Rush Jamie S, Boeving Michael A, Berry William L, Ceresa Brian P
Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky, United States.
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma, United States.
Invest Ophthalmol Vis Sci. 2014 Jul 1;55(8):4691-9. doi: 10.1167/iovs.14-14133.
In many cell types, the E3 ubiquitin ligase, c-Cbl, induces ligand-dependent ubiquitylation of the epidermal growth factor receptor (EGFR) and targets the receptor for lysosomal degradation. The goal of this study was to determine whether c-Cbl is a negative regulator of EGFR in the corneal epithelium and if it can be inhibited to promote corneal epithelial homeostasis.
Expression and activity of c-Cbl were blocked in immortalized human corneal epithelial cells (hTCEpi) using RNAi and pharmacological agents ([4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine] or PP1). Following c-Cbl inhibition, cells were assessed for ligand-dependent receptor ubiquitylation, receptor phosphorylation, and in vitro wound healing. Subsequent experiments used PP1 in hTCEpi cells and monitored in vivo murine corneal epithelial wound healing.
Knockdown and inhibition of c-Cbl decreased ligand-dependent ubiquitylation of the EGFR and prolonged receptor activity as measured by tyrosine phosphorylation. Further, these treatments also increased the extent of ligand-dependent corneal epithelial wound healing in vitro and in vivo.
Manipulating the duration of EGFR activity can enhance the rate of restoration of the corneal epithelial layer. Based on our findings, c-Cbl is a new therapeutic target to enhance EGFR-mediated corneal epithelial homeostasis that bypasses the limitations of previous approaches.
在许多细胞类型中，E3泛素连接酶c-Cbl可诱导表皮生长因子受体（EGFR）发生配体依赖性泛素化，并将该受体靶向溶酶体降解。本研究的目的是确定c-Cbl是否为角膜上皮中EGFR的负调节因子，以及它是否可以被抑制以促进角膜上皮稳态。
使用RNA干扰和药物制剂（[4-氨基-5-（4-甲基苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶]或PP1）在永生化人角膜上皮细胞（hTCEpi）中阻断c-Cbl的表达和活性。在抑制c-Cbl后，评估细胞的配体依赖性受体泛素化、受体磷酸化和体外伤口愈合情况。后续实验在hTCEpi细胞中使用PP1，并监测小鼠角膜上皮体内伤口愈合情况。
通过酪氨酸磷酸化检测发现，敲低和抑制c-Cbl可降低EGFR的配体依赖性泛素化，并延长受体活性。此外，这些处理还增加了体外和体内配体依赖性角膜上皮伤口愈合的程度。
调控EGFR活性的持续时间可提高角膜上皮层的修复速率。基于我们的研究结果，c-Cbl是增强EGFR介导的角膜上皮稳态的新治疗靶点，可绕过先前方法的局限性。
