c-Jun and JunB-dependent functions in skin homeostasis and repair
The skin is a suitable in vivo system to study molecular mechanisms how a given cell is able to execute tissue homeostasis and to adapt itself to environmental cues (e.g. wounding, chemical stress) on the level of gene expression. Altered gene expresion rapidly and appropriately adjusts the balance of critical cellular programs, e.g. cell proliferation, migration and terminal differentiation. For regulation of these complex processes soluble factors play an important role by coordinating the intercellular communication between keratinocytes, fibroblasts and inflammatory cells.
Trans-regulatory functions of c-Jun, JunB and Jun target genes in wound healing
On the left, a schematic representation of the fibroblast-keratinocyte cocultures is shown. In organotypic cocultures keratinocytes grown air-exposed on a collagen matrix containing either wildtype (wt) or genetically modified mouse fibroblasts. Depending on the genotype of the fibroblasts major differences in keratinocyte behaviour to form an intact epithelium can be observed (measured by indicative marker genes; right panel). Middle panel: c-Jun and JunB- dependent factors affecting keratinocyte proliferation, migration and/or differentiation. For experimental details see Szabowski et al (2000) Cell 103, 745-55; Florin et al., 2005.
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We have drawn our attention to fibroblasts of the connective tissue as a source of soluble factors, particularly cytokines, acting in a trans-regulatory fashion to control epithelial cell behaviour. Previously, we have established the concept of c-Jun-dependent cross-talk between keratinocytes and fibroblasts by employing an in vitro 3-dimensional reconstituted skin equivalent,which reflects early molecular events of skin regeneration).Using this approach the consequences of genetic manipulation in murine fibroblasts on proliferation and differentiation of co-cultured primary human keratinocytes was investigated. Indeed, loss of either c-Jun or JunB in fibroblasts affected keratinocyte growth and differentiation and could be attributed, at least in part, to major difference in basal and IL-1-induced expression of KGF and GM-CSF in mutant fibroblasts (Fig. 3). To identify additional AP-1 target genes in fibroblasts, which are involved in the process of cutaneous repair large-scale gene expression profiling of wild type and mutant fibroblasts in response to IL-1 was performed. In addition to previously identified genes, such as the chemo-attractants Gro-1, Mip-2 and Lcn-2 we identified pleiotrophin (PTN), the stromal cell-derived factor 1 (SDF-1) and hepatocyte growth factor (HGF) as novel c-Jun regulated factors, which exert a mitogenic effect on primary human keratinocytes.
c-Jun-dependent migration
The identification of HGF as a novel c-Jun target gene in fibroblasts suggested a critical role of c-Jun in the regulation of keratinocyte migration. In heterologous cocultures expression of HGF in c-Jun-deficient MEFs, in contrast to wildtype fibroblasts, was almost undetectable. Using the heterologous co-culture system with mutant fibroblasts in the absence or presence of exogenously added HGF we aimed at identifying key factors mediating HGF-induced epidermal cell migration. Time-resolved expression kinetics of keratinocytes stimulated with HGF disclosed a genetic network mediating HGF-dependent motility. Carcinoembryogenic antigen-related cell adhesion molecule (CEACAM)-1 and the urokinase plasminogen activator (uPA)/uPA-receptor (uPAR)-pathway were identified as crucial mediators of HGF-induced migration.
To gain further insides into the mechanisms of keratinocyte decision making towards migration, the time resolved expression kinetics of HGF stimulated keratinocytes are currently subjected in a collaborative effort to a system biology based approach. We aim to unravel the network topology, simulate its dynamic behaviour and define the distinct spatio-temporal regulatory network of gene expression after HGF-stimulation that facilitate proper epidermal cell migration in cutaneous wound healing.
JunB functions in skin regeneration and inflammation
Using both in vitro organotypic organ cultures and a conditional mouse knock-out approach JunB was identified as an important regulator of cytokine expression and thus a critical molecular switch of the cutaneous response to injury and stress. Mice lacking JunB in the skin develop normally, indicating that JunB is neither required for cutaneous organogenesis nor homeostasis. Yet, upon wounding or treatment with phorbol ester, JunB-deficiency results in delayed tissue remodeling or pronounced epidermal hyperproliferation, disturbed differentiation and prolonged inflammation, respectively. These phenotypic skin abnormalities were associated with alterations in expression levels and kinetics of important mediators of wound repair, such as GM-CSF, Gro-1, Mip-2 and Lcn-2 in both the dermal and epidermal compartment of the skin. Moreover, a reduced ability of wound contraction of mutant dermal fibroblasts in vitro was observed. Finally, inducible epidermal deletion of JunB and its functional sibling c-Jun in adult mice leads to a phenotype resembling the histological and molecular hallmarks of psoriasis including arthritic lesions. Presumably, loss of JunB in keratinocytes triggers chemokine/cytokine expression resulting in the recruitment of inflammatory immune cells contributing to the psoriasis-like phenotype.