Axel Szabowski

c-Jun and JunB Antagonistically Control Cytokine-Regulated Mesenchymal–Epidermal Interaction in Skin

Interactions between mesenchymal and epithelial cells are responsible for organogenesis and tissue homeostasis. This mutual cross-talk involves cell surface proteins and soluble factors, which are mostly the result of regulated transcription. To elucidate dimer-specific functions of the AP-1 family of transcription factors, we reconstituted skin by combining primary human keratinocytes and mouse wild-type, c-jun(-/-), and junB(-/-) fibroblasts. We have discovered an antagonistic function of these AP-1 subunits in the fibroblast-mediated paracrine control of keratinocyte proliferation and differentiation, and traced this effect to the IL-1-dependent regulation of KGF and GM-CSF. These data suggest that the relative activation state of these AP-1 subunits in a non-cell-autonomous, transregulatory fashion directs regeneration of the epidermis and maintenance of tissue homeostasis in skin.

Function and regulation of AP-1 subunits in skin physiology and pathology

The mouse skin has become the model of choice to study the regulation and function of AP-1 subunits in many physiological and pathological processes in vivo and in vitro. Genetically modified mice, in vitro reconstituted skin equivalents and epidermal cell lines were established, in which AP-1-regulated genetic programs of cell proliferation, differentiation and tumorigenesis can be analysed. Since the epidermis, as our interface with the environment, is subjected to radiation and injury, signal transduction pathways and critical AP-1 members regulating the mammalian stress response could be identified. Regulated expression of important components of the cytokine network, cell surface receptors and proteases, which orchestrate the process of wound healing has been found to rely on AP-1 activity. Here we review our current knowledge on the function of AP-1 subunits and AP-1 target genes in these fascinating fields of skin physiology and pathology.

Increased keratinocyte proliferation by JUN-dependent expression of PTN and SDF-1 in fibroblasts

In skin, fibroblasts of the connective tissue play a decisive role in epidermal homeostasis and repair by contributing to the regulation of keratinocyte proliferation and differentiation. The AP-1 transcription factor subunit JUN plays a crucial role in this mesenchymal-epithelial interplay by regulating the expression of two critical paracrine-acting cytokines, keratinocyte growth factor (KGF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We have performed gene expression profiling of wild-type and Jun–/– mouse embryonic fibroblasts to identify additional players involved in this complex network, and have found pleiotrophin (PTN) and the stromal cell-derived factor 1 (SDF-1) as novel JUN-regulated factors. Both cytokines are expressed by dermal fibroblasts in vivo, as shown by semi-quantitative RT-PCR and in situ hybridization on murine skin sections. Using a heterologous feeder layer co-culture system, we demonstrated that PTN and SDF-1 exert a mitogenic effect on primary human keratinocytes. Moreover, SDF-1-induced keratinocyte proliferation could be specifically inhibited by neutralizing antibodies against SDF-1 or its receptor, CXCR4. Consistent with its role in promoting keratinocyte growth, PTN was upregulated during cutaneous wound healing in vivo. Interestingly, co-cultivation with keratinocytes stimulated PTN expression but repressed SDF-1 production in fibroblasts, demonstrating the complexity of the paracrine regulatory cytokine networks that control skin homeostasis and regeneration.

Organotypic cocultures as skin equivalents: A complex and sophisticated in vitro system

To assess the role of genes required for skin organogenesis, tissue regeneration and homeostasis, we have established in vitro skin equivalents composed of primary cells or cell lines, respectively. In these organotypic cocultures keratinocytes generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The combination of cells derived from mouse and human tissues facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and thus the precise analysis of growth regulatory mechanisms.

