The skin is a constantly renewing organ that undergoes a series of regulated changes as the cells of the basal layer proliferate and differentiate into the anucleate stratum corneum. Failure of regulatory processes leads to abnormal growth in skin diseases such as psoriasis and neoplasia. We propose to define the role of free intracellular calcium, the calcium-binding protein calmodulin, and extracellular calcium in regulation of epidermal proliferation and differentiation. The free intracellular calcium level of keratinocytes is measured using the calcium-binding dye indo-1 and flow cytometry. Changes effected by agents such as TPA, retinoids, cyclosporin and vitamins D will be quantiated. Retinoid acid causes a decrease in the free intracellular calcium concentration of keratinocytes; cell lines free of retinoid binding proteins and inhibitors of calcium transports will be used to study the mechanism. Changes in intracellular calcium will be correlated with changes in cell envelope formation and transglutaminase activity. LTB4 and PGE2 increase the intracellular calcium concentration of keratinocytes and the mechanism will be determined. The calmodulin-binding proteins of the epidermis will be identified using biotinylated calmodulin staining of nitrocellulose blots from epidermal extracts. The effect of TPA, steroids, cyclosporin and vitamin D on the content of these calmodulin-binding proteins and their ability to bind calmodulin will be measured. Monoclonal antibodies we have generated to the calmodulin-binding proteins will be used for subcellular localization of the proteins and functional studies. The calmodulin-dependent protein kinase from epidermis will be isolated and its endogenous substrate (s) identified. The role of extracellular calcium in keratinocyte proliferation and differentiation will be studied. The effect of growth in low calcium medium on intracellular calcium concentration will be measured. Calmodulin binding proteins from low normal calcium keratinocytes will identified using the biotinylated probes and the amount, distribution and binding to calmodulin will be measured. Results of these studies and the technology employed will be applied to disease states such as psoriasis and skin neoplasms.
| Fairley, J A (1990) Intracellular targets of cyclosporine. J Am Acad Dermatol 23:1329-32;discussion 1332-4 |
