Hormones, Growth Factors, and Cell Signaling

Activation of β2-Adrenoceptor Prevents Shiga Toxin 2-Induced TNF-α Gene Transcription

AKIO NAKAMURA, EDWARD J. JOHNS, AKIRA IMAIZUMI, YUKISHIGE YANAGAWA and TAKAO KOHSAKA
JASN November 2001, 12 (11) 2288-2299;

Abstract

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/p44, 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.

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 (5,6,7,8). 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 TNF-α induce expression of Gb3 on the surface of several cells and can upregulate cell sensitivity to Stx cytotoxicity (3,11,12). Importantly, TNF-α has 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 TNF-α production 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.

β2-adrenoceptor agonists have been known to inhibit the production of TNF-α in response to an endotoxin challenge (14,15). Recently, we reported that Stx-2 caused a marked increase in TNF-α protein release from ACHN cells, which could be blunted in the presence of a β2-adrenoceptor agonist, and that the cytotoxic effects of Stx-2 could be prevented when β2-adrenoceptors were activated (16). These findings suggest that the mechanism by which Stx-stimulated TNF-α production might involve intracellular signaling events triggered by β2-adrenoceptors. It would seem that β2-adrenoceptor activation prevents TNF-α production as a consequence of the inhibition of mitogen-activated protein kinase (MAPK, p42/p44) and enhanced cAMP generation (17). The MAPK and cAMP/cAMP-dependent protein kinase (PKA) pathways have been reported to regulate the nuclear binding activity of activating protein—1 (AP-1) and nuclear factor—κB (NF-κB), which involves TNF-α gene transcription (18). However, it remains unclear whether activation of nuclear factors through these pathways is necessary for the induction of TNF-α by Stx.

This study was undertaken to investigate the mechanisms of Stx-2—induced TNF-α gene transcription and the inhibitory mechanisms after β2-adrenoceptor activation. We attempted to clarify the possible roles of cAMP-PKA, p42/p44 MAPK, p38 MAPK, c-Jun N-terminal protein kinase (JNK), AP-1, or NF-κB (p50, p65) in TNF-α gene transcription in ACHN cells, which are an in vitro model of renal tubular epithelial cells.

Materials and Methods

Reagents

Stx-2 was prepared as described previously (19). Dulbecco's modified Eagle's medium (DMEM), glutamine, kanamycin, HEPES, PD098059 (PD), and SB203580 (SB) were obtained from Cosmo Bio Corp. (Tokyo, Japan). Anti β2-adrenoceptor antibody was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Pica Gene basic vector-2 (pGV-B2) and cell lysis buffer were obtained from Toyo Inc. Corp. (Tokyo, Japan). Fetal calf serum (FCS) was purchased from Dainippon Pharmaceutical Corp. (Tokyo, Japan). Transfectam was supplied by Bio Sepra Inc. (Marlborough, MA). The luminometer (Lumat LB953AT) was purchased from Berthold (Wildbad, Germany). Rat TNF-α enzyme-linked immunosorbent assay kit was from Biosource International Inc. (Camarillo, CA). The MAPK (p42/p44) assay kit and isotopes ([α-33P]dCTP, [γ-33P]ATP) were purchased from Amersham Japan (Tokyo, Japan). p38 MAPK and JNK assay kits were purchased from Stratagene (La Jolla, CA). AP-1 and NF—κB (p50, p65) assay kits (NUSHIFT) were purchased from Geneka (Montreal, Quebec, Canada). PAGEL was obtained from ATTO Corp. (Tokyo, Japan). BAS III and BAS2000 were produced by Fuji Photo Film Co. Ltd. (Tokyo, Japan). Unless otherwise stated, reagents were from Sigma Chemical Co. (St. Louis, MO).

Culture Conditions

The ACHN line was obtained from the American Type Culture Collection (ATCC CRL 1611). The cells express the renal tubular marker CD24 as well as CD77, the receptor for Stx (7). Furthermore, the cells were identified by means of immunostaining by using labeling with β2-adrenoceptor antibody-FITC conjugate. The cells were maintained at 37°C in 5% CO2 in DMEM that contained 10% FCS, 0.225% NaHCO3, 0.1 mM nonessential amino acids, and 0.03% glutamine and were cultured on 25-cm2 tissue culture flasks and grown until approximately 80% confluent. After the dishes were washed with phosphate-buffered saline to remove the nonadherent cells, the cells were placed in the medium for 2 h before the Stx-2 challenge.

Cell Viability

Cells were removed from the culture flasks, and plated onto 96-well microtiter plates at a density of 1.5 × 104 cells per well in 200 μl of medium. The cells were incubated for 4, 24, 48 h after the Stx-2 (0 to 512 pg/ml) challenge in either the absence or presence of terbutaline (Ter; 10-6 M to 10-8 M), formoterol (For; 10-6 M), ICI118,551 (ICI; 10-6 M), forskolin (10-5 M), prostaglandin E2 (PG; 10-6 M), PD (10-6 M), SB (10-6 M), H-89 (5 × 10-6 M), and/or KT5720 (KT; 10-5 M). Cell viability was determined by the uptake of neutral red as described previously (20).

