Specific signaling pathways in the regulation of TNF-alpha mRNA synthesis and TNF-alpha secretion in RBL-2H3 mast cells stimulated through the high affinity IgE receptor

Objective: In the present study, we investigated signal transduction pathwa ys involved in TNF-alpha gene expression and TNF-alpha secretion by mast ce lls stimulated through the high affinity IgE receptor (Fc epsilon RI). Materials and Methods: TNF-alpha mRNA steady state levels and TNF-alpha sec retion in the presence of specific pharmacological agents were monitored us ing rat basophilic leukemia cells (RBL-2H3) stimulated through Fc epsilon R I. Relative amounts of TNF-alpha mRNA versus beta-actin levels were quantif ied by RNase protection and RT-PCR assays. TNF-alpha secretion was measured by a current ELISA test. Results: We show that EGTA (5 mM) prevented TNF-alpha mRNA expression and T NF-alpha secretion in antigen-stimulated cells. The protein kinase C (PKC) inhibitor bisindolylmaleimide I substantially blocked TNF-alpha secretion a t 2 mu M but had only a marginal effect on TNF-alpha mRNA expression. The r esults were similar when PKC isoforms were depleted by long-term exposure t o 100 nM phorbol ester (PMA). The PI 3-kinase inhibitor wortmannin blocked TNF-alpha secretion at low doses (EC50 = 13 nM), but only partially affecte d mRNA expression. Conclusions: Our results show that in Fc epsilon RI-stimulated RBL-2H3 cell s calcium mobilization, activation of PKC and PI 3-kinase are necessary for TNF-alpha secretion while for the increased TNF-alpha mRNA expression PKC activity is dispensable and PI 3-kinase activity only partially required.