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• W2068895167 abstract "Tumor necrosis factor α (TNF-α) 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-α on calcium fluxes in FRTL-5 cells. TNF-α per se did not modulate resting [Ca2+]i. However, preincubation (10 min) of the cells with 1–100 ng/ml TNF-α decreased the thapsigargin (Tg)-evoked store-operated calcium entry in a concentration-dependent manner. TNF-α did not inhibit the mobilization of sequestered calcium. To investigate whether the effect of TNF-α 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-α 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-α or C6-ceramide. The effect of TNF-α 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-α for up to 30 min. Thus, one mechanism of action of TNF-α, SMase, and ceramide on thyroid FRTL-5 cells is to inhibit calcium entry. Tumor necrosis factor α (TNF-α) 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-α on calcium fluxes in FRTL-5 cells. TNF-α per se did not modulate resting [Ca2+]i. However, preincubation (10 min) of the cells with 1–100 ng/ml TNF-α decreased the thapsigargin (Tg)-evoked store-operated calcium entry in a concentration-dependent manner. TNF-α did not inhibit the mobilization of sequestered calcium. To investigate whether the effect of TNF-α 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-α 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-α or C6-ceramide. The effect of TNF-α 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-α for up to 30 min. Thus, one mechanism of action of TNF-α, SMase, and ceramide on thyroid FRTL-5 cells is to inhibit calcium entry. tumor necrosis factor α protein kinase C phorbol 12-myristate 13-acetate sphingomyelinase sphingosine sphingosine 1-phosphate dihydroceramide C2 resp C6 intracellular free calcium concentration thapsigargin thyrotropin Jun kinase 1 1,2-sn-dioctanoyl-acylglycerol phosphatidylserine phosphate-buffered saline nuclear factor κB An abundance of reports has shown that the cytokine tumor necrosis factor-α (TNF-α)1 has diverse effects upon several cell systems. TNF-α also potently modulates thyroid function, especially growth and differentiation. In humans, the injection of TNF-α decreases serum triiodothyronine and TSH levels (1van der Poll T. Romijn J.A. Wiersinga W.M. Sauerwein H.P. J. Clin. Endocrinol. & Metab. 1990; 71: 1567-1572Crossref PubMed Scopus (250) Google Scholar), whereas in rats and mice TNF-α decreases both serum triiodothyronine and thyroxine levels and serum TSH levels (2Ozawa M. Sato K. Han D.C. Kawakami M. Tsushima T. Shizume K. Endocrinology. 1988; 123: 1461-1467Crossref PubMed Scopus (119) Google Scholar, 3Pang X.-P. Hershman J.M. Chung M. Pekary A.E. Endocrinology. 1989; 125: 1783-1788Crossref PubMed Scopus (117) Google Scholar). In human thyroid cells in culture, TNF-α decreases the TSH-evoked incorporation of 125I and the secretion of triiodothyronine and thyroxine (4Sato K. Satoh T. Shizume K. Ozawa M. Han D.C. Imamura H. Tsushima T. Demura H. Kanaji Y. Ito Y. Obara T. Fujimoto Y. J. Clin. Endocrinol. & Metab. 1990; 70: 1735-1743Crossref PubMed Scopus (157) Google Scholar). TNF-α attenuates also the production of thyroglobulin and cAMP in these cells (5Rasmussen Å.K. Kayser L. Feldt-Rasmussen U. Bendtzen K. J. Endocrinol. 1994; 143: 359-365Crossref PubMed Scopus (57) Google Scholar). In rat FRTL-5 cells, TNF-α inhibits the TSH-evoked uptake of iodide and inhibits mitogen-evoked cell proliferation (6Zakarija M. McKenzie J.M. Endocrinology. 1989; 125: 1260-1265Crossref PubMed Scopus (44) Google Scholar, 7Patwardhan N.A. Lombardi A. Surgery. 1991; 110: 972-977PubMed Google Scholar, 8Pang X.-P. Yoshimura M. Hershman J.M. Thyroid. 1993; 3: 325-330Crossref PubMed Scopus (38) Google Scholar). Furthermore, TNF-α inhibits the TSH-evoked type I 5′-deiodinase activity, the expression of both the thyroid peroxidase gene and the thyroglobulin gene (9Ongphiphadhanakul O. Fang S.L. Tang K.