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Elevating EGFR-MAPK program by a nonconventional Cdc42 enhances intestinal epithelial survival and regeneration
Xiao Zhang, … , Ivaylo I. Ivanov, Nan Gao
Xiao Zhang, … , Ivaylo I. Ivanov, Nan Gao
Published July 20, 2020
Citation Information: JCI Insight. 2020;5(16):e135923. https://doi.org/10.1172/jci.insight.135923.
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Research Article Gastroenterology Stem cells

Elevating EGFR-MAPK program by a nonconventional Cdc42 enhances intestinal epithelial survival and regeneration

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Abstract

The regulatory mechanisms enabling the intestinal epithelium to maintain a high degree of regenerative capacity during mucosal injury remain unclear. Ex vivo survival and clonogenicity of intestinal stem cells (ISCs) strictly required growth response mediated by cell division control 42 (Cdc42) and Cdc42-deficient enteroids to undergo rapid apoptosis. Mechanistically, Cdc42 engaging with EGFR was required for EGF-stimulated, receptor-mediated endocytosis and sufficient to promote MAPK signaling. Proteomics and kinase analysis revealed that a physiologically, but nonconventionally, spliced Cdc42 variant 2 (V2) exhibited stronger MAPK-activating capability. Human CDC42-V2 is transcriptionally elevated in some colon tumor tissues. Accordingly, mice engineered to overexpress Cdc42-V2 in intestinal epithelium showed elevated MAPK signaling, enhanced regeneration, and reduced mucosal damage in response to irradiation. Overproducing Cdc42-V2 specifically in mouse ISCs enhanced intestinal regeneration following injury. Thus, the intrinsic Cdc42-MAPK program is required for intestinal epithelial regeneration, and elevating this signaling cascade is capable of initiating protection from genotoxic injury.

Authors

Xiao Zhang, Sheila Bandyopadhyay, Leandro Pires Araujo, Kevin Tong, Juan Flores, Daniel Laubitz, Yanlin Zhao, George Yap, Jingren Wang, Qingze Zou, Ronaldo Ferraris, Lanjing Zhang, Wenwei Hu, Edward M. Bonder, Pawel R. Kiela, Robert Coffey, Michael P. Verzi, Ivaylo I. Ivanov, Nan Gao

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Figure 1

Enteroid survival requires Cdc42 for EGF/MAPK signaling.

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Enteroid survival requires Cdc42 for EGF/MAPK signaling.
(A) Propidium i...
(A) Propidium iodide staining (red) showed massive cell death 72 hours after 4-OHT treatment of WT and Cdc42iKO enteroids. (B) After 4-OHT treatment of enteroids, viable enteroids were counted at days 2, 3, and 5 following treatment. Data represent 3 independent experiments, each containing 2 replicates per genotype. (C) WT and Cdc42iKO enteroid sections were stained for cleaved caspase-3 to detect apoptotic cells. Images shown represent 72 hours after treatment. (D) 3D-Glo luminescence assays were used to quantify cell viability in WT and Cdc42iKO enteroids 72 hours after 4-OHT treatment. Data represent 2 independent experiments, each containing 2 replicates per genotype. (E) HEK293 cells were transfected with Flag-tagged Cdc42-V1 or -V2. Mass spectrometry analysis of Flag-Cdc42 immunoprecipitates identified a variant-specific interactome. Venn diagram reveals 910 shared interactors. Functional annotation showed the top 5 most enriched clusters ranked by P value by DAVID Gene Functional Classification Tool. (F) Serum-starved Caco2 control and Cdc42-knockdown cells were analyzed by atomic force microscope. EGF was added to cells 300 seconds after recording of each sample. Young’s modulus (kPa) indicated EGF-induced nanomechanical changes on control Caco2 cell surface, with diminished responses in control cells preincubated with dynasore or in Cdc42-knockdown cells. Graphs represent 4 independent experiments. (G) Western blots for p-ERK1/2 were performed for control and stable Cdc42-knockdown Caco2 cells cultured in regular medium with no treatment. Data were quantified from 2 independent experiments. (H) Phospho-kinase array was used to search for functional pathways downstream of Cdc42. Among 43 kinase targets, overexpressing Cdc42 in HEK293 cells increased levels of p-ERK1/2, p38, and c-Jun, especially in V2-expressing cells compared with empty vector controls. Graph represents mean values from 2 replicates. (I) Western blots were used to determine change of p-ERK1/2 in serum-starved control and Cdc42-V2 expressing cells, following the addition of Wnt3a (100 ng/mL), in time course experiments. (J) Western blots for p-ERK1/2 were used to compare V1 versus V2’s MAPK-activating capabilities. (K) Mutant Cdc42-V2K185R showed a reduced MAPK-activating capability compared with WT Cdc42-V2. Data were quantified from 2 independent experiments in I, J, and K. Please also see Supplemental Figure 1.

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