Ca2+-ATPase inhibitors and PKC activation synergistically stimulate TNF-α production in RBL-2H3 cells

Abstract.

Objective and Design: To investigate the effect of Ca²⁺-ATPase inhibitors on the production of TNF-α in rat basophilic leukemia (RBL-2H3) cells.¶Material: 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-(tert-butyl)-1,4-hydroquinone (MTBHQ) were used.¶Treatment: Cells were treated with TG, CPA, DTBHQ, DTAHQ and MTBHQ for 3 h in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) and released TNF-α from the cells was measured (n >  or = 4).¶Results: All Ca²⁺-ATPase inhibitors (TG, CPA, DTBHQ and DTAHQ) induced TNF-α release in a dose-dependent manner. TNF-α 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-α. TNF-α 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-α, and they work synergistically, 2) that the TNF-α release induced by Ca²⁺-ATPase inhibitors can be regulated at the transcriptional level.