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Chrostek-Grashoff, Anna Magdalena (2007): The Role of Rac1 in the Epidermis and in the Hair Follicle. Dissertation, LMU München: Fakultät für Biologie

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Rac1 is a ubiquitously expressed member of the Rho family of small GTPases, which acts as a molecular switch by shuttling in a highly regulated manner between an active (GTP-bound) and an inactive (GDP-bound) state. Different signalling pathways, which involve integrins, growth factor receptors, cadherins as well as other Rho GTPases, can induce Rac1 activation. Only in the GTP bound form, Rac1 can associate with different effector molecules to initiate cellular responses. Initially described as an important regulator of the actin cytoskeleton, Rac1 was later found to be also involved in the modulation of other processes such as cell adhesion, proliferation, survival, differentiation and migration. In epithelial cells, Rac1 was shown to regulate the formation and maintenance of cadherin dependent cell cell contacts, which are essential for the establishment of the polarized cell morphology. Before this project was initiated, almost all knowledge about the function of Rac1 was based on in vitro studies. As constitutive deletion of the murine rac1 gene leads to early embryonic lethality, mice allowing for a conditional inactivation of the rac1 gene were generated in this study to enable the analysis of the function of Rac1 in selected tissues. To investigate the role of Rac1 in the epidermis and hair follicles and to determine its function in the establishment and maintenance of cell cell contacts between epithelial cells in vivo, mice with a keratinocyte-restricted ablation of the rac1 gene were generated and analyzed. The results obtained in this study showed that the absence of Rac1 in the murine epidermis leads to a progressive hair loss but surprisingly has no effect on the maintenance of the epidermis. The hair loss is caused by the inability of hair follicle keratinocytes to maintain their differentiation state, which leads to the phagocytic removal of the non permanent parts of the hair follicles by infiltrating macrophages. In contrast, differentiation and proliferation of epidermal keratinocytes as well as the formation and maintenance of cell-cell and cell-matrix contacts, and the deposition of the basement membrane in the epidermis are not affected by the loss of Rac1. Biochemical analysis of epidermal lysates demonstrated that the absence of epidermal defects in vivo is not a result of compensatory upregulation of closely related members of the Rho family of GTPases, further indicating that the function of Rac1 in epithelial cells in vivo is limited. Also, the analysis of the formation of the embryoid bodies from Rac1-deficient embryonic stem cells showed that the presence of Rac1 is not required for the establishment of cell cell contacts during differentiation of the polarized primitive ectoderm and for the formation of epithelial sheets, supporting the conclusion that the function of Rac1 in the regulation of cell cell adhesion between epithelial cells is dispensable. However, the re epithelialization after wounding was impaired in the mutant epidermis, demonstrating that Rac1 plays an important role in pathological conditions. The delayed wound closure in the absence of Rac1 is caused by impaired cell migration and proliferation of neo epidermal keratinocytes. Another interesting finding of this study was the observation that, in contrast to the steady state in vivo situation, isolated Rac1 deficent primary keratinocytes display severe defects in cell culture, which lead to their detachment from the matrix. While the initial adhesion is only mildly affected by the lack of Rac1, mutant keratinocytes are unable to spread, show an impaired organization of the actin cytoskeleton and fail to form mature focal adhesions. The differences between in vivo and in vitro effects resulting from the inactivation of the rac1 gene indicate that the function of Rac1 in epithelial cells depends on the complexity of the cellular system and emphasize the importance of performing in vivo studies to fully understand its role. Taken together, the data presented in this study show that Rac1 plays an important role in the maintenance of hair follicles and during epidermal wound healing, but that it is not essential for the homeostasis of the epidermis in physiological conditions and for the formation and maintenance of cell cell contacts between epithelial cells in vivo.