Intestinal Restitution: Progression of Actin Cytoskeleton Rearrangements and Integrin Function in a Model of Epithelial Wound Healing

Abstract

Superficial injury involving the mucosa of the gastrointestinal tract heals by a process termed restitution that involves epithelial sheet movement into the damaged area. The forces that drive epithelial sheet movement are only partially understood, although it is known to involve changes in the morphology of cells bordering the damage, such as the formation of large, flat, cytoplasmic extensions termed lamellae. We investigated the mechanism of epithelial sheet movement by following the response of the actin cytoskeleton and specific integrins (alpha6beta4, alpha6beta1, and alpha3beta1) to wounding. To model this event in vitro, monolayers of T84 cells, well-differentiated colon carcinoma cells, were damaged by aspiration and the ensuing response was analyzed by a combination of time-lapse video microscopy, fluorescence confocal microscopy and antibody inhibition assays. We show that wound healing begins with retraction of the monolayer. alpha6beta4 integrin is localized on the basal surface in structures referred to as type II hemidesmosomes that persist throughout this early stage. We hypothesize that these structures adhere to the substrate and function to retard retraction. Once retraction ceases, the wound is contracted initially by actin purse strings and then lamellae. Purse strings and lamellae produce a pulling force on surrounding cells, inducing them to flatten into the wound. In the case of lamellae, we detected actin suspension cables that appear to transduce this pulling force. As marginal cells produce lamellae, their basal type II hemidesmosomes disappear and the alpha6 integrins appear evenly distributed over lamellae surfaces. Antibodies directed against the alpha6 subunit inhibit lamellae formation, indicating that redistribution of the alpha6 integrins may contribute to the protrusion of these structures. Antibodies directed against the alpha3beta1 integrin also reduce the size and number of lamellae. This integrin's contribution to lamellae extension is most likely related to its localization at the leading edge of emerging protrusions. In summary, wounds in epithelial sheets initially retract, and then are contracted by first an actin purse string and then lamellae, both of which serve to pull the surrounding cells into the denuded area. The alpha6 integrins, particularly alpha6beta4, help contain retraction and both the alpha6 integrins and alpha3beta1 integrin contribute to lamellae formation.

Vitamin A Activation of Transforming Growth Factor-β1 Enhances Porcine Ileum Wound Healing In Vitro

“…As such, the increased activation of TGF-␤ 1 with vitamin A observed in our study may, in part, account for the improved recovery of mucosal epithelium after injury. Various ECM proteins of the basal lamina and epithelial cell integrin expression have been shown to influence epithelial cell migration and differentiation (25,26). Because vitamin A and TGF-␤ 1 modulate ECM protein synthesis and integrin expression (9,27,28), this may be another mechanism by which these compounds modulate wound healing.…”

Identification of an Epitope within Human Integrin α6 Subunit for the Binding of Autoantibody and Its Role in Basement Membrane Separation in Oral Pemphigoid

“…The integrins are involved in wound healing and skin inflammation, and may regulate the balance between proliferation and differentiation (29,30). Changes in the integrin expression, mutation, or dissociation of the subunits lead to several pathological conditions (31,32).…”

Role of Rho family GTPases in epithelial morphogenesis

“…It is noteworthy in this respect that both in the DC and wound healing processes integrins have proven to play a major role. (Harden et al 1996;Lotz et al 1997Lotz et al , 2000Stark et al 1997;Goldfinger et al 1999;Mercurio et al 2001). For example, mutations in the Drosophila integrin receptor ␤-subunit (myospheroid) and in the ␣3 subunit (scab) of the integrin receptor lead to a DC defect (MacKrell et al 1988;Stark et al 1997).…”