The role of the fibronectin synergy site in skin wound healing

Resumen

Upon skin barrier disruption, complex cellular and molecular events are activated to repair the damage and restore skin integrity. In adulthood, the outcome of this process can result in scarring and fibrosis, whereas complete tissue regeneration is observed in fetal wounds and wounds in lower vertebrates and invertebrates .Although there is abundant literature about the factors and mechanisms that determine the endpoint of either scarring/fibrosis or regeneration after tissue injury, the process is still poorly understood. After cutaneous injury, fibronectin (FN) is instantly released and deposited by cells, and represents a major ECM component in all stages of the wound healing process. FN leaks out from injured blood capillaries and provides, together with fibrinogen, the ECM of the forming granulation tissue (GT). In the following hours, this provisional matrix is remodeled and enriched by infiltrated hematopoietic cells, dermal and fascia fibroblasts that migrate into the dermal region of the wound. This newly formed fibrotic tissue provides structural support for migrating cells adhesion. It constitutes the foundation for either the regeneration of the wounded dermis or the formation of a permanent scar and is a key substrate for keratinocytes to re-epithelialize the wound gap in the epidermis. FN harbors the major cell binding motif defined by the arginine-glycine-aspartate (RGD) sequence located in the 10th type III module (FNIII10) and binds α5β1, αIIbβ3 (exclusive of platelets) and αv-class integrins. In the adjacent module (FNIII9), FN contains the so-called synergy motif that binds α5β1/αIIbβ3 integrins but not αv-class integrins and mediates the formation of catch bonds. The mutation of the FN synergy sequence in mice underscored its role in resisting or producing high forces, although up to a certain force threshold, αv-class integrins binding to FN compensate for the loss of the synergy site. In the present work, we investigated the role of FN synergy site during cutaneous wound healing in mice carrying a dysfunctional FN-synergy motif (Fn1syn/syn). The course of fibrotic processes, such as adult skin wound healing, depends on several events that are modulated by mechanical signals and hence, on strong integrin-FN bonds: i) the population of basal keratinocytes that initiates migration from the wound margins towards the center of the wound express and activate α5β1 integrins, and form a sheet that advances by sensing the FN-substrate tension and generating force towards it ii) the GT is infiltrated by migrating dermal fibroblasts that secrete and assemble FN fibrils and fascia fibroblasts that pull their own ECM of FN and collagens iii) in the GT, a number of fibroblasts convert into myofibroblasts in a process regulated by mechanical forces and TGF-β1 signals and iv) further release of latent TGF-β1 from the ECM is induced by integrin-mediated force application to the ECM. We demonstrate that Fn1syn/syn mice show a delayed wound closure at early times. This correlates with the presence of less granulation tissue in the initial stage of healing in Fn1syn/syn wounds, and reduced content of myofibroblasts and FN deposition. In vitro experiments using kidney and dermal fibroblasts derived from Fn1+/+ and Fn1syn/syn mice, and plasma FN (pFN) purified from Fn1+/+ and Fn1syn/syn mice as substrate, reveal that the FN synergy site is important to withstand mechanical tensions required for cell migration and TGF-β1 liberation from the ECMs. At early stages in the epidermis of Fn1syn/syn wounds we observe an increment in the α5β1 integrin expression at the epidermal tongue level compared to wounds from Fn1+/+ mice. When studying keratinocytes behavior in vitro we observe apparently contradictory results during single-cell and collective migration. In single-cell migration, keratinocytes moving on FNsyn as substrates showed delayed wound closure due to lower speeds and decreased directionality. On the contrary, when keratinocytes are allowed to form cell-cell contacts and migrate in a collective way, the presence of FNsyn substrates accelerates the wound closure. This is also demonstrated to be directly linked to the myosin cytoskeleton contraction and the compensatory effects of cell-cell bonds reinforcement. Taken all these results together, we can conclude that the absence of the synergy site is important during the process of skin wound healing at early stages and that the implication is directly linked to processes where the FN-α5β1 bond needs to be reinforced due to high mechanical tension such as TGF-β1 liberation from the ECM and the migration process in cells involved in the healing process.