ErbB3 Control of Pmp22 Regulates Intestinal Barrier Function

Abstract

Background: ErbB3 is a member of the ErbB/EGFR family of receptor tyrosine kinases and is the constitutively-expressed neuregulin (NRG) receptor in the intestinal epithelium. Pathways downstream of ErbB3 have been implicated in regulating tight junctions in other systems, but a role for ErbB3 in barrier function in the intestines has not been described. Here we tested the hypothesis that ErbB3 is required for maintenance of normal barrier function in the intestine. Methods: We generated Villin-Cre;ErbB3 flox/flox mice (ErbB3-IEKO) with ErbB3 deletion in the intestinal epithelium. All comparisons are to ErbB3 flox/flox littermate controls. Mice were given a FITC-dextran 4 kDa (FD4) gavage to assess intestinal permeability. Isolated epithelial ileal samples were collected for RT-qPCR, immunohistochemistry, and bulk RNA-seq analysis. Enteroids were generated and treated with the ErbB3 ligand NRG-1β as well as PD153035 and CP-724714 to inhibit EGFR and ErbB2, respectively. Enteroids were also fixed and stained for whole mount immunofluorescence staining. Caco-2BBe cells were grown on Transwell filters, treated with NRG-1β, and assessed for transepithelial electrical resistance (TEER) and FD4 flux. Results: ErbB3-IEKO mice displayed increased intestinal barrier permeability to FD4 compared to controls (p < 0.01). RNA-seq analysis showed a decrease in the expression of the putative tight junction protein Pmp22 in ErbB3-IEKO mice, while other tight junctional components were unchanged. RT-qPCR analysis of ileal samples showed an 80% Pmp22 downregulation (p < 0.01) with ErbB3 loss compared to controls, with similar results in ErbB3-deficient enteroids. Immunohistochemical analysis confirmed near complete loss of PMP22 in ErbB3-IEKO intestinal epithelium. In vitro, treatment with NRG-1β induced Pmp22 expression (p < 0.001) in control, but not ErbB3-deficient, enteroids. Treatment with PD153035 blocked NRG-1β-driven expression of Pmp22 while treatment with CP-724714 did not, suggesting that EGFR is a critical ErbB3 heterodimer partner in regulating Pmp22. Whole mount staining of enteroids confirmed co-localization of PMP22 with the tight junction protein ZO-1 at the apical junctions. Furthermore, treatment of Caco-2BBe monolayers with NRG-1β increased TEER and reduced FD4 flux, demonstrating active regulation of permeability. Conclusions: We demonstrate that loss of ErbB3 in the intestinal epithelium leads to reduced expression of the tight junction protein PMP22 and impaired barrier function in mice. Co-localization of PMP22 with ZO-1 suggests a role for PMP22 in forming the barrier and may represent a mechanism for regulation of intestinal permeability through the ErbB3-PMP22 axis. Furthermore, increased TEER in Caco-2BBe monolayers following NRG-1β treatment suggests induction of PMP22 may represent a means to enhance barrier function and a potential target for future therapeutic interventions targeting the barrier. Supported by NIH awards R01DK095004 and F31DK131856 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.