Tumor necrosis factor-α, sphingomyelinase, and ceramide inhibit store-operated calcium entry in thyroid FRTL-5 cells

Tumor necrosis factor alpha (TNF-alpha) 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-alpha on calcium fluxes in FRTL-5 cells, TNF-alpha per se did not modulate resting [Ca2+](i). However, preincubation (10 min) of the cells with 1-100 ng/ml TNF-alpha decreased the thapsigargin (Tg)-evoked store-operated calcium entry in a concentration-dependent manner. TNF-alpha did not inhibit the mobilization of sequestered calcium, To investigate whether the effect of TNF-alpha 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 C-2-ceramide and C-6-ceramide analogues, The inactive ceramides dihydro-C-2 and dihydro-C-6 showed only marginal effects, Neither SMase, C-2-ceramide, nor C-6-ceramide affected the release of sequestered calcium. C-2- and C-6-ceramide also decreased the ATP-evoked calcium entry, without affecting the release of sequestered calcium. The effect of TNF-alpha 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, Ive were unable to measure a significant activation of PKC using TNF-alpha or C-6-ceramide, The effect of TNF-alpha was not mediated via activation of either c-Sun 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-alpha for up to 30 min, Thus, one mechanism of action of TNF-alpha; SMase, and ceramide on thyroid FRTL-5 cells is to inhibit calcium entry.