Epithelial RAC1-dependent cytoskeleton dynamics controls cell mechanics, cell shedding and barrier integrity in intestinal inflammation

Abstract

ObjectiveIncreased apoptotic shedding has been linked to intestinal barrier dysfunction and development of inflammatory bowel diseases (IBD). In contrast, physiological cell shedding allows the renewal of the epithelial monolayer without compromising the barrier function. Here, we investigated the role of live cell extrusion in epithelial barrier alterations in IBD.DesignTaking advantage of conditional GGTase and RAC1 knockout mice in intestinal epithelial cells (Pggt1biΔIEC and Rac1iΔIEC mice), intravital microscopy, immunostaining, mechanobiology, organoid techniques and RNA sequencing, we analysed cell shedding alterations within the intestinal epithelium. Moreover, we examined human gut tissue and intestinal organoids from patients with IBD for cell shedding alterations and RAC1 function.ResultsEpithelial Pggt1b deletion led to cytoskeleton rearrangement and tight junction redistribution, causing cell overcrowding due to arresting of cell shedding that finally resulted in epithelial leakage and spontaneous mucosal inflammation in the small and to a lesser extent in the large intestine. Both in vivo and in vitro studies (knockout mice, organoids) identified RAC1 as a GGTase target critically involved in prenylation-dependent cytoskeleton dynamics, cell mechanics and epithelial cell shedding. Moreover, inflamed areas of gut tissue from patients with IBD exhibited funnel-like structures, signs of arrested cell shedding and impaired RAC1 function. RAC1 inhibition in human intestinal organoids caused actin alterations compatible with arresting of cell shedding.ConclusionImpaired epithelial RAC1 function causes cell overcrowding and epithelial leakage thus inducing chronic intestinal inflammation. Epithelial RAC1 emerges as key regulator of cytoskeletal dynamics, cell mechanics and intestinal cell shedding. Modulation of RAC1 might be exploited for restoration of epithelial integrity in the gut of patients with IBD.

Dynamic traction force measurements of migrating immune cells in 3D matrices

“…The data of this study are available upon request from the corresponding author. Rheological measurement of 1.2 mg/ml collagen gels of Batch A were previously published in (36).…”

“…Graphical User Interface for the open-source python package Saenopy (28) that allows to conduct dynamical or static traction force measurements of single cells ( a,b ) or of multicellular aggregates (spheroids, organoids, d ; previously described in (29, 36)). Results can be evaluated and visualized individually or for grouped experiments ( c,e ).…”

Epithelial RAC1 niches in IBD: from barrier integrity to cytoskeletal plasticity

“…In Gut, Martínez-Sánchez et al have addressed the impact of the prenylation enzyme geranylgeranyltransferase-1 (GGT-1) and RAC1 on cell shedding alterations and barrier integrity in IBD. 11 First, they took advantage of murine conditional GGTase and RAC1 knock-out models ( Pggt1b iΔIEC and Rac1 iΔIEC ) to study functional consequences of epithelial specific knockouts. Inhibition of prenylation within IECs ( Pggt1b iΔIEC mice) resulted in increased intestinal permeability and barrier disruption over time accompanied by an inflammatory response in the small intestine.…”

“…In Gut, Martínez-Sánchez et al have addressed the impact of the prenylation enzyme geranylgeranyltransferase-1 (GGT-1) and RAC1 on cell shedding alterations and barrier integrity in IBD 11. First, they took advantage of murine conditional GGTase and RAC1 knock-out models ( Pggt1b iΔIEC and Rac1 iΔIEC ) to study functional consequences of epithelial specific knockouts.…”

The role of Cdc42 Rho GTPase in the cystic fibrosis intestinal epithelial barrier

“…GTPase activity was decreased in inflamed colonic mucosa in IBD patients compared to unaffected regions, with Rac1 inhibition in human intestinal organoids causing actin cytoskeleton disorganization and defects in epithelial cell shedding 82,83 . Notably, increased activity of RhoA, Rac1, and Cdc42 has been associated with colorectal cancer progression and a poorer prognosis [84][85][86] .…”