Activation of β2-adrenoceptor prevents shiga toxin 2-induced TNF-α gene transcription

Abstract

Exposure of renal tubular epithelial cells to shiga toxin 2 (Stx-2) causes cytotoxicity, and the potency of this toxin is enhanced in the presence of tumor necrosis factor-α (TNF-α). It has been shown that Stx-2 induces TNF-α production and that activation of β2-adrenoceptors downregulates TNF-α. However, little is known about the signaling pathway by which β2-adrenoceptor agonists suppress the Stx-2-induced TNF-α gene transcription. The possible signaling components involved in this pathway were investigated. Human adenocarcinoma-derived renal tubular epithelial cells (ACHN) were exposed to Stx-2 in the presence or absence of a β2-adrenoceptor agonist. Mitogen-activated protein kinase (MAPK), activating protein-1 (AP-1), and nuclear factor-κB (NF-κB) were measured to evaluate the regulatory mechanisms involved in TNF-α gene transcription. Stx-2 (4 pg/ml) stimulated MAPK (p42/p447, p38) and AP-1 and increased TNF-α promoter activity by 2.4-fold. The increase in TNF-α was attenuated by both a p42/p44 inhibitor, PD098059 (10-6 M), and a p38 inhibitor, SB203580 (10-6 M), and AP-1-binding activity was inhibited by PD098059. Terbutaline (10-6 M to 10-8 M) suppressed MAPK (p42/p44, p38), NF-κB (p50, p65), and TNF-α promoter activity in a dose-dependent way that was prevented by the β2-adrenoceptor antagonist, ICI118,551. However, inhibition of MAPK (p42/p44) and TNF-α promoter activity was partially prevented by the cAMP-protein kinase (PKA) inhibitors, H-89 (5 × 10-6 M) and KT5720 (10-5 M), whereas the suppression of p38 MAPK or NF-κB (p50) was not blocked by these inhibitors. The suppression of NF-κB (p65) was completely overcome by H-89 or KT5720. In summary, the downregulation of TNF-α transcription by terbutaline was mediated by an inhibitory effect of β2-adrenoceptor activation on MAPK (p42/p44, p38) and NF-κB (p50/p65), which were exerted through a cAMP-PKA pathway and a cAMP-independent mechanism. It is likely that cAMP-PKA and MAPK (p42/p44, p38) may play a critical role in the regulation of the Stx-2-induced TNF-α transcription via β2-adrenoceptor activation.