ErbB3 Control of PMP22 Regulates Barrier Function in the Intestinal Epithelium
Authors: Hsieh Jonathan , Almohazey Dana , Frey Mark 
Published: May 2024

Background: ErbB3 is a member of the ErbB/EGFR family of receptor tyrosine kinases and is the primary neuregulin (NRG) receptor in the intestinal epithelium. Pathways downstream of ErbB3 have been implicated in regulating tight junctions in other systems, but a role in gut barrier function has not been described. PMP22 is a tight junction protein first described in peripheral myelin sheaths, but its role has not been studied in other systems, including the intestine. We tested the hypothesis that ErbB3 is required for maintenance of normal barrier function in the intestine through regulation of PMP22. Methods: We generated Villin-Cre;ErbB3flox/flox mice (ErbB3ΔIE) for ErbB3 deletion in the intestinal epithelium. All comparisons are to ErbB3flox/flox littermate controls. Mice were given FITC-dextran 4 kDa (FD4) gavage to assess gut permeability. Isolated ileal epithelia were collected for qPCR, immunohistochemistry (IHC), and bulk RNA-seq. Enteroids were generated and treated with NRG-1β to activate ErbB3 +/- inhibitors PD153035 (EGFR), CP-724714 (ErbB2), U0126 (MEK), and LY294002 (PI3K), with qPCR assessment of Pmp22 levels. Caco-2bbe cells stably expressing control or PMP22-targeting shRNA constructs were generated and assessed for barrier using TEER measurements and FD4 flux. Results: ErbB3ΔIE intestines showed increased barrier permeability to FD4 compared to controls (p < 0.01). We observed a significant decrease in expression of Pmp22 mRNA in ErbB3ΔIE ileums and enteroids (p < 0.01), while other tight junction components were unchanged. IHC confirmed loss of PMP22 in ErbB3ΔIE ileal epithelium. NRG-1β induced Pmp22 in control but not ErbB3ΔIE ileal enteroids; PD153035 but not CP-724714 blocked this response, suggesting EGFR is the critical heterodimer partner for ErbB3 regulation of PMP22. U0126 significantly reduced Pmp22 expression in enteroids but LY29002 did not, suggesting MAPK/MEK signaling as a key pathway. Whole mount staining of enteroids confirmed co-localization of PMP22 with the tight junction protein ZO-1 at the apical junctions. Treatment of Caco-2bbe Transwell monolayers with NRG-1β increased TEER and reduced FD4 flux, whereas Caco-2bbe cells expressing PMP22 shRNA showed decreased TEER, demonstrating active regulation of barrier permeability. Conclusions: ErbB3 signaling in the intestinal epithelium is required for expression of the tight junction protein PMP22 and barrier function maintenance. Together, disruption of barrier with PMP22 knockdown and co-localization of PMP22 with ZO-1 suggest PMP22 is the effector for ErbB3 regulation of barrier. Since disrupted barrier function contributes to the pathophysiology of conditions like inflammatory bowel disease, these results may point to potential future therapeutic interventions. Supported by NIH awards R01DK095004 and F31DK131856. This is the full abstract presented at the American Physiology Summit 2024 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.