-T. Patwardhan N.A. Braverman L.E. Eur. J. Endocrinol. 1994; 130: 502-507Crossref PubMed Scopus (35) Google Scholar, 10Tan K.-T. Braverman L.E. DeVito W.J. Endocrinology. 1995; 136: 881-888Crossref PubMed Scopus (67) Google Scholar, 11Mori K. Stone S. Braverman L.E. DeVito W.J. Endocrinology. 1996; 137: 4994-4999Crossref PubMed Scopus (13) Google Scholar), and TSH-evoked hydrogen peroxide production (12Kimura T. Okajima F. Kikuchi T. Kuwabara A. Tomura H. Sho K. Kobayashi I. Kondo Y. Am. J. Physiol. 1997; 273: E638-E643Google Scholar). TNF-α binds to two membrane receptors, a 55- and a 75-kDa receptor. Of these two forms, the 55-kDa receptor apparently is the most important (13Wiegmann K. Schutze S. Kampen E. Himmler A. Machleidt T. Kronke M. J. Biol. Chem. 1992; 267: 17997-18001Abstract Full Text PDF PubMed Google Scholar, 14Yanaga F. Watson S.P. FEBS Lett. 1992; 314: 297-300Crossref PubMed Scopus (57) Google Scholar). Binding of TNF-α to FRTL-5 cells has also been reported (3Pang X.-P. Hershman J.M. Chung M. Pekary A.E. Endocrinology. 1989; 125: 1783-1788Crossref PubMed Scopus (117) Google Scholar), although the receptor types have not been characterized. TNF-α activates different sphingomyelinases in cells, resulting in the hydrolysis of sphingomyelin to ceramide and stimulation of the mitogen-activated protein kinase cascade, or the Jun kinase 1 (JNK-1) cascade (15Testi R. Trends Biochem. Sci. 1996; 21: 468-471Abstract Full Text PDF PubMed Scopus (193) Google Scholar). TNF-α may also activate protein kinase C (PKC) (13Wiegmann K. Schutze S. Kampen E. Himmler A. Machleidt T. Kronke M. J. Biol. Chem. 1992; 267: 17997-18001Abstract Full Text PDF PubMed Google Scholar). The ceramide-evoked activation of NFκB is probably important in linking the TNF-α-evoked stimulus to transcriptional activity in the nucleus (16Schütze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Krönke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (971) Google Scholar, 17Yang Z. Costanzo M. Golde D.W. Kolesnick R.N. J. Biol. Chem. 1993; 268: 20520-20523Abstract Full Text PDF PubMed Google Scholar). In human papillary thyroid carcinoma cells TNF-α has been shown to activate NFκB (18Pang X.-P. Ross N.S. Park M. Juillard G.J.F. Stanley T.M. Hershman J.M. J. Biol. Chem. 1992; 267: 12826-12830Abstract Full Text PDF PubMed Google Scholar), indicating that this signaling pathway also is present in thyroid cells. The type of SMase activated upon stimulation is apparently crucial for the ultimate fate of the cells, as the SMase-evoked production of ceramide may lead to activation of apoptosis (via JNK-1), stimulate proliferation (via mitogen-activated protein kinase), or protection against cytotoxicity (15Testi R. Trends Biochem. Sci. 1996; 21: 468-471Abstract Full Text PDF PubMed Scopus (193) Google Scholar, 19Barger S.W. Hörster D. Furukawa K. Goodman Y. Krieglstein J. Mattson M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9328-9332Crossref PubMed Scopus (562) Google Scholar). Of the reported effects of TNF-α on FRTL-5 cells, the inhibition of proliferation (20Chen G. Pekary A.E. Hershman J.M. Endocrinology. 1992; 131: 863-870PubMed Google Scholar), the inhibition of type I 5′deodinase activity (11Mori K. Stone S. Braverman L.E. DeVito W.J. Endocrinology. 1996; 137: 4994-4999Crossref PubMed Scopus (13) Google Scholar), and the inhibition of TSH-evoked production of hydrogen peroxidase (12Kimura T. Okajima F. Kikuchi T. Kuwabara A. Tomura H. Sho K. Kobayashi I. Kondo Y. Am. J. Physiol. 1997; 273: E638-E643Google Scholar) have also been induced by exogenous ceramide, suggesting that these events are the result of the TNF-α-evoked activation of a sphingomyelinase and the hydrolysis of sphingomyelin to ceramide. Other sphingomyelin breakdown products, like sphingosine (SP) and sphingosine 1-phosphate (SPP), potently stimulate proliferation and mobilize sequestered calcium in several cell types (21Spiegel S. Milstein S. J. Membr. Biol. 1995; 146: 225-237Crossref PubMed Scopus (225) Google Scholar). These effects of SP and SPP have also been observed in thyroid FRTL-5 cells (22Törnquist K. Ekokoski E. Biochem. J. 1994; 299: 213-218Crossref PubMed Scopus (33) Google Scholar, 23Okajima F. Tomura H. Sho K. Kimura T. Sato K. Im D.