Assay for TNF-α Release

To confirm the stimulatory effects of Stx-2 on TNF-α release from ACHN cell, cells were exposed to varying concentration of Stx-2 (2 to 512 pg/ml). Furthermore, TNF-α release was assayed up to 24 h after the Stx-2 (4 pg/ml) challenge in either the absence or presence of the β2-adrenoceptor agonists, Ter (10-6 M or 10-7 M) or For (10-6 M), the β2-adrenoceptor antagonist, ICI (10-6 M), the cAMP-elevating agents, forskolin (10-5 M) or PG (10-6 M), and/or the cAMP-PKA inhibitors, H-89 (5 × 10-6 M) or KT (10-5 M). The agonists (activators) or antagonists (inhibitors) were added 10 min or 20 min before the Stx-2 (4 pg/ml) challenge, respectively. To examine the influence of MAPK (p42/p44, p38) inhibition on TNF-α in the cells, the MAPK/extracellular signal—regulated kinase (MEK 1) inhibitor, PD (10-6 M) or the specific p38 inhibitor, SB (10-6 M) was added 20 min before the Stx-2 (4 pg/ml) challenge. TNF-α levels in the incubate were estimated with a commercially available enzyme-linked immunosorbent assay kit.

Assay of MAPK (p42/p44, p38, JNK)

MAPK activities were assayed up to 100 min after the Stx-2 (4 pg/ml) challenge in either the absence or presence of the β2-adrenoceptor agonists, Ter (10-6 M to 10-8 M) or For (10-6 M), the β2-adrenoceptor antagonist, ICI (10-6 M), the cAMP-elevating agents, forskolin (10-5 M) or PG (10-6 M), and/or the cAMP-PKA inhibitors, H-89 (5 × 10-6 M) or KT (10-5 M). The agonists (activators) or antagonists (inhibitors) were added 10 min or 20 min before the Stx-2 (4 pg/ml) challenge, respectively. The assay system is based on the MAPK-catalyzed transfer of the γ-phosphate group of adenosine-5′-triphosphate to a peptide that is selective for MAPK. Cells were lysed in 10 mM Tris, 150 mM NaCl, 2 mM ethylenegly-cotetraacetic acid, 2 mM dithiothreitol, 1 mM orthovanadate, 1 mM phenylmethyl sulfonyl fluoride, and 10 μg/ml aprotinin (pH 7.4) and then precipitated at 25,000 × g for 20 min. The supernatant that contained the cytoplasmic MAPK was retained. The reaction was initiated by the addition of [γ-33P]ATP, and the incorporation of 33P into the peptide at 30°C for 30-min incubation was assayed as a MAPK activity in a scintillation counter.

Electrophoretic Mobility Shift Assay

AP-1 and NF—κB (p50, p65) were assayed 2 h after the Stx-2 (4, 16, 64 pg/ml) challenge in either the absence or presence of the β2-adrenoceptor agonists, Ter (10-6 M to 10-8 M) or For (10-6 M), the β2-adrenoceptor antagonist, ICI (10-6 M), and/or the cAMP-PKA inhibitor, H-89 (5 × 10-6 M). The agonists or antagonists were added 10 min or 20 min before the Stx-2 challenge, respectively. To examine the influence of MAPK (p42/p44, p38), inhibition on AP-1 and NF—κB (p50, p65)—binding activities in the cells, PD (10-6 M) or SB (10-6 M) was added 20 min before the Stx-2 challenge. Nuclear extracts from ACHN cells were prepared by using methods described previously (21). The nuclear proteins were stored in aliquots at -70°C. Double-stranded oligonucleotides that contained AP-1 and NF—κB (p50, p65) consensus—binding sites were radiolabeled by using T4 polynucleotide kinase and [γ33P]-ATP and were then purified by centrifugation over a G-25 purification column. Specific oligonucleotide probes, NF-κB (p50) probe (5′-GCCATGGGGGGATCCCCGAAGTCC-3′), NF-κB (p65) probe (5′-AGCTTGGGGTATTTCCAGCCG-3′), AP-1 probe (5′- CGCTTGATGAGTCAGCCGGAA-3′) and its mutation probe, NF-κB (p50) (5′-GCCATGGGCCGATCCCCGAAGTCC-3′), NF-κB (p65) (5′-AGCTTGGCATAGGTCCAGCCG-3′), AP-1 (5′-CGCTTGATGACCCAGCCGGAA-3′), were radiolabeled and used to examine the specificity of the nuclear factor binding. Five micrograms of nuclear protein were incubated with the 33P-labeled probes for 20 min at 4 to 10°C according to the manufacturer's instructions. The samples were loaded onto a 5% polyacrylamide (38:2) gel (PAGEL) with Tris-glycine buffer, and electrophoresis was performed at 100 V for 1 h. The gel was vacuum dried and exposed on an imaging plate (BAS III), and the incorporated radioactivity was measured with a Bioimage analyzer (BAS 2000). The specificity of the reaction was determined by competition reactions in which a 100-fold molar excess of unlabeled AP-1 or NF-κB oligonucleotide probe was added to the binding reaction. The composition of activated AP-1 or NF-κB was analyzed by using a supershift assay. In the study, 2 μl of antibodies reactive to the human c-Fos, c-Jun, p50, and p65 proteins were incubated with the reaction mixture for 20 min and then added to radiolabeled AP-1 or NF-κB probe.

