Epidermal Rac1 regulates the DNA damage response and protects from UV-light-induced keratinocyte apoptosis and skin carcinogenesis.

Non-melanoma skin cancer (NMSC) is the most common type of cancer. Increased expression and activity of Rac1, a small Rho GTPase, has been shown previously in NMSC and other human cancers; suggesting that Rac1 may function as an oncogene in skin. DMBA/TPA skin carcinogenesis studies in mice have shown that Rac1 is required for chemically induced skin papilloma formation. However, UVB radiation by the sun, which causes DNA damage, is the most relevant cause for NMSC. A potential role of Rac1 in UV-light-induced skin carcinogenesis has not been investigated so far. To investigate this, we irradiated mice with epidermal Rac1 deficiency (Rac1-EKO) and their controls using a well-established protocol for long-term UV-irradiation. Most of the Rac1-EKO mice developed severe skin erosions upon long-term UV-irradiation, unlike their controls. These skin erosions in Rac1-EKO mice healed subsequently. Surprisingly, we observed development of squamous cell carcinomas (SCCs) within the UV-irradiation fields. This shows that the presence of Rac1 in the epidermis protects from UV-light-induced skin carcinogenesis. Short-term UV-irradiation experiments revealed increased UV-light-induced apoptosis of Rac1-deficient epidermal keratinocytes in vitro as well as in vivo. Further investigations using cyclobutane pyrimidine dimer photolyase transgenic mice revealed that the observed increase in UV-light-induced keratinocyte apoptosis in Rac1-EKO mice is DNA damage dependent and correlates with caspase-8 activation. Furthermore, Rac1-deficient keratinocytes showed reduced levels of p53, γ-H2AX and p-Chk1 suggesting an attenuated DNA damage response upon UV-irradiation. Taken together, our data provide direct evidence for a protective role of Rac1 in UV-light-induced skin carcinogenesis and keratinocyte apoptosis probably through regulating mechanisms of the DNA damage response and repair pathways.

Deletion of epidermal Rac1 inhibits HPV-8 induced skin papilloma formation and facilitates HPV-8- and UV-light induced skin carcinogenesis.

Overexpression and increased activity of the small Rho GTPase Rac1 has been linked to squamous cell carcinoma of the epidermis and mucosa in humans. Targeted deletion of Rac1 or inhibition of Rac1 activity in epidermal keratinocytes reduced papilloma formation in a chemical skin carcinogenesis mouse model. However, a potential role of Rac1 in HPV- and UV-light induced skin carcinogenesis has not been investigated so far, solar UV radiation being an important carcinogen to the skin.To investigate this, we deleted Rac1 or modulated its activity in mice with transgenic expression of Human papilloma virus type-8 (HPV-8) in epidermal keratinocytes. Our data show that inhibition or deletion of Rac1 results in reduced papilloma formation upon UV-irradiation with a single dose, whereas constitutive activation of Rac1 strongly increases papilloma frequency in these mice. Surprisingly, we observed that, upon chronic UV-irradiation, the majority of mice with transgenic expression of HPV-8 and epidermis specific Rac1 deletion developed squamous cell carcinomas. Taken together, our data show that Rac1 exerts a dual role in skin carcinogenesis: its activation is, on one hand, required for HPV-8- and UV-light induced papilloma formation but, on the other, suppresses the development of squamous cell carcinomas.

Epidermal RelA specifically restricts contact allergen-induced inflammation and apoptosis in skin.

Strong inhibition of NF-κB signaling in the epidermis results in spontaneous skin inflammation in mice and men. As there is evidence for linkage between polymorphisms within the NF-κB signaling pathway and human inflammatory skin phenotypes, we asked whether partial functional inhibition of NF-κB signaling in epidermal keratinocytes can modulate clinically relevant skin inflammation. We therefore mutated rela specifically in the epidermis of mice (RelA(E-MUT) mice). These mice show no inflammatory phenotype. Induction of contact allergy, but not croton oil-induced irritant dermatitis, resulted in stronger ear swelling and increased epidermal thickness in RelA(E-MUT) mice. Both contact allergen and croton oil treatment led to increased expression of calgranulins A and B (S100A8/A9) in RelA(E-MUT) mice. Epidermal hyperproliferation in RelA(E-MUT) mice was non-cell autonomous as cultured primary epidermal keratinocytes from RelA(E-MUT) mice showed reduced proliferation compared with controls. These results demonstrate that epidermal RelA specifically regulates delayed-type hypersensitivity-induced skin inflammation. In addition, we describe here an essential but nonspecific function of RelA in the protection of epidermal keratinocytes from apoptosis. Our study identifies functions of NF-κB signaling in the epidermis and corroborates a specific role of epidermal keratinocytes in the regulation of skin inflammation.

A function for Rac1 in the terminal differentiation and pigmentation of hair.

The small GTPase Rac1 is ubiquitously expressed in proliferating and differentiating layers of the epidermis and hair follicles. Previously, Rac1 was shown to regulate stem cell behaviour in these compartments. We have asked whether Rac1 has, in addition, a specific, stem-cell-independent function in the regulation of terminal hair follicle differentiation. To address this, we have expressed a constitutively active mutant of Rac1, L61Rac1, only in the basal epidermal layer and outer root sheath of mice possessing an epidermis-specific deletion of endogenous Rac1, which experience severe hair loss. The resulting 'rescue' mice exhibited a hair coat throughout their lives. Therefore, expression of Rac1 activity in the keratin-14-positive compartment of the skin is sufficient for the formation of hair follicles and hair in normal quantities. The quality of hair formed in rescue mice was, however, not normal. Rescue mice showed a grey, dull hair coat, whereas that of wild-type and L61Rac1-transgenic mice was black and shiny. Hair analysis in rescue mice revealed altered structures of the hair shaft and the cuticle and disturbed organization of medulla cells and pigment distribution. Disorganization of medulla cells correlates with the absence of cortical, keratin-filled spikes that normally protrude from the cortex into the medulla. The desmosomal cadherin Dsc2, which normally decorates these protrusions, was found to be reduced or absent in the hair of rescue mice. Our study demonstrates regulatory functions for Rac1 in the formation of hair structure and pigmentation and thereby identifies, for the first time, a role for Rac1 in terminal differentiation.

