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.
Li
L
L
Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Etiology, National Cancer Institute, Bethesda, MD 20892-4255, USA.
Tennenbaum
T
T
Yuspa
S H
SH
D000818:Animals; D000943:Antigens, Differentiation / Q000032:analysis / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology*; D002132:Calcium Radioisotopes / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D013997:Time Factors
D000943:Antigens, Differentiation; D002132:Calcium Radioisotopes; D002614:Chelating Agents; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium