Identification of Wnt-5a Receptors Important in Diabetic and Non-Diabetic Corneal Epithelial Wound Healing

Investigative Ophthalmology & Visual Science, Journal Year: 2025, Volume and Issue: 66(2), P. 64 - 64

Published: Feb. 25, 2025

Purpose: Persistent epithelial alterations such as delayed wound healing are a key feature of diabetic corneal disease. Previously, we reported that epigenetic changes in the cornea led to suppression Wnt-5a, and addition Wnt-5a accelerated healing. In this study, set determine which Wnt receptor(s) mediated induced stimulation Methods: Human limbal cells (LECs) were isolated from postmortem non-diabetic donor eyes for single-cell RNA sequencing (scRNA-seq) DNA methylation analysis. These analyses validated by qRT-PCR, western blot, or immunostaining tissue sections. Cultured primary LECs transfected with small interfering (siRNA) specific receptors evaluate their role scratch presence absence 200 ng/mL Wnt-5a. Results: Single-cell analysis revealed differential gene expression receptors, ROR2, MCAM, FZD5, FZD6, FZD7. arrays showed hypomethylation ROR2 promoter versus 41.3% (**P < 0.01) resulting increased protein expression. Non-diabetic siRNA knockdown but not FZD7, RYK significantly decreased approximately 50% (*P 0.05) control siRNA. LECs, inhibited 40% FZD5 partially blocked could be restored Conclusions: seems mediate mainly through receptor tyrosine kinase like orphan 2 Frizzled-5 serving possible co-receptor smaller effect.

Language: Английский

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Structure and function of the ROR2 cysteine-rich domain in vertebrate noncanonical WNT5A signaling

eLife, Journal Year: 2024, Volume and Issue: 13

Published: May 23, 2024

The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, metastatic diseases. domain(s) mechanisms required for function, however, remain unclear. We solved crystal structure extracellular cysteine-rich (CRD) Kringle (Kr) domains found that, unlike other CRDs, CRD lacks signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism ligand reception. Functionally, we showed CRD, but not domains, is minimally sufficient promote signaling, mutations in adjacent Kr impair secretion function. Moreover, using function-activating -perturbing antibodies against Frizzled (FZ) family WNT receptors, demonstrate involvement FZ WNT5A-ROR signaling. Thus, acts via its potentiate function super-complex includes transduce signals.

Language: Английский

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Non-canonical WNT5A-ROR signaling: New perspectives on an ancient developmental pathway

Current topics in developmental biology/Current Topics in Developmental Biology, Journal Year: 2023, Volume and Issue: unknown, P. 195 - 227

Published: Jan. 1, 2023

Language: Английский

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Structural and Functional Insights into Dishevelled-Mediated Wnt Signaling

Cells, Journal Year: 2024, Volume and Issue: 13(22), P. 1870 - 1870

Published: Nov. 11, 2024

Dishevelled (DVL) proteins precisely control Wnt signaling pathways with many effectors. While substantial research has advanced our understanding of DVL's role in pathways, key questions regarding its regulatory mechanisms and interactions remain unresolved. Herein, we present the recent advances perspectives on how DVL regulates signaling. The experimentally determined conserved domain structures conjunction AlphaFold-predicted are used to understand regulation. We also summarize various diseases provide insights into further directions for DVL-mediated mechanisms. These findings underscore importance as a pharmaceutical target or biological marker diseases, offering exciting potential future biomedical applications.

Language: Английский

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An efficient method for immortalizing mouse embryonic fibroblasts

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 30, 2024

Abstract Mouse embryonic fibroblasts (MEFs) derived from genetically modified mice are a valuable resource for studying gene function and regulation. The MEF system can also be combined with rescue studies to characterize the of mutant genes/proteins, such as disease-causing variants. However, primary MEFs undergo senescence soon after isolation passaging, making long-term genetic manipulations difficult. Previously described methods immortalization often inefficient or alter physiological properties cells. Here, we describe an optimized protocol immortalizing via CRISPR-mediated deletion Tp53 gene. This method is highly efficient consistently generates immortalized MEFs, iMEFs, within 14 days. Importantly, iMEFs closely resemble parent cell populations, individual cloned expanded subsequent manipulation characterization. We envision that this adopted immortalize other mouse types. Key Features CRISPR-based knockout enables in under 2 weeks. Immortalization requires Neon electroporator another comparable electroporation transfect cells CRISPR constructs.

Language: Английский

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Novel biomarkers and drug correlations of non-canonical WNT signaling in prostate and breast cancer

Discover Oncology, Journal Year: 2024, Volume and Issue: 15(1)

Published: Sept. 30, 2024

Language: Английский

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