SUSPENSION-INDUCED MURINE KERATINOCYTE DIFFERENTIATION IS MEDIATED BYCALCIUM

Modulating extracellular Ca2+ (Ca-o) and suspension culture are two fr equently used methods to induce maturation of cultured human and mouse keratinocytes, To determine if the two methods share a common mechani sts, changes in Ca2+ metabolism were studied in suspension cultures of mouse keratinocytes. Spontaneously detached and suspension-cultured k eratinocytes in 0.05 mM Ca2+ medium express markers of suprabasal diff erentiation, while 0.05 mM Ca2+ is not permissive for marker expressio n by attached keratinocytes. Intracellular free Ca2+ (Ca-i) increased rapidly after placing keratinocytes in suspension in 0.05 mM Ca2+, rea ching levels up to 3- to 4-fold higher than Ca-i in attached cells aft er 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 w ell as depletion of intracellular Ca2+-stores. Diffrentiation marker e xpression and terminal differentiation were inhibited in suspension-cu ltured keratinocytes by preventing the rise of Ca-i using either 2-bis (o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid to chelate intracel lular Ca2+ or ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetr aacetic acid to reduce Ca-o. Together, these results indicate that a r ise in Ca-i is a common mechanism controlling differentiation in cultu red mouse keratinocytes, and suspension of keratinocytes enhances Ca2 transport and alters intracellular Ca2+ sequestration producing a ris e in Ca-i.