Table of Contents

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 expression 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.

The role of JNK in cutaneous wound healing

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During maintenance of epithelial thickness or regeneration after wounding epidermal keratinocytes require a delicate balance between proliferation, migration and differentiation. The decision to enter either one of the genetic programs controlling these processes is regulated by both intra- and intercellular regulatory networks, the latter ones being mediated by soluble factors such as cytokines and growth factors. Intercellular communications initiate intracellular signal transduction pathways and, at their receiving end, a program of specific target genes regulating the status of the recipient cells.
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, by employing an in vitro co-culture system composed of primary human keratinocytes and genetically modified mouse fibroblasts we have defined c-Jun/AP-1 regulated genes in fibroblasts, such as KGF, GM-CSF, SDF-1 and HGF, whose gene products affect keratinocyte proliferation and migration.
Using this co-culture system, we have recently observed that deficiency for the MAP kinase family member c-jun N-terminal kinase (JNK) in fibroblasts strongly affects the genetic program adjusting the balance of keratinocyte proliferation and differentiation. Thus, the present project is focusing on the signaling cascade downstream of the JNK, such as the identification of critical JNK substrates distinct from c-Jun. Moreover, novel target genes controlled by JNK activity, particularly growth factors and cytokines will be identified via acquisition and modeling of time-resolved microarray data (collaboration: Busch/Eils – WP1). Finally, cutaneous wound healing experiments will be performed using newly generated transgenic mouse models carrying a dermal-specific JNK1 deletion in the presence or absence of the second member of JNK protein family, JNK2. These experiments will define the importance of JNK function as well as JNK-regulated gene programs for skin homeostasis and wound healing.

Identification of gene regulatory networks involved in the transition between proliferation and differentiation of kerationcytes

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The cutaneous response to injury and stress comprises a temporary, tightly controlled change in the balance between epidermal proliferation and differentiation. To define the underlying gene regulatory network dynamics, which control a keratinocyte´s decision to enter the terminal differentiation program, we will generate time-resolved microarray data from keratinocytes, which were driven into differentiation via different methods (e.g. shift of calcium concentration, conditioned medium from mesenchymal cells, specific chemokines). Analysis of these data by methods of system biology enables us to establish an in silico model of keratinocyte differentiation (collaboration: Busch/Eils/Grabe – WP1). This will allow predictions on the possible regulatory function of currently unknown hub-proteins or pathways critical for the initiation of keratinocyte differentiation. In order to further improve this model, we will perform several rounds of refinement by functional analyses (gain-of-function and loss-of-function approaches) of individual proteins of interest. Taken together, these results will enable us to gain deeper insight into the cellular principles and regulatory networks controlling the differentiation process. Importantly, these studies may allow to predict ontime-dependent combinatory application of external factors initiating distinct signalling pathways inside the cell, by which the decision of the keratinocytes can be manipulated. Finally, we will transfer the insights to a preclinical situation: the pro-regeneratory effects of the combinatory treatment will be studied in an in vivo wound healing model (collaboration: G. Germann – WP5).