Suspension-induced murine keratinocyte differentiation is mediated by calcium

Modulating extracellular Ca²⁺ (Ca(o)) 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 mM Ca²⁺ medium express markers of suprabasal differentiation, while 0.05 mM Ca²⁺ is not permissive for marker expression by attached keratinocytes. Intracellular free Ca²⁺ (Ca(i)) increased rapidly after placing keratinocytes in suspension in 0.05 mM Ca²⁺, reaching levels up to 3-to 4-fold higher than Ca(i) in attached cells after 4-5 h. In suspended cells, the increase in Ca(i) 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 Ca(i) using either 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid to chelate intracellular Ca²⁺ or ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid to reduce Ca(o). Together, these results indicate that a rise in Ca(i) 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 Ca(i).