Impaired epidermal wound healing in vivo upon inhibition or deletion of Rac1.

To address the functions of Rac1 in keratinocytes of the basal epidermal layer and in the outer root sheath of hair follicles, we generated transgenic mice expressing a dominant inhibitory mutant of Rac, N17Rac1, under the control of the keratin 14 promoter. These mice do not exhibit an overt skin phenotype but show protracted skin wound re-epithelialization. Investigation into the underlying mechanisms revealed that in vivo both proliferation of wound-edge keratinocytes and centripetal migration of the neo-epidermis were impaired. Similar results were obtained in mice with an epidermis-specific deletion of Rac1. Primary epidermal keratinocytes that expressed the N17Rac1 transgene were less proliferative than control cells and showed reduced ERK1/2 phosphorylation upon growth factor stimulation. Adhesion, spreading, random migration and closure of scratch wounds in vitro were significantly inhibited on collagen I and, to a lesser extent, on fibronectin. Stroboscopic analysis of cell dynamics (SACED) of N17Rac1 transgenic and control keratinocytes identified decreased lamella-protrusion persistence in connection with increased ruffle frequency as a probable mechanism for the observed impairment of keratinocyte adhesion and migration. We conclude that Rac1 is functionally required for normal epidermal wound healing and, in this context, exerts a dual function - namely the regulation of keratinocyte proliferation and migration.

Localized inflammatory skin disease following inducible ablation of I kappa B kinase 2 in murine epidermis.

Skin inflammation is a complex process that involves interactions between various cell types residing in different skin compartments. Using mice with conditionally targeted I kappa B kinase 2 (IKK2) alleles, we have previously shown that epidermal keratinocytes can play a dominant role in the initiation of an inflammatory reaction. In order to investigate long-term consequences of IKK2 deletion in adult skin, we have generated mice with floxed IKK2 alleles in which expression of a Tamoxifen-inducible Cre recombinase construct is targeted to epidermal keratinocytes (K14-Cre-ER(T2)IKK2(fl/fl) mice). K14-Cre-ER(T2)IKK2(fl/fl) mice are born normally and do not show signs of a skin disease until the age of 6 months. Deletion of IKK2 can be observed after Tamoxifen application to the back skin or spontaneously, without Tamoxifen application, in mice older than 6 months. This deletion is accompanied by dramatic, localized skin changes that are characterized by invasion of inflammatory cells, hair follicle disruption, and pseudoepitheliomatous hyperplasia of the epidermis, but not by tumor formation. The hyperplastic epithelium shows increased phosphorylation of signal transducer and activator of transcription 3 and extracellular signal-regulated protein kinase 1/2, typical features of psoriatic epidermis. Our results identify a primary role for IKK2 in the development of skin inflammation and confirm its requirement for the maintenance of skin homeostasis.

Ras-induced spreading and wound closure in human epidermal keratinocytes.

Although it is known that growth factor signaling cascades are active during epithelial wound healing, signals that regulate reepithelialization after wounding are not very well characterized. The small GTP binding protein Ras is a molecular switch involved in the regulation of signals originating from different growth factor receptors. We have investigated consequences of its activation in primary human keratinocytes. We provide evidence that activation of Ras can lead to shape changes of keratinocytes caused by rearrangements of the actin cytoskeleton that result in membrane protrusion and ruffling. Similar shape changes were found in the migrating tip of newly formed epithelium in mouse wounds. These cytoskeletal changes occur independently of keratinocyte terminal differentiation, and they can determine the speed of wound epithelialization in vitro. Using various mutant constructs and specific pharmacological inhibitors, we found that the effects of activated Ras on the cytoskeleton of keratinocytes are mediated by a phosphatidylinositol 3 kinase-independent activation of Rac. Our results suggest that growth factor-induced, Ras-mediated changes of keratinocyte shape may be an important mechanism that determines the speed of wound epithelialization.

Regulation of keratinocyte shape, migration and wound epithelialization by IGF-1- and EGF-dependent signalling pathways.

Adult epidermal keratinocytes migrate by crawling, a process that requires protrusion of the plasma membrane at the front of the cell and contraction of the cell body at the rear. We have found that epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF-1) influence keratinocyte shape differently. Whereas IGF-1 stimulates membrane protrusion and facilitates cell spreading, EGF induces contraction of keratinocytes. The effects of each growth factor on keratinocyte shape are mediated by distinct signal transduction pathways: EGF stimulates the activity of the classical mitogen-activated protein kinase pathway and IGF-1 stimulates phosphatidylinositol-3-kinase. Activation of these kinases is both necessary and sufficient to induce cell shape changes upon growth factor treatment. In addition, IGF-1-stimulated keratinocyte spreading depends on the activation of Rho family proteins. In vitro assays of wound re-epithelialization show that both growth factors stimulate migration of keratinocytes, and the activity of the respective signalling pathways is required for this re-epithelialization process. When added simultaneously, IGF-1 and EGF have additive effects on wound epithelialization. Our results show that IGF-1 and EGF can influence different components of the keratinocyte migration machinery that determines the speed of wound epithelialization.