-S. Akbar M. Kondo Y. Endocrinology. 1997; 138: 220-229Crossref PubMed Scopus (62) Google Scholar, 24Törnquist K. Saarinen P. Vainio M. Ahlström M. Endocrinology. 1997; 138: 4049-4057Crossref PubMed Scopus (34) Google Scholar). Recent reports also show that SP attenuated store-operated calcium entry (25Orlati S. Cavazzoni M. Rugolo M. Cell Calcium. 1996; 20: 399-407Crossref PubMed Scopus (4) Google Scholar, 26Mathes C. Fleig A. Penner R. J. Biol. Chem. 1998; 273: 25020-25030Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Sphingosines and ceramides seem to have mostly opposite effects on cellular proliferation. As both SP and SPP mobilize sequestered calcium and stimulate calcium entry in FRTL-5 cells, two important events in the initiation of proliferation, we thought that it would be of interest to investigate whether ceramides could have any effect on the regulation of calcium fluxes in these cells. Our results showed that, in FRTL-5 cells, TNF-α, SMase, and ceramides potently attenuated calcium entry. Thus, one mechanism of action of TNF-α on thyroid cells is an inhibition of calcium entry. Culture medium, serum, and hormones needed for the cell culture was purchased from Life Technologies, Inc., Biological Industries (Beth Haemek, Israel), and Sigma. Culture dishes were obtained from Falcon Plastics (Oxnard, CA) or from Greiner (Germany). GF109203X, N-acetylsphingosine (C2-ceramide), and N-hexanoylsphingosine (C6-ceramide) and the inactive forms N-acetylsphinganine (dihydro-C2) and N-hexanoylsphinganine (dihydro-C6) were purchased from Biomol (Plymouth Meeting, PA). Phenylmethylsulfonyl fluoride, 1,2-sn-dioctanoyl-acylglycerol (DAG), phosphatidylserine (PS), Triton X-100, staurosporin, phorbol 12-myristate 13-acetate (PMA), and sphingomyelinase were all purchased from Sigma. Human recombinant tumor necrosis factor-α, calphostin C, and D609 were from Alexis Corp. (Laufelfingen, Switzerland). The anti-active c-Jun N-terminal kinase was from Promega (Madison, WI). The p38 kinase inhibitor SB203580 was from Calbiochem. Fura 2-AM and bisoxonol were purchased from Molecular Probes, Inc. (Eugene, OR). Thapsigargin was from LC Services Corp. (Woburn, MA). Lipid standards (bovine brain sphingomyelinase, ceramide, and sphingosine) were obtained from Sigma. All other chemicals used were of reagent grade. Whatman P81 phosphocellulose paper was purchased from Whatman (UK). [γ-32P]ATP is a product of Amersham Corp. (UK). Bovine TSH was a generous gift from Dr. A. F. Parlow (NHPP, NIDDK, National Institutes of Health). Rat thyroid FRTL-5 cells were a generous gift of Dr. Egil Haug (Akers Hospital, Oslo, Norway). The cells were grown in Coon's modified Ham's F-12 medium, supplemented with 5% calf serum, and six hormones (27Ambesi-Impiombato F.S. Parks L.A.M. Coon H.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3455-3459Crossref PubMed Scopus (974) Google Scholar) (insulin, 10 μg/ml; transferrin, 5 μg/ml; hydrocortisone, 10 nm; the tripeptide Gly-l-His-l-Lys, 10 ng/ml; TSH, 0.3 milliunits/ml; somatostatin, 10 ng/ml) in a water-saturated atmosphere of 5% CO2 and 95% air at 37 °C. Before an experiment, cells from one donor culture dish were harvested with a 0.2% trypsin solution and plated onto plastic 100- or 35-mm culture dishes. The cells were grown for 7–8 days before an experiment, with 2–3 changes of the culture medium. Fresh medium was always added 24 h prior to an experiment. In the current-clamp experiments, the cells were grown on round coverslips in 24-well culture dishes. The medium was aspirated, and the cells were harvested with HEPES-buffered saline solution (HBSS, in