Growth and differentiation stimuli induce different and distinct increases in intracellular free calcium in human keratinocytes

Abstract

The effect of growth and differentiation stimuli on intracellular free calcium ([Ca²⁺]_i) in cultured human keratinocytes was investigated using micro-spectrofluori-metric techniques and the calcium-sensitive dye FURA-2. The mean [Ca²⁺]_i of keratinocytes in 70 ΜM 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-Β (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.