纤连蛋白/Lumikine 通过非经典 Smad 独立的 TGFβ/TBRs 信号上调 EGFR 配体表达促进角膜上皮清创愈合。

Lumican/Lumikine Promotes Healing of Corneal Epithelium Debridement by Upregulation of EGFR Ligand Expression via Noncanonical Smad-Independent TGFβ/TBRs Signaling.

机构信息

Department of Ophthalmology, University of Cincinnati, Cincinnati, OH 45267, USA.

Department of Environmental Health, University of Cincinnati, Cincinnati, OH 45267, USA.

出版信息

Cells. 2024 Sep 24;13(19):1599. doi: 10.3390/cells13191599.

DOI:10.3390/cells13191599

PMID:39404363

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11475839/

Abstract

The synthetic peptide of lumican C-terminal 13 amino acids with the cysteine replaced by an alanine, hereafter referred to as lumikine (LumC13: YEALRVANEVTLN), binds to TGFβ type I receptor/activin-like kinase5 (TBR1/ALK5) in the activated TGFβ receptor complex to promote corneal epithelial wound healing. The present study aimed to identify the minimum essential amino acid epitope necessary to exert the effects of lumikine via ALK5 and to determine the role of the Y (tyrosine) residue for promoting corneal epithelium wound healing. This study also aimed to determine the signaling pathway(s) triggered by lumican-ALK5 binding. For such, adult knockout () mice (~8-12 weeks old) were subjected to corneal epithelium debridement using an Agerbrush. The injured eyes were treated with 10 µL eye drops containing 0.3 µM synthetic peptides designed based on the C-terminal region of lumican for 5-6 h. To unveil the downstream signaling pathways involved, inhibitors of the Alk5 and EGFR signaling pathways were co-administered or not. Corneas isolated from the experimental mice were subjected to whole-mount staining and imaged under a ZEISS Observer to determine the distance of epithelium migration. The expression of EGFR ligands was determined following a scratch assay with HTCE (human telomerase-immortalized cornea epithelial cells) in the presence or not of lumikine. Results indicated that shorter LumC-terminal peptides containing EVTLN and substitution of Y with F in lumikine abolishes its capability to promote epithelium migration indicating that Y and EVTLN are essential but insufficient for Lum activity. Lumikine activity is blocked by inhibitors of Alk5, EGFR, and MAPK signaling pathways, while EGF activity is only suppressed by EGFR and MAPK inhibitors. qRT-PCR of scratched HTCE cells cultures treated with lumikine showed upregulated expression of several EGFR ligands including epiregulin (EREG). Treatment with anti-EREG antibodies abolished the effects of lumikine in corneal epithelium debridement healing. The observations suggest that Lum/lumikine binds Alk5 and promotes the noncanonical Smad-independent TGFβ/TBRs signaling pathways during the healing of corneal epithelium debridement.

摘要

具有半胱氨酸被丙氨酸取代的亮氨酸聚糖 C 端 13 个氨基酸的合成肽，以下称为亮氨酸聚糖（LumC13：YEALRVANEVTLN），与激活的 TGFβ 受体复合物中的 TGFβ 型 I 受体/激活素样激酶 5（TBR1/ALK5）结合，以促进角膜上皮伤口愈合。本研究旨在鉴定通过 ALK5 发挥亮氨酸聚糖作用所需的最小必需氨基酸表位，并确定促进角膜上皮伤口愈合的 Y（酪氨酸）残基的作用。本研究还旨在确定亮氨酸聚糖-ALK5 结合触发的信号通路。为此，对成年基因敲除（）小鼠（~8-12 周龄）使用 Agerbrush 进行角膜上皮清创。受伤的眼睛用含有 0.3 µM 基于亮氨酸 C 端区域设计的合成肽的 10 µL 滴眼液处理 5-6 小时。为了揭示所涉及的下游信号通路，共施用或不施用 Alk5 和 EGFR 信号通路抑制剂。从实验小鼠中分离出角膜，进行全角膜染色，并在 ZEISS Observer 下成像，以确定上皮迁移的距离。在存在或不存在亮氨酸聚糖的情况下，用人端粒酶永生化角膜上皮细胞（HTCE）进行划痕试验，确定 EGFR 配体的表达。结果表明，包含 EVTLN 的较短亮氨酸聚糖 C 端肽和亮氨酸聚糖中 Y 被 F 取代会使其丧失促进上皮迁移的能力，表明 Y 和 EVTLN 是亮氨酸活性所必需的，但不足以发挥亮氨酸的作用。亮氨酸聚糖活性被 Alk5、EGFR 和 MAPK 信号通路抑制剂阻断，而 EGF 活性仅被 EGFR 和 MAPK 抑制剂抑制。用亮氨酸聚糖处理划痕 HTCE 细胞培养物的 qRT-PCR 显示几种 EGFR 配体（包括表皮调节素（EREG））的表达上调。用抗 EREG 抗体处理可消除亮氨酸聚糖对角膜上皮清创愈合的作用。这些观察结果表明，亮氨酸聚糖在角膜上皮清创愈合过程中与 Alk5 结合并促进非经典 Smad 非依赖性 TGFβ/TBRs 信号通路。