The Contributions of ESRP1 to the Functions of the Intestinal Epithelium

Abstract

Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2) are RNA binding proteins expressed exclusively in epithelial cells. They direct a splicing program necessary for maintaining important epithelial cell characteristics, including cell-cell adhesion, anchorage to the basement membrane, and cell-cell communication. ESRP1 and 2 have been studied for their importance in development. The loss of ESRP1 causes a series of craniofacial defects called cleft lip and cleft palate, while the loss of both ESRP1 and ESRP2 result in more severe versions of these defects, several epithelial organ formation defects, and a skin barrier defect which causes significant water loss. ESRP1 is known for its role in craniofacial development, epidermal barrier development, and cancer progression. However, its role in other epithelial organs where it is highly expressed, such as the large intestine, remains understudied. Mice with a hypo-morphic mutation of Esrp1, termed triaka, exhibited decreased intestinal wound healing and increased intestinal permeability. Thus, we hypothesized that ESRP1 contributes to intestinal homeostasis and intestinal barrier integrity by sustaining tight junction localization and intestinal cell proliferation. This thesis project sought to investigate the functions of ESRP1 in maintaining intestinal homeostasis using mouse colon organoids and mouse colon organoid-derived monolayers as a model. Upon the deletion of ESRP1 and subsequent mechanical dissociation of the organoids, we observed a significant decrease in organoid re-formation. Esrp1 KO organoids exhibited no change in organoid cell proliferation. However, they did exhibit a decrease in the expression of Lgr5, an intestinal stem cell marker and receptor for R-spondin. LGR5 helps to maintain the stem cell niche by promoting the Wnt signalling cascade through binding to R-spondin, a Wnt agonist. Thus, its downregulation in Esrp1 KO organoids suggests that ESRP1 helps sustain of the intestinal stem cell niche by maintaining the response of the intestinal epithelium to the Wnt signalling cascade. As R-spondin is produced by subepithelial stromal cells, this would suggest that ESRP1 is necessary for proper epithelial-mesenchymal communication in the intestine. This is ultimately similar to its observed role in craniofacial development. Through investigating the role of ESRP1 in maintaining intestinal barrier integrity, ZO-1 staining showed that tight junctions are still able to assemble properly in Esrp1 KO organoids but become more diffuse in Esrp1 KO monolayers. The loss of ESRP1 resulted in a slight decrease in the barrier integrity of the organoid-derived monolayers. This contrasts with published findings in other cell lines, suggesting that the dependence of epithelial barrier integrity on ESRP1 may vary based on tissue and the chosen model of the epithelium. These findings will provide a solid foundation for further investigations into the role of ESRP1 in maintaining intestinal homeostasis, which will enable future research to uncover its connection to pathological conditions such as Inflammatory Bowel Disease.