Reporter Plasmid and Luciferase Assay

pGV-B2-TNFprom was generated in which a rat TNF-α promoter (22) drives the expression of luciferase of pGV-B2 (pica gene basic vector-2) and prepared as described in our previous manuscript (23). pGV-B2-TNFprom (0.7 kb) contains potential recognition sites for several transcription factors, including AP-1 and NF-κB, and comparison of the region with human TNF-α promoter revealed a high degree of conversion (24). Transfection of pGV-B2-TNFprom into the cells was performed by using Transfectam according to the manufacturer's recommendations. For the luciferase assay, the cells were incubated for 3 h with 10% FCS-DMEM after the Stx-2 (4 pg/ml) challenge in either the absence or presence of the β2-adrenoceptor agonist, Ter (10-6 M or 10-7 M), the cAMP-PKA stimulators, forskolin (10-5 M) or PG (10-6 M), the MAPK (p42/p44, p38) inhibitors, PD (10-6 M) or SB (10-6 M), the NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC; 1.5 × 10-4 M), and/or the cAMP-PKA inhibitors, H-89 (5 × 10-6 M) or KT (10-5 M). The agonists (stimulators) or antagonists (inhibitors) were added 10 min or 20 min before the Stx-2 challenge, respectively. The luciferase activities were normalized on the basis of β-galactosidase activities, which were assayed as described previously (23).

Statistical Analyses

Statistical analyses were undertaken by using nonparametric analysis with the Kruskal-Wallis test followed by the Mann-Whitney U test. Results are expressed as mean ± SE of the mean.

Results

Effect of Stx-2 Stimulation and β2-Adrenoceptor Activation on Cell Viability

As shown in Figure 1A (line graph), up to 4 h incubation with 2 to 64 pg/ml Stx-2 had no detectable effect on ACHN cell viability (98 to 102% control). Figure 1A (bar graph) indicates that all treatments had little or no effect on the degree of cytotoxicity. This indicates that Stx-2 (4 to 16 pg/ml) did not have a cytotoxic effect on the cells after a 2-h exposure (some data not shown). This approach was used for analysis of the Stx-induced kinase activity, nuclear binding activity, or TNF-α synthesis. In contrast, Figure 1B and 1C (line graphs) show that 24 to 48 h exposure to >2 pg/ml Stx-2 caused a reduction in cell viability, which was significantly attenuated by Ter (10-6 M) over the dose range of 2 to 128 pg/ml Stx-2. The Ter inhibition of Stx-2 (4 pg/ml)—induced cell cytotoxicity at 24 h was completely abolished by the β2-adrenoceptor antagonist, ICI (10-6 M), and the inhibitors of the cAMP-PKA pathway, H-89 (5 × 10-6 M) and KT (10-5 M), but the Ter inhibition at 48 h was incompletely blocked by the cAMP-PKA inhibitors (Figure 1B and 1C, bar graphs). These findings suggest that Stx-2 (4 pg/ml)—induced cell cytotoxicity and its modulation by β2-adrenoceptor activation seem to involve not only cAMP-PKA pathway but some other factors.

Figure 1.
Figure 1.

Effects of Stx-2 stimulation and β2-adrenoceptor activation on cell viability. Adenocarcinoma-derived renal tubular epithelial cells (ACHN) were incubated with different concentrations of Stx-2 as indicated (line graph). Bar graphs indicate cytotoxic responses to 4 pg/ml Stx-2 and its modulation by terbutaline (Ter), ICI118,551 (ICI), H-89, and/or KT5720 (KT). After incubation for 4 h (A), 24 h (B), and 48 h (C), viable cells were estimated by using the neutral red assay and presented as the ratio against untreated cells. Data are mean ± SE from four experiments. *P < 0.05 versus cells exposed with Stx-2 alone (control). §P < 0.05 versus cells exposed with 4pg/ml Stx-2 plus Ter (10-6 M).

Effect of Stx-2 Stimulation and β2-Adrenoceptor Activation on TNF-α Release

Stx-2—induced TNF-α release at 2 h was significantly increased by the dose of >4 pg/ml (P < 0.05), whereas 2 pg/ml Stx-2 was not able to increase TNF-α release at 2 h from the ACHN cells (data not shown). Therefore, the minimal dose (4 pg/ml) that significantly stimulated TNF-α release was used for this study. Figure 2A shows the time course of the effect of 4 pg/ml Stx-2 on TNF-α protein supernatant levels in the ACHN cell cultures when incubated alone and in the presence of β2-adrenoceptor agonist and MAPK (p42/p44, p38) inhibitors. After 4 h of exposure to 4 pg/ml Stx-2, there was a sharp increase in TNF-α release of some six-fold, which was maintained after 24 h. Ter (10-6 M), SB (10-6 M), and PD (10-6M) significantly suppressed these responses (P < 0.05). In Figure 2B, it can been seen that the level of Stx-2 (4pg/ml)—induced TNF-α release at 2 h was suppressed in a dose-dependent way by 10-6 M and 10-7 M Ter (P < 0.05). The inhibitors of the cAMP-PKA pathway, H-89 (5 × 10-6 M) and KT (10-5 M), and the β2-adrenoceptor antagonist, ICI (10-6 M), significantly blocked the inhibitory action of Ter on Stx-2 (4 pg/ml)—mediated TNF-α release (all P < 0.05). Forskolin (10-5 M), PG (10-6 M), and For (10-6 M) also suppressed the release of TNF-α (P < 0.05). These findings indicate that β2-adrenoceptor activation during an Stx-2 (4 pg/ml) challenge prevented TNF-α release through the cAMP-PKA pathway.

Figure 2.
Figure 2.

Effects of Shiga toxin 2 (Stx-2) and β2-adrenoceptor activation on tumor necrosis factor—α (TNF-α) release. (A) Time-related increase in TNF-α release from ACHN cells after exposure to 4 pg/ml Stx-2. The addition of Ter, PD098059 (PD), and SB203580 (SB) suppressed these responses. ○, control; [UNK], Stx-2; ▴, Stx-2 + Ter (10-6 M); ♦, Stx-2 + PD (10-6 M); ▪, Stx-2 + SB (10-6 M). (B) The inhibitory effect of Ter or formoterol (For) on TNF-α release from the cells 2 h after the Stx (4 pg/ml) challenge was due to activation of β2-adrenoceptor and cAMP generation as they were blunted by ICI (10-6 M), H-89 (5 × 10-6 M), and KT (10-5 M). Both forskolin and prostaglandin E2 (PG) had an inhibitory action on TNF-α release activated by Stx-2. Data are mean ± SE from four experiments. 1, Stx-2; 2, Stx-2 + Ter (10-6 M); 3, Stx-2 + Ter (10-7 M); 4, Stx-2 + Ter (10-6 M) + H-89; 5, Stx-2 + Ter (10-6 M) + KT; 6, Stx-2 + Ter (10-6 M) + ICI; 7, Stx-2 + For (10-5 M); 8, Stx-2 + PG (10-6 M); 9, Stx-2 + For (10-6 M). *P < 0.05 versus cells exposed to Stx-2.

Effect of Stx-2 Stimulation and β2-Adrenoceptor Activation on MAPK Activity

Figure 3A illustrates the time course of the action of 4 pg/ml Stx-2 and/or Ter (10-6 M to 10-8 M) on MAPK (p42/p44) activity in the cells. MAPK (p42/p44) activity was stimulated by the Stx-2 challenge, beginning after the initial exposure, reaching a peak level of some six-fold at 20 min, and then returning to the baseline at 60 min. In the presence of Ter, the peak level of Stx-2—induced p42/p44 activity was suppressed in a dose-dependent manner. The comparisons of p42/p44 activity at 20 min among these groups are shown as bar graphs in Figure 3B. H-89 (5 × 10-6 M) and KT (10-5 M) overcame the inhibitory action of Ter on Stx-2—mediated MAPK (p42/p44) activity (P < 0.05). However, the magnitude of the block was small, and the MAPK (p42/p44) activity was still significantly suppressed by 10-6 M Ter (H-89, 44% inhibition; KT, 42% inhibition; both P < 0.05). On the other hand, the addition of ICI (10-6 M) significantly blocked the inhibitory action of Ter on Stx-2—mediated p42/p44 MAPK activity (P < 0.05), indicating that the inhibitory action of Ter on the Stx-2—mediated p42/p44 activity was dependent on activation of β2-adrenoceptors. Forskolin (10-5 M), PG (10-6 M), and For (10-6 M) also significantly suppressed Stx-2—mediated p42/p44 MAPK activity (P < 0.05). Importantly, suppression of Stx-2—induced p42/p44 MAPK activity by β2-adrenoceptor activation was partly mediated by the cAMP-PKA pathway, but it was apparent that other mechanisms were also involved in the suppression.

Figure 3.
Figure 3.

Effects of Stx-2 and β2-adrenoceptor activation on MAPK (p42/p44) activity. (A) 4pg/ml Stx-2 increased p42/p44 activity, but this response was blocked by Ter (10-6 M to 10-8 M) in a dose-dependent manner. ○, control; [UNK], Stx-2; ▴, Stx-2 + Ter (10-6 M); ♦, Stx-2 + Ter (10-7 M); ▪, Stx-2 + Ter (10-8 M). (B) The inhibitory effect of Ter or For on p42/p44 activity in the cells at 20 min after the Stx (4pg/ml) challenge was prevented by ICI (10-6 M) but partially prevented by H-89 (5 × 10-6 M) and KT (10-5 M). Forskolin or PG was able to suppress Stx-induced p42/p44 activity. The data are expressed as relative changes (fold) to level at time 0 in each group. Data are mean ± SE from four experiments. 1, control; 2, Stx-2; 3, Stx-2 + Ter (10-6 M); 4, Stx-2 + Ter (10-6 M) + H-89; 5, Stx-2 + Ter (10-6 M) + KT; 6, Stx-2 + Ter (10-6 M) + ICI; 7, Stx-2 + Forskolin (10-5 M); 8, Stx-2 + PG (10-6 M); 9, Stx-2 + For (106 M). *P < 0.05 versus cells exposed with Stx-2 alone. †P < 0.05 versus cells exposed without any treatment (control). §P < 0.05 versus cells exposed with Stx-2 + Ter (10-6 M).

Figure 4A presents the time course of the effect of 4 pg/ml Stx-2 and/or Ter (10-6 M to 10-8 M) on p38 MAPK activity in the cells. p38 MAPK activity was stimulated by the Stx-2 challenge, beginning some 40 min after the initial exposure, reaching a peak level of some 2.7-fold at 60 min, and then returning toward the baseline at 100 min. In the presence of Ter, the peak level of Stx-2—induced p42/p44 activity was suppressed in a dose-dependent manner. In Figure 4B, the comparisons of p38 MAPK activity at 60 min among these groups are shown as bar graphs, and it is evident that Ter (10-6 M) and For (10-6 M) significantly suppressed the Stx-2—mediated p38 MAPK activity (P < 0.05). The addition of ICI (10-6 M) significantly blocked the inhibitory action of Ter on Stx-2—mediated p38 MAPK activity (P < 0.05). This would be consistent with Ter suppressing the Stx-2—mediated p38 MAPK activity via β2-adrenoceptor activation. Forskolin (10-5 M) or PG (10-6 M) was unable to suppress the Stx-2—induced p38 MAPK activity. Furthermore, H-89 (5 × 10-6 M) and KT (10-5 M) were unable to block the inhibitory action of Ter on Stx-2—mediated p38 MAPK activity. Thus, the inhibitory effect of the β2-adrenoceptor agonist on p38 MAPK activity was independent of the cAMP-PKA pathway. The time course of action of 4 pg/ml Stx-2 with or without Ter (10-6 M to 10-8 M) on JNK activity in the cells was examined. Neither Stx-2 nor β2-adrenoceptor activation were able to influence JNK activity in the cells (data not shown).

Figure 4.
Figure 4.

Effects of Stx-2 and β2-adrenoceptor activation on p38 MAPK activity. (A) 4pg/ml Stx-2 increased p38 MAPK activity, but this response was blocked by Ter (10-6 M to 10-8 M) in a dose-dependent manner. ○, control; [UNK], Stx-2; ▴, Stx-2 + Ter (10-6 M); ♦, Stx-2 + Ter (10-7 M); ▪, Stx-2 + Ter (10-8 M). (B) The inhibitory effect of Ter or For on p38 MAPK activity in the cells at 60 min after the Stx (4pg/ml) challenge was prevented by ICI (10-6 M) but not by H-89 (5 × 10-6 M) or KT (10-5 M). Forskolin or PG could not suppress Stx-induced p38 MAPK activity. The data are expressed as relative changes (fold) to level at time 0 in each group. Data are mean ± SE from four experiments. 1, control; 2, Stx-2; 3, Stx-2 + Ter (10-6 M); 4, Stx-2 + Ter (10-6 M) + H-89; 5, Stx-2 + Ter (10-6 M) + KT; 6, Stx-2 + Ter (10-6 M) + ICI; 7, Stx-2 + Forskolin (10-5 M); 8, Stx-2 + PG (10-6 M); 9, Stx-2 + For (10-6 M). *P < 0.05 versus cells exposed with Stx-2 alone. †P < 0.05 versus cells exposed without any treatment (control).

Effect of Stx-2 Stimulation and β2-Adrenoceptor Activation on AP-1 and NF-κB Activity

To address the question of whether Stx-2 stimulation and β2-adrenoceptor activation could lead to nuclear AP-1— and NF-κB translocation, an electrophoretic mobility shift assay was performed on nuclear extracts from the cell. The AP-1—binding activity of the nuclear extracts was markedly enhanced after 60-min treatment with 4pg/ml Stx-2 and reached a peak level at 2 h, after which it was sustained, but NF-κB (p50, p65) was not induced by this Stx-2 (4pg/ml) challenge (data not shown). In Figure 5, a competition analysis was performed to investigate the sequence specificity of the protein-DNA interaction in the cell that was exposed by Stx-2 (4 pg/ml). Furthermore, to define the subunit composition of the Stx-2—induced AP-1— and NF-κB—binding complexes, supershift assays were performed by using selective antibodies specifically for c-Fos, c-Jun, NF-κB/p50, and NF-κB/p65. Incubation with antibodies to c-Fos elicited a supershift complex, whereas the antibody to c-Jun did not alter the gel-shift band, which suggests that Stx-2 induced an AP-1 binding complex containing c-Fos. For the anti-p50 antibody, the supershifted complex showed only a moderate extension. The situation with the p65, was that the supershifted bands could be easily and clearly detected. Therefore, the results suggest that the NF-κB complex contained p65 and small amounts of p50 proteins. Figures 6A and 7A show that AP-1—binding activity in response to Stx-2 (4, 16, 64 pg/ml) was increased in a dose-dependent manner and that Ter (10-8 M) was unable to change the AP-1 activity. Furthermore, the high doses of Ter (10-6 M or 10-7 M) and For (10-6 M) did not alter the AP-1—binding activity (Figure 8A). Figures 6B and 7B show that NF-κB (p50)—binding activity was not induced after the Stx-2 (4, 16, 64 pg/ml) challenge. By contrast, Ter (10-8 M) was able to downregulate NF-κB (p50)—binding activity in both the presence and absence of Stx-2 (Figure 6B). Moreover, the high dose of Ter (10-6 M or 10-7 M) significantly suppressed NF-κB (p50)—binding activity in a concentration-related way (Figure 8B). This response was blocked by ICI (10-6 M) but not by H-89 (5 × 10-6 M). Thus the suppression of NF-κB (p50)—binding activity by Ter was mediated by β2-adrenoceptors and was independent of the cAMP-PKA pathway. Figure 6C and 7C show that NF-κB (p65)—binding activity could not be induced by the Stx-2 (4, 16, 64 pg/ml) challenge. The high doses of Ter (10-6 M or 10-7 M) were able to suppress NF-κB (p65)—binding activity significantly and in a concentration-related way (Figure 8C), whereas the low dose of Ter (10-8 M) could not modify NF-κB (p65)—binding activity in either the presence or absence of Stx-2 (Figure 6C). These responses were blocked by ICI (10-6 M) and H-89 (5 × 10-6 M). Thus, the suppression of NF-κB (p65)—binding activity by the high dose of β2-adrenoceptor agonist was mediated by β2-adrenoceptor activation and was dependent on the cAMP-PKA pathway. To investigate which pathways of MAPK are involved in the regulation of Stx-2—induced AP-1— and NF-κB—binding activities, SB (10-6 M) and PD (10-6 M) were added to the cells exposed to 16 pg/ml Stx-2. The data in Figure 7 indicate that AP-1—binding activity induced by the Stx-2 challenge was significantly suppressed by PD but not by SB, which suggests that the Stx-2—induced AP-1 was influenced by the p42/p44 MAPK cascade. On the other hand, NF-κB (p50, p65)—binding activity was not significantly suppressed by either PD or SB, which suggests that NF-κB (p50, p65) was not dependent on a MAPK pathway.

Figure 5.
Figure 5.

Competition analysis and supershift assay of AP-1—, NF-κB (p50)—, and NF-κB (p65)—binding activity in the nuclei of ACHN. AP-1—oligonucleotide (left), NF-κB (p50)—oligonucleotide (middle), and NF-κB (p65)—oligonucleotide (right) were used for competition experiments. Wild-type: wild-type oligonucleotide, Mutant: mutant oligonucleotide. Supershift assay was performed by using specific anti—c-Fos and anti—c-Jun (left), anti-p50 (middle), or anti-p65 (right). ACHN cells were stimulated with 4 pg/ml Stx-2 for 2 h, after which the nuclear proteins were collected and extracted.

Figure 6.
Figure 6.

The effect of Stx-2 and β2-adrenoceptor activation on AP-1—, NF-κB (p50)—, and NF-κB (p65)—binding activity in the nuclei of ACHN. Stx-2 (0, 4, 16 pg/ml) was added to ACHN cells after 10 min of pretreatment with Ter (10-8 M). The arrows show the AP-1—binding activity (A), NF-κB (p50)—binding activity (B), and NF-κB (p65)—binding activity (C) during the following 2 h, which were analyzed by using electrophoretic mobility shift assay (EMSA). Results shown are representative of three separate experiments.

Figure 7.
Figure 7.

Modulation of Stx-2—mediated AP-1—, NF-κB (p50)—, and NF-κB (p65)—binding activity by a specific p38 MAPK inhibitor or a MEK-1 inhibitor. Stx-2 (4, 16, 64 pg/ml) was added to ACHN cells after 20 min of pretreatment with SB (10-6 M) or PD (10-6 M). The arrows indicate the AP-1—binding activity (A), NF-κB (p50)—binding activity (B), and NF-κB (p65)—binding activity (C) during the following 2 h, which were analyzed by using EMSA. Results shown are representative of three separate experiments.

Figure 8.
Figure 8.

Regulation of Stx-2—mediated AP-1—, NF-κB (p50)—, and NF-κB (p65)—binding activity by β2-adrenoceptor activation and cAMP-PKA cascade. Stx-2 (4 pg/ml) was added to ACHN cells 10 min or 20 min after pretreatment with the β2-adrenoceptor agonist, Ter (10-7 M and 10-6 M) or For (10-6 M), the β2-adrenoceptor antagonist, ICI (10-6 M), and/or the cAMP-PKA inhibitor, H-89 (5 × 10-6 M). The arrows indicate the AP-1—binding activity (A), NF-κB (p50)—binding activity (B), and NF-κB (p65)—binding activity (C) during the following 2 h, which were analyzed using EMSA. Results shown are representative of three separate experiments.

Regulation of TNF-α Promoter Activity

Figure 9A shows that TNF-α promoter activity was stimulated by the Stx-2 challenge, reaching a peak level of some 2.4-fold at 2 h and then returning toward basal at 3 h. In Figure 9B, the comparisons of TNF-α promoter activity at 2 h among these groups are given as bar graphs. Ter (10-6 M or 10-7 M) significantly suppressed Stx-2—mediated TNF-α promoter activity in a dose-dependent manner (P < 0.05). PD (10-6 M) and SB (10-6 M) significantly suppressed the Stx-2—induced TNF-α promoter activity (both P < 0.05). Forskolin (10-5 M) and PG (10-6 M) also depressed Stx-2—mediated TNF-α promoter activity (P < 0.05). H-89 (5 × 10-6 M) and KT (10-5 M) blocked the inhibitory action of Ter on Stx-2—mediated TNF-α promoter activity (P < 0.05), but the magnitude of the block was small and the promoter activity was still suppressed significantly by 10-6 M Ter (H-89, 25% inhibition; KT, 24% inhibition; both P < 0.05). Thus β2-adrenoceptor activation and MAPK (p42/p44 and p38) inhibition resulted in suppression of the Stx-2—induced TNF-α promoter activity. The cAMP-PKA pathway was partially involved in the suppression. Under these conditions, in which the cells were not treated with Stx-2, TNF-α promoter activity was significantly inhibited (P < 0.05) by Ter in a concentration-dependent manner by approximately 70% and 59% at 10-6 M and 10-7 M, respectively (Figure 9C). The Ter suppression of TNF-α promoter activity was abolished by the addition of H-89 (5 × 10-6 M) and KT (10-5 M), which was most likely caused by cAMP-PKA pathway activation. The NF-κB inhibitor, PDTC (1.5 × 10-4 M), also significantly suppressed TNF-α promoter activity in the cells (P < 0.05). Therefore, the constitutive production of TNF-α in the cell was regulated through cAMP-PKA and NF-κB activation. However, the inhibition of MAPK (p42/p44, p38) activities by PD (10-6 M) and SB (10-6 M) did not attenuate the constitutive expression of TNF-α gene in the cell.

Figure 9.
Figure 9.

TNF-α promoter activities expressed by pGV-B2-TNFprom in ACHN cells. (A) Time course of TNF-α promoter activity in the cells after the administration of 4pg/ml Stx-2. (B) Ter (10-6 M or 10-7 M) significantly inhibited TNF-α promoter activity in the cells 2 h after the Stx (4pg/ml) challenge, but the inhibitory effect of Ter was only partially overcome by H-89 (5 × 10-6 M) and KT (10-5 M). PD, SB, forskolin, and PG could suppress the Stx-induced TNF-α promoter activity. 1, Stx-2; 2, Stx-2 + Ter (10-6 M); 3, Stx-2 + Ter (10-7 M); 4, Stx-2 + PD (10-6 M); 5, Stx-2 + SB (10-6 M); 6, Stx-2 + Forskolin (10-5 M); 7, Stx-2 + PG (10-6 M); 8, Stx-2 + Ter (10-6 M) + H-89; 9, Stx-2 + Ter (10-6 M) + KT. (C) In the cells untreated with Stx-2, Ter was able to suppress TNF-α promoter activity at 2 h, and this effect was blocked by H-89 (5 × 10-6 M) and KT (10-5 M). PD and SB were unable to suppress the TNF-α promoter activity, whereas PDTC (1.5 × 10-4 M) could prevent the activity in the cell. The data are expressed as the fold activation that was calculated by dividing normalized luciferase activity (luciferase/β-galactosidase) in the cells by the level of activity in the cells untreated with any regimens. Data are mean ± SE from four experiments. 1, control; 2, Ter (10-6 M); 3, Ter (10-7 M); 4, Ter (10-6 M) + H-89; 5, Ter (10-6 M) + KT; 6, PDTC; 7, PD (10-6 M); 8, SB (10-6 M). *P < 0.05 versus cells exposed to 4pg/ml Stx-2 alone. †P < 0.05 versus cells exposed with no treatment (control). §P < 0.05 versus cells exposed with 4 pg/ml Stx-2 + Ter (10-6 M).

Discussion

The concept has been put forward that TNF-α produced by macrophage (25) and proximal tubule cells (10) in response to Stx may have a direct impact on the degree of Stx-mediated cytotoxicity (9). However, the signal transduction mechanism whereby TNF-α is produced as a result of Stx stimulation was unknown. In this study, we have demonstrated that Stx-2 induces both p42/p44 MAPK/AP-1 and p38 MAPK pathways and, subsequently, TNF-α production in the ACHN cells. p42/p44 MAPK phosphorylates Elk-1 and activates a serum response element, which leads to c-Fos induction, which is a component of AP-1 (26). The AP-1 site is present on the TNF-α promoter, and AP-1 plays an important role in the transcriptional regulation of TNF-α (27). It has been reported that activation of p42/p44 MAPK and the subsequent TNF-α production is mediated via the Ras/Raf-1 pathway in RAW 264.7 macrophage cells (28). Therefore, we speculated that a tyrosine kinase might mediate the Stx-2—induced TNF-α production through the Ras/MAPK/AP-1 pathway. The view was supported by the observation that the MEK1 inhibitor, PD, which selectively inhibited Stx-2—induced p42/p44 MAPK phosphorylation and AP-1 activation in this study, had an inhibitory effect on Stx-2—induced TNF-α promoter activity and protein release from the cells. These results indicate that p42/p44 MAPK and AP-1 activation are both involved in the important pathway that mediates the Stx-2—induced TNF-α production in the cells. On the other hand, Stx-2 did not influence the levels of JNK activation, c-Jun induction, or NF-κB (p50 and p65) translocation, which suggests that JNK and NF-κB activation are probably not involved in the major pathway whereby Stx-2 leads to TNF-α gene transcription. This study also indicates that p38 MAPK in the cells was stimulated by Stx-2. It is also evident that SB inhibited Stx-2—induced TNF-α promoter activity and protein release from the cells. However, neither DNA binding of NF-κB nor Stx-2—induced AP-1 was affected by SB. In addition, the p38 MAPK signaling pathway was not influenced at a time when the cAMP-PKA pathway was activated. Thus, Stx-2 induces p38 MAPK activation with the subsequent initiation of TNF-α gene transcription through routes other than those that use AP-1, NF-κB (p50 and p65), or cAMP-PKA.

An additional observation that arises from this study is that Stx-2—induced TNF-α gene transcription and protein release were depressed by β2-adrenoceptor activation. Importantly, however, this was exerted through both the cAMP-PKA cascade and a cAMP-independent pathway. Previous investigators (15,29,30) have reported that the inhibition of TNF-α production by β2-adrenoceptor activation was dependent on an increase in intracellular cAMP levels. This view is consistent with our study in that the depressed TNF-α protein release in response to Stx-2 after exposure to β2-adrenoceptor agonists could be abolished by the cAMP-PKA inhibitor, H-89, or KT. Moreover, at the transcriptional level, one component of the mechanisms that inhibit TNF-α gene transcription could be attributed to an action of intracellular cAMP. The constitutive activity of TNF-α promoter in the cells was suppressed by the NF-κB inhibitor, PDTC, but not by the MAPK inhibitors, PD and SB. Thus, the suppression of TNF-α gene transcription in the absence of Stx-2 appeared to be mediated by NF-κB (p65) translocation via a cAMP-PKA pathway. Conversely, in the presence of Stx-2, the suppression of TNF-α gene transcription is partially mediated by MAPK as well as NF-κB translocation via a cAMP-PKA pathway. These observations would support the reports of Olivier et al. (31) and Zhong et al. (32), who found that an increase in intracellular cAMP concentration decreased the NF-κB—mediated function of TNF-α gene transcription. Furthermore, Burgering et al. (33) demonstrated that elevated cAMP could block MAPK activation of oncogenic Ras proteins. Together, these pieces of evidence suggest that the cAMP pathway leads to an inhibition of Stx-2—induced MAPK (p42/p44) activation that indirectly suppresses TNF-α gene transcription, whereas an inhibition of NF-κB (p65) translocation by intracellular cAMP would lead to a decrease in TNF-α promoter activity. Interestingly, Zhong et al. (32) reported that cAMP-PKA not only regulates activation of NF-κB, but also that of AP-1. However, Grassl et al. (34) found that AP-1 DNA-binding activity was enhanced after treatment of cAMP. We were, however, unable to detect a significant change in AP-1—binding activity after β2-adrenoceptor activation. The reasons for this discrepancy between their results and our data in this study are unclear. It is evident from our findings that the inhibition of MAPK (p42/p44) by β2-adrenoceptor stimulation did not regulate AP-1 induction in these cells.

Conversely, MAPK (p38) and NF-κB (p50) were found to be involved in the suppression of TNF-α gene transcription by a cAMP-independent pathway. Moreover, the Ter-induced downregulation of MAPK (p42/p44) activity was only partly abolished by the addition of H-89 or KT, which suggests that the inhibition of MAPK (p42/p44) activity by β2-adrenoceptor activation was partially independent of the cAMP-PKA pathway. Together, these findings would suggest that β2-adrenoceptor stimulation during the Stx-2 challenge is able to prevent TNF-α gene transcription not only as a consequence of the enhanced cAMP generation but also as a consequence of MAPK (p42/p44 and p38) and NF-κB (p50) inhibition. This view would be consistent with the report of Seldon et al. (35), who found that the inhibitory effects of β2-adrenoceptor agonists on TNF-α gene transcription and/or translation could be regulated by both a cAMP-PKA cascade and a cAMP-independent mechanism. Intracellular signaling pathways other than cAMP-PKA could also be involved in the modulation of MAPK and NF-κB by β2-adrenoceptors. Crespo et al. (36) reported that signaling that arises from β2-adrenoceptor stimulation leading to MAPK activation involves an initiating signal that requires βγ subunits of G protein and a Gαs-induced inhibitory signal mediated by the cAMP-PKA pathway. However, we were unable to observe an elevation of MAPK as a result of exposure of the cells to the β2-adrenoceptor agonist alone (17).

Stx consists of an A subunit of 32 kD associated with five B subunits, each of 7.5 kD. The B subunits bind specifically to the cell surface Gb3 receptor, also known as CD77, and facilitate the entry of the holotoxin into susceptible cells (37). ACHN cells express the receptor for Stx, Gb3/CD77, and binding to the CD77 antigen induces cell cytotoxicity/apoptosis (11). Taga et al. (38) have reported that ligand binding to CD77 causes an increase of intracellular Ca2+ concentration and a rapid increase in cAMP in Burkitt lymphoma cell, which seems to be a critical step in the apoptotic process. However, in ACHN cells, intracellular cAMP generation was not found to be elevated by the addition of Stx-2 (data not shown), and the activation of the cAMP-PKA pathway via β2-adrenoceptor appears to be part of a protective response against Stx-2—induced cell cytotoxicity. Katagiri et al. (39) showed that Stx binding to Gb3 causes activation of the Src family tyrosine kinase Yes. Whether these intracellular signaling pathways are part of the process that leads TNF-α gene transcription and induces cell death is still unclear. Hughes et al. (10) examined the effect of protein synthesis inhibitor, cycloheximide, on TNF-α mRNA and protein release in human glomerular epithelial cells and suggested that the mechanism of Stx-1—induced TNF-α production was, in part, independent of global inhibition of protein synthesis or cytotoxicity. We were able to demonstrate that Stx binding to the Gb3/CD77 receptor stimulated TNF-α production in ACHN cells through both the p42/p44MAPK/AP-1 pathway and the p38MAPK pathway. However, it has to be accepted that other intracellular signaling pathways induced by Stx may be involved in the activation of TNF-α gene transcription. The mechanisms that transduce the signal mediated by Stx binding to Gb3/CD77 on ACNH require further examination. In the renal tubule, it is quite clear that β2-adrenoceptors are present and able to influence tubular function (40). We have also confirmed in this study that functional β2-adrenoceptors are expressed on the ACHN cells. In an in vivo study, it has been found that β2-adrenoceptors are distributed predominantly in the outer and inner stripe of the outer medulla in the rat kidney (41). A significant inhibitory effect of β2-adrenoceptor agonists on lipopolysaccharide-induced TNF-α mRNA has been observed in the medullary region of rat (42). However, it is unclear whether β2-adrenoceptor agonists are able to inhibit TNF-α production in the human kidney in response to Stx. Therefore, the importance of β2-adrenoceptor stimulation as a means whereby TNF-α can be inhibited must be investigated further by using human tissue and then in humans. It may offer a therapeutic approach to overcome the impairment of renal tubular function in HUS.

In summary, we found that Stx-2 induced p42/p44 MAPK/AP-1 and p38 MAPK activation with subsequent TNF-α gene transcription. The β2-adrenoceptor agonist inhibited NF-κB (p50, p65) activation and Stx-2—induced p42/p44 MAPK and p38 MAPK activation, which, in turn, was correlated with reduction in TNF-α gene transcription. The inhibitory effect was regulated by both a cAMP-PKA pathway and a cAMP-independent mechanism.

Acknowledgments

This study was supported by grants from the Human Science Foundation in Japan. We thank Prof. Donald Kohan, Division of Nephrology, University of Utah, for his comments regarding the manuscript. Part of this work was presented in two abstract forms in the 32nd and 33rd annual meetings of the American Society of Nephrology, November 1999 and October 2000.

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Activation of β2-Adrenoceptor Prevents Shiga Toxin 2-Induced TNF-α Gene Transcription
AKIO NAKAMURA, EDWARD J. JOHNS, AKIRA IMAIZUMI, YUKISHIGE YANAGAWA, TAKAO KOHSAKA
JASN Nov 2001, 12 (11) 2288-2299;
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