Ca2+-ATPase inhibitors and PKC activation synergistically stimulate TNF-alpha production in RBL-2H3 cells.
OBJECTIVE
To investigate the effect of Ca2+-ATPase inhibitors on the production of TNF-alpha in rat basophilic leukemia (RBL-2H3) cells.


METHODS
Two Ca2+-ATPase inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), and three hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ), 2,5-di-(tert/amyl)-1,4-hydroquinone (DTAHQ), 2-(tertbutyl)-1,4-hydroquinone (MTBHQ) were used.


METHODS
Cells were treated with TG, CPA, DTBHQ, DTAHQ and MTBHQ for 3 h in the presence of 12-Otetradecanoylphorbol-13-acetate (TPA) and released TNF-alpha from the cells was measured (n > or = 4).


RESULTS
All Ca2--ATPase inhibitors (TG, CPA, DTBHQ and DTAHQ) induced TNF-alpha release in a dose-dependent manner. TNF-alpha release was inhibited by treatment with protein kinase C inhibitors (staurosporine, Ro31-8220, calophostin C) (p < or = 0.05). In contrast, MTBHQ, which does not induce increases in [Ca2+]i, did not induce the release of TNF-alpha. TNF-alpha release induced by DTBHQ and CPA was inhibited by treatment with actinomycin-D, the immunosuppressant FK506 and the glucocorticoid dexamethasone (p < or = 0.01).


CONCLUSIONS
These results suggest 1) that [Ca2+]i increase and subsequent activation of protein kinase C is necessary for the release of TNF-alpha, and they work synergistically, 2) that the TNF-alpha release induced by Ca2+-ATPase inhibitors can be regulated at the transcriptional level.
Teshima
R
R
Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Tokyo, Japan. rteshima@nihs.go.jp
Onose
J
J
Ikebuchi
H
H
Sawada
J
J
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D003609:Dactinomycin / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006873:Hydroquinones / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism* / Q000473:pathology; D011493:Protein Kinase C / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D016559:Tacrolimus / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000187:drug effects*
D004791:Enzyme Inhibitors; D006873:Hydroquinones; D007211:Indoles; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin; C019359:2,5-di-tert-butylhydroquinone; D019284:Thapsigargin; D003907:Dexamethasone; C018855:2-tert-butylhydroquinone; D011493:Protein Kinase C; D000252:Calcium-Transporting ATPases; D016559:Tacrolimus; C000543:cyclopiazonic acid