Growth and differentiation stimuli induce different and distinct increases in intracellular free calcium in human keratinocytes.
The effect of growth and differentiation stimuli on intracellular free calcium ([Ca2+]i) in cultured human keratinocytes was investigated using micro-spectrofluorimetric techniques and the calcium-sensitive dye FURA-2. The mean [Ca2+]i of keratinocytes in 70 microM calcium medium was 104 +/- 3 nM (mean +/- SEM), significantly lower than the transformed keratinocyte line SVK14 (128 +/- 2 nM). When cultured in 2.0 mM calcium medium the [Ca2+]i increased in both normal and transformed keratinocytes to 135 +/- 4 nM and 180 +/- 4 nM, respectively. Keratinocytes grew more slowly in the absence of EGF, but [Ca2+]i was unaltered. Stimulation with EGF (10 ng/ml) induced, over 4 min, a large transient rise in [Ca2+]i up to 230 nm, due to an influx of extracellular calcium. Heterogeneity of keratinocytes was observed with 46% (n = 13) responding, but confluent or differentiated keratinocytes did not respond. TGF--beta (1 ng/ml) reduced cell growth without inducing differentiation and was not associated with any change in [Ca2+]i. The phorbol ester TPA (50 nM) induced irreversible growth arrest and terminal differentiation and increased the [Ca2+]i from 102 +/- 2 nM to 126 +/- 3 nM at 2 h, an effect similar to that of 2 mM extracellular calcium. Addition of 500 nM TPA was associated with a rise in [Ca2+]i, over several minutes to a plateau of 200-300 nM, due to release from internal stores and an influx of extracellular calcium. In normal human keratinocytes an increase in [Ca2+]i appears to be an early event in differentiation, whether induced by calcium or TPA, but not during growth inhibition without differentiation.
Sharpe
G R
GR
University Department of Dermatology, Royal Victoria Infirmary, Newcastle upon Tyne, UK.
Fisher
C
C
Gillespie
J I
JI
Greenwell
J R
JR
D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004815:Epidermal Growth Factor; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium