title: Tumor necrosis factor-alpha, sphingomyelinase, and ceramide inhibit store-operated calcium entry in thyroid FRTL-5 cells.
abstract: 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 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-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, we were unable to measure a significant activation of PKC using TNF-alpha or C6-ceramide. The effect of TNF-alpha 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-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.
authors: Department of Biology, Abo Akademi University, BioCity, 20520 Turku, Finland. kid.tornqvist@abo.fi
authors: K
authors: K
authors: Törnquist
authors: A M
authors: AM
authors: Malm
authors: M
authors: M
authors: Pasternack
authors: R
authors: R
authors: Kronqvist
authors: S
authors: S
authors: Björklund
authors: R
authors: R
authors: Tuominen
authors: J P
authors: JP
authors: Slotte
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002518:Ceramides / Q000494:pharmacology*; D004261:DNA Replication / Q000187:drug effects; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013110:Sphingosine / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D015220:Calcium Channels; D002518:Ceramides; D004791:Enzyme Inhibitors; D014409:Tumor Necrosis Factor-alpha; C101954:N-caproylsphingosine; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D013108:Sphingomyelin Phosphodiesterase; D013110:Sphingosine; D002118:Calcium