millimolar concentrations: NaCl, 118; KCl, 4.6; glucose, 10; CaCl2, 1.0; HEPES, 20; pH 7.2) lacking Ca2+ but containing 0.02% EDTA and 0.1% trypsin. After washing the cells three times by pelleting, the cells were incubated with 1 μm Fura 2-AM for 30 min at 37 °C. Following the loading period, the cells were washed twice with HBSS buffer and incubated for at least 10 min at room temperature and washed once again. Fluorescence was measured with a Hitachi F2000 fluorimeter. The excitation wavelengths were 340 and 380 nm, and emission was measured at 510 nm. The signal was calibrated by addition of 1 mmCaCl2 and Triton X-100 to obtain maximal fluorescence. Chelating extracellular Ca2+ with 5 mm EGTA and the addition of Tris-base was used to elevate pH above 8.3 to obtain minimal fluorescence. [Ca2+]i was calculated as described by Gryenkiewicz et al. (28Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar), using a computer program designed for the fluorimeter with a K d value of 224 nm for Fura 2. Cells grown on 35-mm dishes were labeled withl-[3-3H]serine (5 μCi/ml) for 48 h in 6H medium. The plates were then washed twice with PBS (in millimolar concentrations; NaCl, 137; KCl, 2.7; Na2HPO4, 8, KH2PO4, 1.5; pH 7.4) and incubated for 1 h in serum-free Ham's F-12 medium in a water bath at 37 °C. Then TNF-α (final concentration 100 ng/ml) was added, and the plates were incubated for 3–30 min. In some experiments SMase (final concentration 100 milliunits/ml diluted in Ham's F-12) was added to the plates, and the plates were incubated for 30 min. After the incubation, the plates were rapidly washed with ice-cold PBS and then frozen. Lipids were extracted by two 20-min incubations in 2 ml of hexane:propanol (3:2, v/v) on a shaker at room temperature. For protein measurements, the proteins were hydrolyzed in 1 ml of 0.1 mNaOH and determined according to Lowry et al. (29Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). The lipid extracts were pipetted to glass tubes and dried in a gentle stream of air. The dried lipids were dissolved in 70 μl of hexane:2-propanol. Sphingomyelin was determined by application of the lipid extract to plastic-backed TLC plates (Whatman). The lipids were separated using chloroform:methanol:concentrated acetic acid:water (50:30:8:3) (30Skipski V.P. Peterson R.F. Barclay M. Biochem. J. 1964; 90: 374-378Crossref PubMed Scopus (930) Google Scholar). After detection using iodine vapor, the appropriate bands were cut, and radioactivity was measured in a scintillation counter. Ceramide and sphingosine were separated using high performance TLC plates (Merck), and the lipids were separated by two elutions using chloroform:methanol:2 n NH4OH (40:10:1) (31Sambasivarao K. McCluer R.H. J. Lipid Res. 1963; 4: 106-108Abstract Full Text PDF PubMed Google Scholar). The plates were allowed to dry between the separate runs. The lipids were detected using iodine vapor, and the appropriate bands were scraped into scintillation vials, and the radioactivity was determined. Immediately before exposure of the cells, the 6H medium was removed from the wells and the treatment was started by adding 0H medium containing various concentrations of the substances. Protein kinase C (PKC) activity was measured by the method of Kikkawa et al. (32Kikkawa U. Minakuchi R. Takai Y. Nishizuka Y. Methods Enzymol. 1983; 99: 288-298Crossref PubMed Scopus (246) Google Scholar) and Roskoski (33Roskoski Jr., R. Methods Enzymol. 1983; 99: 3-6Crossref PubMed Scopus (691) Google Scholar), with some modifications (34Tuominen R. McMillian M. Ye H. Hudson P. Stachowiak M. Hong J. J Neurochem. 1992; 58: 1652-1658Crossref PubMed Scopus (40) Google Scholar). The experiments were terminated by removing the medium and washing the cells three times with an ice-cold Ca2+-free salt solution (in millimolar concentrations: NaCl, 145; KCl, 5.2; NaH2PO4, 1; glucose, 11.2; HEPES, 15; pH 7.4). The cells were scraped from the plates and homogenized by sonication (2 × 15 s) in an ice-cold lysis buffer (containing in millimolar concentrations: EDTA, 2; phenylmethylsulfonyl fluoride, 1; Tris-HCl, 20; pH 7.5, and 50 μg/ml leupeptin). Homogenates were centrifuged for 60 min at 100,000 ×g at 4 °C. The supernatant served as the soluble fraction. The pellet was dispersed into the same buffer containing 0.1% Triton X-100, and the homogenate was incubated on ice for 60 min. The mixture was centrifuged at 100,000 × g for 60 min at 4 °C. This supernatant constituted the particulate PKC activity. The protein content in both subcellular fractions was measured according to Bradford (35Bradford M.M. Anal. Biochem. 1976; 72: 238-259Crossref PubMed Scopus (215575) Google Scholar). In the PKC assay, the final reaction mixture (100 μl) contained in millimolar concentrations the following: Tris-HCl, 35; pH 7.5; mm EGTA, 0.25; EDTA, 0.5; MgCl2, 6; phenylmethylsulfonyl fluoride, 0.25; PKC-specific substrate peptide FKKSFKL-NH2, 34 nm (36Chakravarthy B.R. Bussey A. Whitfield J.F. Sikorska M. Williams R.E. Durkin J.P. Anal. Biochem. 1991; 196: 144-150Crossref PubMed Scopus (113) Google Scholar); CaCl2, 1; and 0.1 mm [32P]ATP (100–200 cpm/pmol). The mixture also contained leupeptin (12.5 μg/ml), phosphatidylserine (PS, 40 μg/ml), and diacylglycerol (DAG, 8 μg/ml). PKC activity was calculated as the difference in the activity in the presence and absence of CaCl2, PS, and DAG. The activity in the absence of CaCl2, PS, and DAG was the same as that obtained when only PS and DAG were omitted. The reaction was started by adding protein (0.7–1.5 μg). The samples were incubated for 5 min at 30 °C, and the reaction was stopped by spotting 25 μl of each reaction mixture onto Whatman P81 phosphocellulose paper (1.5 × 1.5 cm). The papers were washed three times in 75 mm phosphoric acid. After air-drying, the radioactivity measured was determined. The results are expressed as nanomoles of inorganic phosphate incorporated to substrate peptide/mg of protein/min. SDS-polyacrylamide gel electrophoresis was run using a minigel apparatus (Midget Electrophoresis Unit, Pharmacia, Sweden). Proteins (1 and 3 μg per well for soluble and particulate proteins, respectively) were loaded onto 8% polyacrylamide-SDS gels and separated according to molecular weight. The proteins were electrophoretically transferred to methylcellulose membranes. The membranes were incubated three times for 15 min at 45 °C in Tween/TBS (TTBS, containing in millimolar concentrations: NaCl, 500; Tris-base, 20; pH 7.5, and 0.1% Tween 20) containing 5% fat-free dry milk and 15 min in TTBS. Then the membranes were incubated for 2 h with 1:4000–1:80,000 dilution of rabbit polyclonal anti-rat PKC antibodies that recognize α, β1, β2, γ, δ, ε, and τ subtypes of PKC (37Wetsel W. Khan W. Merchenthaler I. Rivera H. Halpern A. Phung H. Negro-Vilar A. Hannun Y. J. Cell Biol. 1992; 117: 121-133Crossref PubMed Scopus (394) Google Scholar). A horseradish peroxidase-labeled goat anti-rabbit antibody (Bio-Rad) was used as the secondary antibody, and the immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Amersham Corp., UK). The localization of immunoreactive proteins was compared with those of prestained molecular weight markers (Life Technologies, Inc.). The cells were grown on 60- or 100-mm plates as described above. The cells were harvested as above and were allowed to rest for 30 min at 37 °C in HBSS. After incubation with TNF-α for 1–30 min, the cells were centrifuged and extracted in ice-cold lysis buffer (containing in mm concentrations: NaCl, 100; Na3VO4, 2; EDTA, 2; Tris-base, 20 mm, pH 8.0; and 3% Nonidet P-40). A sample of the extract was mixed with an equal volume of boiling SDS buffer (glycerol, 20%; 2-mercaptoethanol, 10%; SDS, 4%; bromphenol blue, 0.02%; Tris-base, 0.125 mm, pH 6.8). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels. The proteins were transferred electrophoretically to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). The membrane was incubated with 5% nonfat dry milk for 1 h at room temperature in Tris-buffered saline (TBS, in mm concentrations: NaCl, 500; Tris-base, 20, pH 7.5) to block the remaining binding sites. The blots were incubated with anti-active JNK antibody (1:5000) diluted in TBS containing 5% nonfat dry milk at 4 °C overnight. The blots were then incubated with peroxidase-conjugated anti-rabbit antibody (1:10 000) for 2 h at room temperature, and the proteins were detected using the ECL Western blotting detection kit according to the manufacturer's instructions. The cells were plated onto 35-mm dishes and grown in 6H medium for 2–3 days. Then the cells were washed twice with PBS and grown in 0H (Coon's medium without hormones or serum) containing 0.2% bovine serum albumin for 2 days. The medium was then changed to 0H/bovine serum albumin containing the appropriate concentrations of the test compounds and [3H]thymidine (0.4 μCi/ml), and the cells were incubated for 24 h (38Valente W.A. Vitti P. Kohn L.D. Brandi M. Toccafondi R. Tramontano D. Azou S.M. Ambesi-Impiombato F.S. Endocrinology. 1983; 112: 71-79Crossref PubMed Scopus (192) Google Scholar). The cells were washed twice with cold PBS solution and once with cold 5% trichloroacetic acid. The trichloroacetic acid-insoluble precipitate was dissolved in 0.1 n NaOH, and the radioactivity was measured by scintillation counting. The results are expressed as the means ± S.E. Statistical analysis was made using Student's t test for paired observations. When three or more means were tested, analysis of variance was used. Previous studies have shown that TNF-α and C2- and C6-ceramides potently inhibit both the TSH- and the insulin-evoked incorporation of [3H]thymidine in DNA, i.e. DNA synthesis (6Zakarija M. McKenzie J.M. Endocrinology. 1989; 125: 1260-1265Crossref PubMed Scopus (44) Google Scholar, 7Patwardhan N.A. Lombardi A. Surgery. 1991; 110: 972-977PubMed Google Scholar, 8Pang X.-P. Yoshimura M. Hershman J.M. Thyroid. 1993; 3: 325-330Crossref PubMed Scopus (38) Google Scholar, 20Chen G. Pekary A.E. Hershman J.M. Endocrinology. 1992; 131: 863-870PubMed Google Scholar). We confirmed these results and further showed that SMase also inhibited the incorporation of [3H]thymidine in response to TSH and insulin (data not shown). The inactive ceramides dihydro-C2 and dihydro-C6 had only marginal effects (data not shown). In FRTL-5 cells, as well as in other cell types, changes in [Ca2+]i are probably important events in the initialization of cell proliferation (39Takada K. Amino N. Tada H. Miyai K. J. Clin. Invest. 1990; 86: 1548-1555Crossref PubMed Scopus (60) Google Scholar, 40Törnquist K. Ekokoski E. Dugué B. J. Cell. Physiol. 1996; 166: 241-248Crossref PubMed Scopus (45) Google Scholar). We thus investigated the effect of TNF-α on [Ca2+]i and on the entry of calcium in FRTL-5 cells. TNF-α (100 ng/ml, the highest dose tested) did not per se affect [Ca2+]i in these cells (data not shown). To avoid any possible effects of TNF-α on receptor-mediated events, we activated calcium entry by stimulating the cells with the Ca2+ATPase inhibitor thapsigargin (Tg) (41Thastrup O. Cullen P.J. Drøbak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2997) Google Scholar). Tg activates a rapid store-operated calcium entry in FRTL-5 cells (42Törnquist K. Biochem. J. 1993; 290: 443-447Crossref PubMed Scopus (55) Google Scholar). Pretreatment of the cells with TNF-α for 10–30 min potently attenuated the Tg-evoked calcium entry in both a calcium-containing buffer and in a calcium-free buffer in a concentration-dependent manner (Fig. 1). We also observed that TNF-α did not inhibit the Tg-evoked mobilization of sequestered calcium. In cells pretreated with 100 ng/ml TNF-α for 10 min, the Tg-evoked release of intracellular calcium was 178 ± 15 nm, compared with 155 ± 10 nm in control cells. These experiments were performed in a