Specific signaling pathways in the regulation of TNF-α mRNA synthesis and TNF-α secretion in RBL-2H3 mast cells stimulated through the high affinity IgE receptor
- 138 Downloads
Objective: In the present study, we investigated signal transduction pathways involved in TNF-α gene expression and TNF-α secretion by mast cells stimulated through the high affinity IgE receptor (FcɛRI).¶Materials and Methods: TNF-α mRNA steady state levels and TNF-α secretion in the presence of specific pharmacological agents were monitored using rat basophilic leukemia cells (RBL-2H3) stimulated through FcɛRI. Relative amounts of TNF-α mRNA versus β-actin levels were quantified by RNase protection and RT-PCR assays. TNF-α secretion was measured by a current ELISA test.¶Results: We show that EGTA (5 mM) prevented TNF-α mRNA expression and TNF-α secretion in antigen-stimulated cells. The protein kinase C (PKC) inhibitor bisindolylmaleimide I substantially blocked TNF-α secretion at 2 μM but had only a marginal effect on TNF-α mRNA expression. The results were similar when PKC isoforms were depleted by long-term exposure to 100 nM phorbol ester (PMA). The PI 3-kinase inhibitor wortmannin blocked TNF-α secretion at low doses (EC50 = 13 nM), but only partially affected mRNA expression.¶Conclusions: Our results show that in FcɛRI-stimulated RBL-2H3 cells calcium mobilization, activation of PKC and PI 3-kinase are necessary for TNF-α secretion while for the increased TNF-α mRNA expression PKC activity is dispensable and PI 3-kinase activity only partially required.
Unable to display preview. Download preview PDF.