J Biol Chem, Vol. 274, Issue 14, 9370-9377, April 2, 1999

Tumor Necrosis Factor-, Sphingomyelinase, and Ceramide Inhibit Store-operated Calcium Entry in Thyroid FRTL-5 Cells

Kid Törnquist^§, Anna-Maria Malm^§, Michael Pasternack^**, Robert Kronqvist, Sonja Björklund, Raimo Tuominen^§§, and J. Peter Slotte

From the  Department of Biology and the  Department of Biochemistry and Pharmacy, Åbo Akademi University, BioCity, 20520 Turku, Finland and the ^§§ Department of Pharmacology and Toxicology, Institute of Biomedicine, the  Department of Biosciences, Division of Animal Physiology, and the ^** Institute of Biotechnology, ^§ University of Helsinki, and the Minerva Foundation Institute for Medical Research, 00250 Helsinki, Finland

Tumor necrosis factor  (TNF-) is a potent inhibitor of proliferation in several cell types, including thyroid FRTL-5 cells. As intracellular free calcium ([Ca²⁺]_i) is a major signal in activating proliferation, we investigated the effect of TNF- on calcium fluxes in FRTL-5 cells. TNF- per se did not modulate resting [Ca²⁺]_i. However, preincubation (10 min) of the cells with 1-100 ng/ml TNF- decreased the thapsigargin (Tg)-evoked store-operated calcium entry in a concentration-dependent manner. TNF- did not inhibit the mobilization of sequestered calcium. To investigate whether the effect of TNF- on calcium entry was mediated via the sphingomyelinase pathway, the cells were pretreated with sphingomyelinase (SMase) prior to stimulation with Tg. SMase inhibited the Tg-evoked calcium entry in a concentration-dependent manner. Furthermore, an inhibition of calcium entry was obtained after preincubation of the cells with the membrane-permeable C₂-ceramide and C₆-ceramide analogues. The inactive ceramides dihydro-C₂ and dihydro-C₆ showed only marginal effects. Neither SMase, C₂-ceramide, nor C₆-ceramide affected the release of sequestered calcium. C₂- and C₆-ceramide also decreased the ATP-evoked calcium entry, without affecting the release of sequestered calcium. The effect of TNF- and SMase was inhibited by the kinase inhibitor staurosporin and by the protein kinase C (PKC) inhibitor calphostin C but not by down-regulation of PKC. However, we were unable to measure a significant activation of PKC using TNF- or C₆-ceramide. The effect of TNF- was not mediated via activation of either c-Jun N-terminal kinase or p38 kinase. We were unable to detect an increase in the ceramide (or sphingosine) content of the cells after stimulation with TNF- for up to 30 min. Thus, one mechanism of action of TNF-, SMase, and ceramide on thyroid FRTL-5 cells is to inhibit calcium entry.