Early signals for keratinocyte differentiation: role of Ca2+-mediated inositol lipid metabolism in normal and neoplastic epidermal cells

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Abstract
Differentiation of cultured keratinocytes is regulated by the Ca2+ concentration of the culture medium. Below 0.1 mM Ca2+, a monolayer of basal cells is formed which fully differentiates in response to a rise in medium Ca2+. A role for protein kinase C in this differentiation program has been suggested because phorbol esters induce epidermal differentiation in cells grown in reduced Ca2+ medium, and exogenously added phospholipase C (which increases cellular diacylglycerol) mimics phorbol ester action. These findings suggested that the external Ca2+ signal may lead to protein kinase C activation via stimulation of cellular phospholipase C activity. The effect of the external Ca2+ signal on phospholipase C was studied in cultures prelabeled with [3H] inositol. Within 2 min after addition of Ca2+ to 1 mM, an increase in inositol phosphates was measured. This correlated with a decrease in radiolabeled phosphoinositides, suggesting that these were the source of the increased inositol phosphates. After 3 h in 1 mM Ca2+ medium, each of the inositol phosphates remained increased to 130–140% of control levels. Inositol phosphate metabolism in neoplastic epidermal cells was quantitatively similar to normal cells in response to the Ca2+ signal. Stimulation of phosphatidylinositol (PIP) metabolism appears to be mediated by a rise in intracellular free Ca2+ because Ca2+ ionophores A23187 and ionomycin also cause a similar rise in inositol phosphate levels. Phorbol esters did not increase PIP turnover but instead stimulated phosphatidylcholine metabolism. The induction of epidermal differentiation by phorbol esters was enhanced by ionomycin, suggesting that both protein kinase C activation, elevation of intracellular calcium and PIP turnover were important components of the signal for epidermal differentiation. These results demonstrate that the second messenger system for Ca2+ -mediated keratinocyte differentiation may be through a direct effect on phospholipase C activity.