Magnetoterapia

The most obvious consequence of the rac1 gene deletion in the murine epidermis was an almost complete loss of fur (3.1.3) caused by an impaired growth and progressive deterioration of the HFs. While the morphology of HFs shortly after birth was not obviously affected by the absence of Rac1, within 2 weeks the lower parts of HFs became increasingly distorted and lost their integrity (3.2.1), which was accompanied by the loss of expression of epithelial and HF-specific markers in those regions (3.2.2). Changes in the differentiation state of cells in the lower parts of HFs were followed by the infiltration of macrophages, which removed the non-permanent parts of HFs (3.2.3). No reconstitution of anagen HFs was observed afterwards. However, infundibula, SGs and most likely bulge regions were preserved throughout the whole

life span of mutant mice.

It is interesting to note, that, although Rac1 protein was clearly lost in the epidermis of

rac1fl/fl,cre mice at least at day 3 after birth, no obvious changes in the HF morphology were visible until later stages of the morphogenesis, indicating that Rac1 is not crucial for the downward growth of the developing HF as well as for the initial differentiation of ORS, IRS and HS. However, the subsequent loss of HF layer-specific markers in the non-permanent part of the HF shows that Rac1 is required for the maintenance of HF keratinocytes in the differentiated state. The loss of HF-identity most likely reflects changes in gene expression, which could be either a direct result of the absence of Rac1 signalling or an indirect consequence of the loss of cell-cell and cell-ECM adhesion between the Rac1-deficient cells.

The direct involvement of Rac1 in the regulation of gene expression was demonstrated before in cell culture experiments. Rac1 was shown to act as a potent stimulator of MAPK signalling cascades by activating JNK and p38 (Coso et al., 1995; Minden et al., 1995). Activated JNK translocates to the nucleus, where it phosphorylates transcription factors such as c-Jun, while p38 is known to activate both transcription factors (e.g. ATF-2) and other kinases implicated in the regulation of gene expression (e.g. MAPKAP kinase 2) (Gupta et al., 1995).

Rac1 was also implicated in the integrin-dependent activation of NF-κB signalling pathway (Reyes-Reyes et al., 2001), which was previously shown to play an important role in the HF morphogenesis (Schmidt-Ullrich et al., 2001). In unstimulated cells, NF-κB is sequestered in the cytoplasm by binding to members of the IκB family. Upon activation, IκB becomes degraded and NF-κB translocates to the nucleus, where it can induce the transcription of many target genes.

Although no significant differences in the phosphorylation levels of JNK and p38 as well as no changes in the amount of IκB-α were detected in epidermal lysates from rac1fl/fl,cre mice (3.4.2), it is possible that Rac1 plays a role in the activation of these signalling pathways specifically in HF keratinocytes. Such differences would most likely escape detection in whole epidermal lysates, where contribution of HF cells is rather low. The activation of p38 or JNK signalling pathways in mutant HFs could be analysed in more detail by IF staining of skin sections with antibodies specifically recognizing phosphorylated forms of these proteins. The activation of NF-κB signalling in the absence of Rac1 could be further studied by crossing

rac1fl/fl,cre mice with NF-κB reporter mice (Schmidt-Ullrich et al., 1996).

The lack of Rac1 could also indirectly influence the HF-specific gene expression by affecting the cell-cell and cell-ECM adhesion. This notion is supported by the observation that the loss of epithelial-specific markers in the absence of Rac1 seemed to be preceded by

morphological defects (Fig. 3.10, Fig. 3.19) and the loss of cell polarity (Fig. 3.18).

Although no obvious defects in the formation of cell-cell or cell-ECM contacts were observed in the epidermis of rac1fl/fl,cre mice (3.3), it is possible that Rac1 is more critical for their maintenance in the HF. A more detailed ultrastructural analysis of cell-cell contacts in the lower parts of HFs would be certainly helpful to further address this point.

During formation of the IRS, “Flügelzellen” from the Huxley layer of the IRS penetrate through the lower, less differentiated part of the Henle layer and come in contact with CL keratinocytes in a process apparently dependent on the formation of lamellipodia (Langbein et al., 2003). Loss of Rac1 might impair this migration, thus contributing to the malformation of HFs during morphogenesis.

The exact molecular mechanism causing the infiltration of macrophages is not clear. Since no increase in apoptosis was observed in the lower parts of mutant HFs (Fig. 3.23), it is possible that the alterations of gene expression in Rac1-deficient HF keratinocytes lead to the secretion of macrophage attracting substances. This possibility could be investigated using an anti- inflammatory/immunosuppressive drug, such as dexamethasone, which would allow to determine if production of inflammatory cytokines and chemokines is involved in the skin inflammation of rac1fl/fl,cre mice. If successful, blocking of macrophage infiltration would also allow for a more detailed analysis of changes in the HF morphology.

Macrophage mediated removal of HFs is sometimes observed under normal conditions (Eichmuller et al., 1998). It is possible that Rac1 is involved in the regulation of this process and its absence leads to an abnormal induction of such a programmed organ deletion.

The lack of the HF re-growth observed after the removal of the non-permanent parts of HFs can have different reasons. It is possible that the Rac1-mediated signalling plays an important role in the activation of bulge stem cells, which is necessary for the initiation of anagen (Stenn and Paus, 2001; Blanpain and Fuchs, 2006). Alternatively, the severe deterioration of the HFs and the lack of a proper involution phase (catagen), which normally leads to the retraction of the DP within the close proximity of the bulge, might have resulted in a separation of the DP fibroblasts from the HFs. Since anagen is induced by the crosstalk between DP cells and HF stem cells that would result in the inability of Rac1-deficient HFs to cycle.