CELL BIOLOGY OF MELANOCYTE MIGRATION AND LOCALIZATION

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My long term objectives are to determine the molecular mechanisms which underlie melanocyte migration and localization in the epidermis. Melanocyte migration and localization in the epidermis are critical for establishing normal pigmentation during cutaneous development, and for repigmentation of skin after trauma. The experiments outlined in this proposal will test the hypotheses that migration of melanocytes is determined by specific receptor-ligand interactions and that the localization of melanocytes in the epidermis is determined, in part, by integrin receptors. We have shown that fetal and neonatal melanocytes express integrins and that integrin receptors mediate melanocyte attachment to fibronectin. We have also shown that fetal and neonatal melanocytes interact differently with fibronectin and express different amounts of integrins, suggesting that developmental regulation of melanocyte-matrix interactions exist. Finally, we have shown that TGF- beta, bFGF and stem cell factor (SCF) regulate integrin expression in melanocytes, and have shown that treatment of melanocytes with TGF-beta and SCF result in alterations in melanocyte affinity for extracellular matrix (ECM) proteins. Because HGF, a melanocyte mitogen, has been shown to directly affect melanocyte migration, experiments to determine the mechanisms of this phenomenon will be performed. Specific aim 1 will focus on defining the matrix proteins, receptors, and regulatory factors which control fetal and neonatal melanocyte migration. Melanocyte migration on extracellular matrix proteins will be evaluated by Boyden chamber assays and time-lapse videomicroscopy in a planar model; the role of melanocyte integrins and glycosaminoglycans and of TGF-beta, bFGF, SCF and HGF in melanocyte migration will then be determined. Once we have defined the parameters that control migration in this model, we will study melanocyte migration in collagen cells, which recapitulates the environment of migrating melanocytes in fetal life. In each experiment, fetal and neonatal melanocytes will be compared to identify developmental differences which may be important for regulating migration. We developed a skin equivalent (SE) model to study melanocyte-keratinocyte interactions in fetal and neonatal epidermis, and have shown that melanocyte number and position is determined by keratinocytes, and that these factor(s) are probably developmentally regulated. Integrins alpha2 and alpha3 have recently been shown to function as developmentally regulated cell-cell receptors for keratinocytes. Specific aim 2 focuses on determining the role of these keratinocyte integrins in the localization and number of melanocytes in fetal and neonatal epidermis. Initial experiments will involve blocking studies with anti-integrin antibodies on keratinocyte-melanocyte co-cultures. To confirm and extend these observations, we will transfect keratinocytes with integrins alpha2 and alpha3 and determine the effect on melanocyte attachment to these keratinocytes. Finally, to evaluate the role of integrins on melanocyte number and position in the stratified epidermis, we will construct SEs with the transfected keratinocytes and determine the effect of increased keratinocyte integrin expression on melanocyte-keratinocyte interactions.