Abstract
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.
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