calcium-free buffer to avoid any interference of Tg-evoked calcium entry. To investigate whether the observed effect of TNF-α was due to activation of a sphingomyelinase, we preincubated the cells with the phosphatidylcholine-phospholipase C inhibitor D609. Previous investigations have shown that D609 effectively inhibits TNF-α-evoked events (16Schütze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Krönke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (971) Google Scholar). However, we observed that D609 was a very potent modulator of calcium entry in FRTL-5 cells (data not shown). Furthermore, D609 also mobilized sequestered calcium in our cells (data not shown). Thus, D609 is apparently not a suitable compound for studies using intact cells, as its effects on calcium fluxes probably will affect a multitude of cellular events. Activation of protein kinases, including PKC, is an important part of the signaling cascade evoked by TNF-α (15Testi R. Trends Biochem. Sci. 1996; 21: 468-471Abstract Full Text PDF PubMed Scopus (193) Google Scholar). We preincubated the cells with 200 nm staurosporin for 10 min prior to addition of 100 ng/ml TNF-α. In these cells, we were unable to detect any TNF-α-evoked inhibition of calcium entry in cells stimulated with Tg (Fig. 2). We next investigated the effect of the PKC inhibitor calphostin C, and we treated the cells with 100 nm calphostin C for 10 min prior to addition of 100 ng/ml TNF-α. In these cells, the effect of TNF-α on the Tg-evoked increase in [Ca2+]i was abolished (Fig. 2). We have also shown earlier that stimulating FRTL-5 cells with the phorbol ester PMA attenuates store-operated calcium entry (42Törnquist K. Biochem. J. 1993; 290: 443-447Crossref PubMed Scopus (55) Google Scholar). In the present study, pretreatment with 200 nm PMA significantly decreased the plateau level of [Ca2+]i after readdition of calcium to cells stimulated with Tg in a calcium-free buffer (Fig. 2). Pretreatment of the cells with both PMA and 100 ng/ml TNF-α decreased both the transient increase in [Ca2+]i as well as the new plateau level of [Ca2+]i (Fig. 2). In addition, in these cells the increase in the plateau level of [Ca2+]i was of lower magnitude than in cells treated with PMA only, suggesting an additive effect of PMA and TNF-α on calcium entry (Fig. 2). To investigate further whether the effect of TNF-α on calcium entry was mediated via activation of SMase, we preincubated the cells with different concentrations of exogenous SMase for 30 min. As shown in Fig. 3, SMase inhibited calcium entry in a concentration-dependent manner very similar to that of TNF-α. Furthermore, SMase did not affect the amount of sequestered calcium. In cells treated with SMase (1 units/ml) for 30 min, the increase in [Ca2+]i evoked by Tg in a calcium-free buffer was 130 ± 23 nm, compared with 155 ± 10 nm in control cells. In the next series of experiments, the cells were incubated with 200 nm staurosporin for 10 min prior to addition of SMase (1 units/ml for 30 min). In these experiments, pretreatment with staurosporin totally abolished the effect of SMAse (Fig.4), in a manner similar to what was observed in cells treated with both staurosporin and TNF-α. To investigate whether the effect of SMase was mediated via activation of PKC, we pretreated the cells with 100 nm calphostin C. In these experiments calphostin C also abolished the effect of SMase (Fig.4). However, in cells in which PKC was down-regulated by preincubating the cells with 2 μm PMA for 24 h, SMase (1 milliunit/ml for 30 min) still attenuated the Tg-evoked calcium entry (Fig. 4). To investigate whether the observed effect of SMase was due to inhibition o" @default.
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• W2068895167 title "Tumor Necrosis Factor-α, Sphingomyelinase, and Ceramide Inhibit Store-operated Calcium Entry in Thyroid FRTL-5 Cells" @default.
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