The role of E-cadherin mediated cell-cell contact in melanocyte homeostasis

Background Melanocytes are highly specialised cells within the skin, where they act as a protective shield against harmful UV rays in a process called the tanning response. Melanocytes are part of the Epidermal Melanin Unit, in which each melanocyte exists in a symbiotic relationship with ca. 36 keratinocytes at the basement membrane of the epidermis. Sun exposure triggers the production of the pigment melanin within the melanocytes, and this melanin is transferred to the surrounding mitotically active keratinocytes in order to protect them from UV induced DNA damage. On the other hand keratinocytes secrete factors that control the survival, differentiation and growth of melanocytes in response to UV radiation. In fact keratinocytes are mainly responsible for the regulation of melanocyte homeostasis and they ensure that melanocytes stay localised within the Epidermal Melanin Unit and that differentiation is favoured over proliferation. In order to function within this symbiotic relationship an accurate communication between melanocytes and keratinocytes is absolutely essential and direct cell-cell contact is a prerequisite for this communication. At the molecular level the major mediators of cell-cell contacts are cadherins and they regulate cellular functions through multi-protein complexes. Any disturbance of the melanocyte-keratinocyte interaction, and this means the controlled contact between these two cell types, can create a pathological situation. The best-known example for such a situation is the loss of the cell-cell contact protein E-cadherin in melanocytes. This loss results in deregulated melanocyte proliferation and contributes to the transformation into malignant melanoma, the most deadly form of skin cancer. Challenge Elucidating the mechanisms regulating the concerted actions between melanocytes and keratinocytes under physiological conditions will allow to identify dysregulations in pathological situations. Therefore it is important to fully comprehend how cell-cell contact with keratinocytes contributes to the regulation of melanocyte homeostatsis. The main focus in the investigation of melanocyte regulation over the past years has been on the direct effects of keratinocyte-derived growth and differentiation factors on melanocyte monocultures. This does not reflect the physiological situation in which melanocytes adhere to keratinocytes via E-cadherin during growth-factor exposure. Therefore more complex cell systems are required to accurately investigate keratinocyte-mediated regulation of melanocyte function. In addition, although E-cadherin is known to be critical for the malignant transformation of melanocytes, the function and regulation of E-cadherin and its associated proteins in melanocytes is greatly unknown. Approach This project will identify cell-cell contact dependent effects on melanocyte homeostasis within a system that closely recapitulates the human tissue. I will set up a melanocyte-keratinocyte co-culture system, in which melanocytes will be exposed to keratinocyte-derived factors in the context of cell-cell contact to keratinocytes. A technique will be applied that allows monitoring the formation of E-cadherin complexes in the cell-cell contacts. By manipulating the cells (e.g. using specific inhibitors of cellular signalling networks), the molecular signals involved in the regulation of E-cadherin complexes will be identified. Furthermore, the function of E-cadherin in melanocyte homeostasis will be dissected by analysing cellular signalling activities in melanocytes after E-cadherin depletion. This approach will allow uncovering the molecular basis of important control mechanisms in a physiological context. These mechanisms are often lost in the case of disease. Understanding the molecular basis will help to efficiently interfere with a pathological situation and to design drugs that can restore the lost control, ultimately helping to cure the patient.

Melanocyte homeostasis within the skin is regulated by keratinocyte-derived growth factors, but keratinocytes also control melanocytes through so far unknown E-cadherin dependent mechanisms. The proposed research will analyse the function and regulation of E-cadherin adhesion complexes during melanocyte proliferation, differentiation and migration in a context that recapitulates the human tissue. I will set up a melanocyte-keratinocyte co-culture system, in which melanocytes will be exposed to keratinocyte-derived factors in the context of cell-cell contact to keratinocytes. Within this system E-cadherin mediated downstream signalling in melanocytes will be examined. After RNAi mediated knock down of E-cadherin and its 'core' complex components p120 catenin, beta-catenin and alpha-catenin in melanocytes, cellular signalling activities (e.g. MAP kinase, PI3-kinase, Rho) will be analysed by immunofluorescence using phospho-specific antibodies. Identified downstream effcetors will be functionally characterised. In order to monitor the E-cadherin 'core' complex during melanocyte proliferation, differentiation or migration in the co-culture system, a Protein Fragment Complementation assay (PCA) or Bimolecular Fluorescene Complementation (BiFC) will be performed. In the PCA/BiFC strategy protein-protein interactions are measured by fusing each of the proteins of interest to fragments of a reporter protein (here GFP). In the case of an interaction the reporter folds into an active structure and a GFP signal will be visible at the site of complex formation. This allows not only to detect the formation of E-cadherin complexes in situ but also to monitor the dynamics of the complexes during various cellular processes. By using inhibitors and siRNAs targeting relevant signalling pathways (e.g. SRC kinase) the molecular signals involved in the formation of the detected E-cadherin complexes will be identified.