Antagonizing c-Cbl Enhances EGFR-Dependent Corneal Epithelial Homeostasis

Rush, Jamie S.; Boeving, Michael A.; Berry, William L.; Ceresa, Brian P.

doi:10.1167/iovs.14-14133

Cited by 14 publications

(15 citation statements)

References 39 publications

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“…The effect on EGF-induced cell migration was verified by suppression of HDAC6 activity by trichostatin A or of its expression by siRNA (Wang et al, 2010a). Extending the duration of EGFR activity by inhibiting its negative regulator, E3 ubiquitin ligase (c-CbI), enhanced the rate of restoration of the corneal epithelial layer both in vitro and in vivo (Rush et al, 2014). In vivo studies in rats with type 1 diabetes mellitus (DM1) showed that a significant delay in corneal epithelial wound healing was correlated with altered EGFR signaling pathways through phosphatidylinositol 3-kinase (PI3K)–Akt and ERK, as well as their downstream BAD signaling pathways in migratory epithelium (Xu and Yu, 2011).…”

Section: Corneal Epithelial Wound Healingmentioning

confidence: 99%

Progress in corneal wound healing

Ljubimov

Saghizadeh

2015

Progress in Retinal and Eye Research

642

554

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Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and nanocarriers for corneal drug delivery are discussed. Attention is also paid to problems in wound healing understanding and treatment, such as lack of specific epithelial stem cell markers, reliable identification of stem cells, efficient prevention of haze and stromal scar formation, lack of data on wound regulating microRNAs in keratocytes and endothelial cells, as well as virtual lack of targeted systems for drug and gene delivery to select corneal cells.

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Section: Corneal Epithelial Wound Healingmentioning

confidence: 99%

Progress in corneal wound healing

Ljubimov

Saghizadeh

2015

Progress in Retinal and Eye Research

642

554

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“…The molecular mechanisms that regulate the magnitude, duration, and specificity of EGFR⅐ effector communication are less understood, but these factors determine receptor specific cell biology such as cell proliferation, migration, and differentiation. Further, alterations in regulatory mechanisms are associated with cell transformation (6) and have been targeted to promote receptor activity to enhance wound healing (7).…”

mentioning

confidence: 99%

Proteomics reveals novel protein associations with early endosomes in an epidermal growth factor–dependent manner

Gosney¹,

Wilkey

Merchant

et al. 2018

Journal of Biological Chemistry

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The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is an integral component of proliferative signaling. EGFRs on the cell surface become activated upon EGF binding and have an increased rate of endocytosis. Once in the cytoplasm, the EGF·EGFR complex is trafficked to the lysosome for degradation, and signaling is terminated. During trafficking, the EGFR kinase domain remains active, and the internalized EGFR can continue signaling to downstream effectors. Although effector activity varies based on the EGFR's endocytic location, it is not clear how this occurs. In an effort to identify proteins that uniquely associate with the internalized, liganded EGFR in the early endosome, we developed an early endosome isolation strategy to analyze their protein composition. Post-nuclear supernatant from HeLa cells stimulated with and without EGF were separated on an isotonic 17% Percoll gradient. The gradient was fractionated, and early endosomal fractions were pooled and immunoisolated with an EEA1 mAb. The isolated endosomes were validated by immunoblot using antibodies against organelle-specific marker proteins and transmission EM. These early endosomes were also subjected to LC-MS/MS for proteomic analysis. Five proteins were detected in endosomes in a ligand-dependent manner: EGFR, RUFY1, STOML2, PTPN23, and CCDC51. Knockdown of RUFY1 or PTPN23 by RNAi indicated that both proteins play a role in EGFR trafficking. These experiments indicate that endocytic trafficking of activated EGFR changes the protein composition, membrane trafficking, and signaling potential of the early endosome.

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“…Due to technical limitations, previous in vivo studies have only provided indirect evidence that antagonizing ubiquitylation will promote corneal epithelial wound healing and homeostasis 39 . However, this study provides direct evidence that the inhibition of c‐Cbl can prevent EGFR desensitization as indicated by the slowed rate of EGFR degradation and sustained receptor phosphorylation.…”

Section: Discussionmentioning

confidence: 79%

“…For these compounds to be successful therapeutic agents, it is important to understand the substrate specificity, what stages of membrane trafficking are impacted, and how cell‐biology is affected. Although the inhibition of EGFR ubiquitylation in corneal epithelial cells has been shown to enhance tissue regeneration, 39 this study in corneal epithelial cells is the first to define the contribution of c‐Cbl in controlling EGFR endocytic trafficking and signaling. We are now positioned to study the biological consequences using in vitro (cell proliferation, migration, viability) and in vivo (corneal epithelial development and wound healing).…”

Section: Discussionmentioning

confidence: 97%

Knockout of c‐Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation

Crotchett

Ceresa

2021

Pharmacology Res & Perspec

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Epidermal growth factor receptor (EGFR) activity is necessary and sufficient for corneal epithelial homeostasis. However, the addition of exogenous Epidermal Growth Factor (EGF) does not reliably restore the corneal epithelium when wounded. This is likely due to high levels of endogenous EGF in tear fluid as well as desensitization of the EGFR following ligand stimulation. We hypothesize that preventing receptor downregulation is an alternative mechanism to enhance EGFR signaling and promote the restoration of compromised corneas. Ligand‐dependent EGFR ubiquitylation is associated with the targeted degradation of the receptor. In this manuscript, we determine whether knockout of c‐Cbl, an E3 ubiquitin ligase that ubiquitylates the EGFR, is sufficient to prolong EGFR phosphorylation and sustain signaling. Using CRISPR/Cas9 gene editing, we generated immortalized human corneal epithelial (hTCEpi) cells lacking c‐Cbl. Knockout (KO) cells expressed the other E3 ligases at the same levels as the control cells, indicating other E3 ligases were not up‐regulated. As compared to the control cells, EGF‐stimulated EGFR ubiquitylation was reduced in KO cells, but not completely abolished. Similarly, EGF:EGFR trafficking was slowed, with a 35% decrease in the rate of endocytosis and a twofold increase in the receptor half‐life. This resulted in a twofold increase in the magnitude of EGFR phosphorylation, with no change in duration. Conversely, Mitogen Activating Protein Kinase (MAPK) phosphorylation did not increase in magnitude but was sustained for 2–3 h as compared to control cells. We propose antagonizing c‐Cbl will partially alter receptor ubiquitylation and endocytic trafficking but this is sufficient to enhance downstream signaling.

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Antagonizing c-Cbl Enhances EGFR-Dependent Corneal Epithelial Homeostasis

Abstract: 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.

Cited by 14 publications

References 39 publications

Progress in corneal wound healing

Progress in corneal wound healing

Proteomics reveals novel protein associations with early endosomes in an epidermal growth factor–dependent manner

Knockout of c‐Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation

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