Suspension-induced murine keratinocyte differentiation is mediated by calcium

Modulating extracellular Ca2+ (Cao) and suspension culture are two frequently used methods to induce maturation of cultured human and mouse keratinocytes. To determine if the two methods share a common mechanism, changes in Ca2+ metabolism were studied in suspension cultures of mouse keratinocytes. Spontaneously detached and suspension- cultured keratinocytes in 0.05 microM Ca2+ medium express markers of suprabasal differentiation, while 0.05 microM Ca2+ is not permissive for marker expression by attached keratinocytes. Intracellular free Ca2+ (Cai) increased rapidly after placing keratinocytes in suspension in 0.05 microM Ca2+, reaching levels up to 3- to 4-fold higher than Cai in attached cells after 4-5 h. In suspended cells, the increase in Cai was associated with a 2- to 6- fold increase in Ca2+ transport across plasma membrane as well as depletion of intracellular Ca2+ -stores. Differentiation marker expression and terminal differentiation were inhibited in suspension-cultured keratinocytes by preventing the rise of Cai using either 1,2-bis (o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid to chelate intracellular Ca2+ or ethyleneglycol-bis (beta-aminoethyl ether)- N,N,N',N' -tetraacetic acid to reduce Cao. Together these results indicate that a rise in CAi is a common mechanism controlling differentiation in cultured mouse keratinocytes, and suspension of keratinocytes enhances Ca2+ transport and alters intracellular Ca2+ sequestration producing a rise in Cai.