸化。进行划痕试验以评价核心蛋白聚糖对EGF介导的hCSF迁移的影响。在RTK印迹中，Dcn在暴露10分钟内引起EGFR快速磷酸化，从而确定其作为hCSF中EGFR的生物配体的作用。在划痕试验中，长期暴露于Dcn导致EGFR完全消失，并抑制hCSF迁移，表明Dcn与EGFR结合导致EGFR下调。免疫染色研究表明，对hCSF的Dcn处理使Dcn-EGFR复合物内化，当用AG 1478抑制剂处理时，Dcn-EGFR复合物不需要酪氨酸激酶活性，并且使复合物共定位于核周区域。接下来，我们发现Dcn-EGFR复合物不遵循EEA 1抗体所揭示的典型早期内体内化，而是结合针对晚期内体降解的CD 63抗体。我们还发现Dcn通过阻止其与Rab 11结合来调节EGFR再循环，Rab 11是一种用于再循环内体的特异性抗体。此外，用药理学抑制剂甲基-β-环糊精和氯丙嗪预处理并补充Dcn的hCSF表明EGFR通过小窝介导的途径运输。这些结果表明，Dcn作为EGFR的生物配体，并通过EGFR下调来调节hCSF迁移，从而在角膜伤口愈合中发挥重要作用。
Decorin (Dcn), a small leucine-rich proteoglycan, is involved in the regulation of corneal wound healing. Epidermal growth factor receptor (EGFR) plays a critical role in corneal fibroblasts proliferation, migration and extracellular matrix (ECM) modulation upon injury or infection. The present study aimed to investigate the mechanistic role of Dcn in EGFR internalization to the regulation of corneal stromal fibroblasts (CSFs) migration, a key step in the corneal wound healing. Human corneal stromal fibroblasts (hCSF) cultures were generated from donor corneas. At 70% confluence, cells were switched to serum-free conditions for 48 h and then treated with decorin (250 nM) in the presence or absence of EGF (100 ng/ml) for various time points (10–60 min). Cell lysates were subjected to proteome array analysis screening for 42 different phosphorylated human receptor tyrosine kinases (RTKs), immunocytochemistry, and western blots to analyze EGFR phosphorylation. The scratch-wound assay was performed to evaluate the effects of decorin on EGF-mediated hCSF migration. Dcn caused a rapid EGFR phosphorylation within 10 min of exposure in RTK blot defining its role as a biological ligand for EGFR in hCSFs. Prolonged exposure to Dcn caused complete disappearance of EGFR and inhibition of the hCSF migration in the scratch wound assay suggesting Dcn binding to EGFR causes EGFR down-regulation. Immunostaining studies indicated that Dcn-treatment to hCSFs internalizes Dcn-EGFR complex, which does not require tyrosine kinase activity when treated with the AG1478 inhibitor and co-localizes the complex to the perinuclear region. Next, we found that Dcn-EGFR complex does not follow canonical early endosome internalization as revealed by the EEA1 antibody instead binds to the CD63 antibody directed for degradation by the late endosome. We also found that Dcn regulates the EGFR recycling by preventing its binding to Rab11, a specific antibody for recycling endosome. Further, hCSFs-pretreated with pharmacological inhibitors, methyl-β-cyclodextrin and chlorpromazine and supplemented with Dcn suggested EGFR trafficking via the caveolae-mediated pathway. These results suggest that Dcn acts as a biological ligand for EGFR and modulates hCSF migration via EGFR down-regulation, thus playing a vital role in corneal wound healing.
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影响因子：
4.8
作者：
Chaurasia SS;Lim RR;Lakshminarayanan R;Mohan RR
通讯作者：
Mohan RR
影响因子：
17.8
作者：
Ljubimov AV;Saghizadeh M
通讯作者：
Saghizadeh M
影响因子：
1.6
作者：
Donnelly, Kevin S.;Giuliano, Elizabeth A.;Mohan, Rajiv R.
通讯作者：
Mohan, Rajiv R.
影响因子：
4.8
作者：
Iozzo, RV;Moscatello, DK;Eichstetter, I
通讯作者：
Eichstetter, I
影响因子：
64.8
作者：
BORDER, WA;NOBLE, NA;RUOSLAHTI, E
通讯作者：
RUOSLAHTI, E