| Background: The essential functions of the skin involve the homeostatic mechanisms of thermoregulation, maintenance of fluid balance, and protection against a variety of environmental insults. The loss of skin due to burns or trauma exposes the body to severe stress, impairing or even eliminating the many vital functions this organ performs. Both skin maintenance under steady-state conditions and tissue repair after injury are dependent on tissue-specific stem cells. The regeneration of the epidermis after wounding involves activation, migration and proliferation of epidermal stem cells (ESCs) from the surrounding epidermis. The regulating mechanisms of ESCs remain unclear.Several different cytokines and extracellular proteins have been identified that contribute to the proliferation and activation of ESCs [e.g. epidermal growth factor, keratinocyte growth factor and stromal cell-derived factor-1]. It is now appreciated that the focal adhesions (FAs) proteins can serve as a point of convergence for signals emanating from stimulated growth factor receptors. they can bind focal adhesion kinase, and is responsible for the recruitment of Rac1, a member of the Rho family of small GTPases, to FAs. Therefore, Rac1 could be a crucial factor in the development of ESCs.Rac1 is classified as a member of the Rho family of small GTPases, which function as regulated switches, cycling between inactive GDP- bound state and active GTP-bound state. Primarily, Rac1 has been found to control the organization of actin cytoskeleton components, but subsequently Rac1 has been identified as playing in the control of numerous other cellular processes, including cell growth and lamella protrusion. A rapid depletion of stem cells after deletion of Rac1 from adult mouse epidermis was reported. All above indicate a key role of Rac1 in ESCs regulation. our study focus on finding the relationship between them, especially during wound healing.Objection: To explore the role of Rac1 in ESCs regulation during wound healing.Methods: To investigate the relationship of Rac1 and ESC phenotypes, we induced either the dominant negative isoform or constitutively active mutant of human Rac1 in ESC using a retroviral vector. Putative human ESCs were enriched based on collagen type IV adhesiveness and analyzed after lentivirus infection. Alterations on proliferation, differentiation and migration were detected in Rac1 mutant-expressing ESCs. At last, epidermis regeneration on 3D skin equivalent was observed by HE staining.Results:1. ESCs were enriched based on collagen type IV adhesiveness. Approximately 50% of these rapid adherent cells (RAC) formed large colonies within 2 weeks. Almost all cells from a single colony wereα6briCD71dim, suggesting a ESCs phenotype.2. Recombinant mutants were confirmed using restriction enzymes and DNA sequencing. A more than 90% infectious efficiency of lentivirus was detected by FACS. Rac1 expression was observed under confocal laser scanning microscope.3. Measured by MTT assay, the proliferation of ESCs was positively correlated to Rac1 function.4. There was no difference found in cell cycle profile of ESCs that expressed different Rac1 mutants.5. Both the adhesiveness on matrix and surfaceα6/β1 integrin expression were positively correlated to Rac1 function. Inhibition of attachment was more pronounced when cells were plated on collagen I instead of collagen IV or fibronectin, indicating different roles of matrix in ESCs development.6. Cell spreading of ESCs was enhanced by the expression of constitutively active mutant of Rac1.7. The upregulation of ESCs transwell migration was more pronounced under SDF-1 effect than DK-SFM, indicating a more important role of Rac1 under stress.8. Confirming by wound scrap experiment, Rac1 could act on ESCs and therefore regulated the skin repair.9. The downregulation of Rac1 function inhibited the colony-forming efficiency of ESCs, while promoted their differentiation.10. The epidermis regeneration on 3D skin equivalent was better developed when constitutively active mutant of Rac1 expressed in ESCs, indicating a positive correlation between Rac1 and ESCs. Conclusions:1. ESCs can be selected and cultured in vitro successfully. A steady high level of Rac1 expression in ESCs can be achieved by lentivirus infection.2. Significant alterations of both differentiation and adhesion were found in Rac1 mutant-expressing ESCs. Along with the change detected in the clonogenicity assay, these results suggest that Rac1 helps determine the fate of ESCs by regulating their exit from the stem cell compartment.3. The proliferation and migration of ESCs were positively correlated to Rac1 function, while their differentiation was negatively correlated. Since all these three cellular processes were essential to wound healing, these results indicated a active role of Rac1 in skin repair.4. The epidermis regeneration on 3D skin equivalent were tightly regulated by Rac1, therefore Rac1 might play a key role in wound healing process. |