Function of AP-1 target genes in mesenchymal-epithelial cross-talk in skin

An increasing number of examples on the importance of mesenchymal-epithelial interactions in physiological (e.g. embryonic development) and pathological (tumourigenesis) processes have been described. This is best illustrated in the skin, where the well-controlled balance of keratinocyte proliferation and differentiation forms the basis for a proper histoarchitecture of the epidermis. Here, a double paracrine loop of cytokines, which are synthesised and secreted by cells of the epidermis (keratinocytes) and the underlying dermis (fibroblasts) seems to play a major role. The aim of this commentary is to review research that has investigated the role of specific subunits of transcription factor AP-1 (Jun/Fos) in this regulatory network. Using an in vitro skin equivalent model strong evidence was provided for a critical and specific function of c-Jun and JunB in mesenchymal-epithelial interaction in the skin by regulating the expression of interleukin-1 (IL-1)-induced keratinocyte growth factor (KGF) and GM-CSF in fibroblasts. These factors, in turn, adjust the balance between proliferation and differentiation of keratinocytes ensuring proper architecture of the epidermis. This commentary will summarise our current knowledge on the molecular mechanisms underlying AP-1-dependent mesenchymal-epithelial interactions and discuss the physiological relevance of these in vitro findings in skin physiology and pathology.

Identification of novel AP-1 target genes in fibroblasts regulated during cutaneous wound healing

Mesenchymal–epithelial interactions are increasingly considered to be of vital importance for epithelial homeostasis and regeneration. In skin, the transcription factor AP-1 was shown to be critically involved in the communication between keratinocytes and dermal fibroblasts. After skin injury, the release of IL-1 from keratinocytes induces the activity of the AP-1 subunits c-Jun and JunB in fibroblasts leading to a global change in gene expression. To identify AP-1 target genes in fibroblasts, which are involved in the process of cutaneous repair, we performed gene expression profiling of wild-type, c-jun- and junB-deficient fibroblasts in response to IL-1, mimicking the initial phase of wound healing. Using a 15K cDNA collection, over 1000 genes were found to be Jun-dependent and additional 300 clones showed IL-1 responsiveness. Combinatorial evaluation allowed for the dissection of the specific contribution of either AP-1 subunit to gene regulation. Besides previously identified genes that are involved in cutaneous repair, we have identified novel genes regulated during wound healing in vivo and showed their expression by fibroblasts on wound sections. The identification of novel Jun target genes should provide a basis for understanding the molecular mechanisms underlying mesenchymal–epithelial interactions and the critical contribution of AP-1 to tissue homeostasis and repair.

Gene network dynamics controlling keratinocyte migration

Translation of large‐scale data into a coherent model that allows one to simulate, predict and control cellular behavior is far from being resolved. Assuming that long‐term cellular behavior is reflected in the gene expression kinetics, we infer a dynamic gene regulatory network from time‐series measurements of DNA microarray data of hepatocyte growth factor‐induced migration of primary human keratinocytes. Transferring the obtained interactions to the level of signaling pathways, we predict in silico and verify in vitro the necessary and sufficient time‐ordered events that control migration. We show that pulse‐like activation of the proto‐oncogene receptor Met triggers a responsive state, whereas time sequential activation of EGF‐R is required to initiate and maintain migration. Context information for enhancing, delaying or stopping migration is provided by the activity of the protein kinase A signaling pathway. Our study reveals the complex orchestration of multiple pathways controlling cell migration.

AP-1-Controlled Hepatocyte Growth Factor Activation Promotes Keratinocyte Migration via CEACAM1 and Urokinase Plasminogen Activator/ Urokinase Plasminogen Receptor

Keratinocyte migration is essential for the rapid closure of the epidermis in the process of wound healing. Mesenchymal cell-derived hepatocyte growth factor (HGF) is a central regulator of this process. However, the molecular mechanisms and relevant genes that facilitate this cellular response are still poorly defined. We used heterologous cocultures combining primary human keratinocytes and genetically modified murine fibroblasts to identify key factors mediating HGF-induced epidermal cell migration. The absence of c-Jun activity in fibroblasts completely abolished the expression of HGF in these cells and consequently altered the behavior of keratinocytes. Time-resolved expression series of keratinocytes stimulated with HGF disclosed target genes regulating HGF-dependent motility. In addition to well-established HGF-dependent wound healing-associated genes, carcinoembryogenic antigen-related cell adhesion molecule (CEACAM)-1 and the urokinase plasminogen activator (uPA)/uPA-receptor (uPAR) pathway were identified as possible mediators in HGF-induced keratinocyte migration. The functional relevance of CEACAM-1 and uPA/uPAR on epidermal cell motility was demonstrated using the HaCaT cell culture model. In conclusion, the distinct spatiotemporal regulation of genes by HGF is essential for proper epidermal cell migration in cutaneous wound healing.