Activation of 2-Adrenoceptor Prevents Shiga Toxin 2-Induced TNF- Gene Transcription

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– (TNF). It has been shown that Stx-2 induces TNFproduction and that activation of 2-adrenoceptors downregulates TNF. However, little is known about the signaling pathway by which 2-adrenoceptor agonists suppress the Stx-2–induced TNFgene 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 2-adrenoceptor agonist. Mitogen-activated protein kinase (MAPK), activating protein–1 (AP-1), and nuclear factor– B (NFB) were measured to evaluate the regulatory mechanisms involved in TNFgene transcription. Stx-2 (4 pg/ml) stimulated MAPK (p42/p44, p38) and AP-1 and increased TNFpromoter activity by 2.4-fold. The increase in TNFwas 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), NFB (p50, p65), and TNFpromoter activity in a dose-dependent way that was prevented by the 2-adrenoceptor antagonist, ICI118,551. However, inhibition of MAPK (p42/p44) and TNFpromoter 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 NFB (p50) was not blocked by these inhibitors. The suppression of NFB (p65) was completely overcome by H-89 or KT5720. In summary, the downregulation of TNFtranscription by terbutaline was mediated by an inhibitory effect of 2-adrenoceptor activation on MAPK (p42/p44, p38) and NFB (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 TNFtranscription via 2adrenoceptor activation. Hemolytic uremic syndrome (HUS) is characterized by renal failure. Thrombocytopenia and hemolytic anemia and shiga toxin (Stx)-producing Escherichia coli are responsible for the majority of cases of HUS in childhood (1, 2). There are two forms of Stx (also known as verocytotoxins): VT-1 and VT-2. It has been reported that induction of the globotriaosylceramide (Gb3) receptor, known to be the functional receptor for Stx, is one mechanism by which inflammatory mediators increase susceptibility to Stx (3). The major pathogenesis of HUS has been ascribed to initial endothelial and vascular damage. However, evidence of primary renal tubular cell damage in HUS has been reported from some studies in humans. The receptor sites for Stx binding in normal kidney sections are most prominent in renal cortical tubules (4), probably in the distal tubule. Renal biopsy studies early in the course of HUS have suggested a direct action on the proximal tubules, and cultured epithelial cells from this region express very high levels of Gb3 (5–8). These reports imply that renal tubular impairment contributes to the development of HUS. Hughes et al. (9) have indicated that proximal tubules are exquisitely sensitive to Stx-1 cytotoxicity and that inflammatory factors can increase their responsiveness to the toxin through a variety of mechanisms. They furthermore indicated that Stx-1 stimulates the production of inflammatory cytokines, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF) by the proximal tubule (10). It has been found that IL-1 and TNFinduce expression of Gb3 on the surface of several cells and can upregulate cell sensitivity to Stx cytotoxicity (3,11,12). Importantly, TNFhas been shown to enhance Stx-mediated apoptosis in a human renal tubular epithelium– derived cell line, ACHN (7). In an additional report that used a mouse model of HUS, it was found that Stx induced TNF synthesis within the kidney and at the same time increased the renal sensitivity to the toxic effects of TNF (13). One explanation may be that Stx stimulation of TNFproduction in the renal tubule increases its sensitivity to the tubular cytotoxic effect of Stx. However, the mechanism by which Stx is able to stimulate TNF production remains uncertain at present. Received December 12, 2000. Accepted May 7, 2001. Correspondence to Dr. Akio Nakamura, Department of Paediatrics, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173, Japan. Phone: 03-3964-1211 (ext. 1480); Fax: 03-3579-8212; E-mail: [email protected]