Evidence That Angiotensin II and Lipoxygenase Products Activate c-Jun NH2-Terminal Kinase
Abstract The effect of angiotensin II (Ang II) to activate c-Jun amino-terminal kinase (JNK) was studied in a Chinese hamster ovary fibroblast cell line overexpressing the rat vascular type-1a Ang II receptor (CHO-AT1a). Ang II treatment induced a time-dependent activation of JNK. Ang II (10−7 mol/L) activated JNK activity, with a peak at 30 minutes (9.39±2.52-fold, n=7, P<.02 versus control), which was maintained until 3 hours (2.7±0.65-fold, n=3, P<.02 versus control). Ang II–induced JNK activation at 30 minutes was inhibited by a specific lipoxygenase (LO) pathway inhibitor, cinnamyl-3,4-dihydroxy-α-cyanocinnamate (1 μmol/L) by 87.5% (n=4, P<.01 versus Ang II–induced JNK activity). The direct addition of 12-HETE also induced a time-dependent JNK activation. 12-HETE (10−7 mol/L) activated JNK activity, with a peak at 10 minutes (3.43±0.87-fold, n=6, P<.02 versus control), which remained elevated until 1 hour. These results suggest that the LO pathway is a mediator of Ang II–induced JNK activation. 15-HETE can also activate JNK at 5 minutes, but this activity was reduced at 30 minutes and could not be seen at 1 hour, indicating that the time course was different from that seen with 12-HETE. N-Acetylcysteine (NAC), an antioxidant, was used to perturb intracellular reactive oxygen intermediate (ROI) levels to assess the role of endogenous ROIs in regulating JNK activity. Pretreatment of cells with 500 μmol/L NAC for 1 hour attenuated ≈50% of Ang II–induced JNK activation, suggesting that ROIs, at least partially, mediate Ang II–induced JNK activation. Furthermore, 12-HETE–induced JNK activation was reduced by ≈90% by NAC. Finally, pertussis toxin completely blocked 12-HETE–induced JNK activation, suggesting that Gi-protein signaling participates in 12-HETE–induced effects. These results suggest that LO activation plays a role in mediating Ang II–induced JNK activation in part by altering the redox tone and Gi-protein signaling of cells.
Also known as SAPK, JNK is a member of the MAP kinase family,1 2 which is linked to cellular growth, inflammation, and apoptosis.3 The relevance of this signal in growth responses was recently demonstrated by Olson et al,4 who demonstrated a clear role of the small GTP-binding proteins, Rac and JNK, in cell cycle progression through G1. There is also evidence showing that the activation of the JNK cascade may be growth inhibitory and that JNK activation by growth factors could provide a negative modulatory signal to limit the mitogenic response.5 Therefore, JNK may play a growth-promoting or -inhibitory role, depending on the model studied. New emerging data suggest that JNK activation plays an important role in ischemia and reperfusion in the perfused heart,6 as well as in inflammation and atherosclerosis.3
c-Jun, ATF-2, and Elk-1 have been identified as the downstream substrates of JNK. Upstream, JNK is activated by a specific SAPK/ERK kinase, which is, in turn, activated by a MEKK.7 New data indicate that small GTP-binding proteins, such as Rac and Cdc42, may mediate JNK and p38 activation through another kinase known as PAK.8 9 10 A novel group of ste20-like kinases, called the mixed lineage kinase (MLK) family, has also been shown to mediate Rac and Cdc42 activation of p46sapk and p38mapk. However, the identity of the PAK substrate that couples this kinase to the JNK/SAPK and p38 pathways is not known.
Ang II is a growth factor and potent vasoconstrictor. The growth-inducing action of Ang II is primarily linked to type-1 receptor activation. One report has shown that Ang II can activate JNK in rat liver epithelial cells.11 New data also indicate that JNK plays an important role in modulating serum deprivation–induced apoptosis and that Ang II can maintain growth by increasing ERK1/2 activity and decreasing JNK activity in vascular smooth muscle cells.12
It has been demonstrated that Ang II is a potent mitogen for CHO-AT1a cells, which have been transfected with Ang II type-1 receptor cDNA.13 We have shown that initial responses to Ang II type-1 receptor activation involve phospholipid metabolism and arachidonic acid release.14 We also recently demonstrated that Ang II induces a biphasic activation of ERK1, with the early peak at 5 minutes and late sustained peak at 3 hours. Furthermore, we found that the late sustained peak of ERK activity was linked to the cell proliferation and activation of the 12-LO pathway of arachidonate metabolism.15 In the present study, we have evaluated the role of Ang II type-1 receptor activation on JNK activity. Furthermore, we have studied whether lipids derived from 12-LO activation mediate this Ang II response on JNK. The results support the hypothesis that 12-LO products participate in JNK activation in part by altering redox tone and Gi-protein signaling.
Materials and Methods
Human Ang II was from Peninsula Laboratories Inc. HAM’s F-12 medium and FBS were supplied by Irvine Scientific. BSA (fatty acid free), NAC, leupeptin, and aprotinin were from Sigma Chemical Co. CDC, 12-HETE, and 15-HETE were from Biomol. The GST–c-Jun1/79 plasmid was kindly provided by Dr Michael Karin (University of California at San Diego). JNK1 antibody was from Santa Cruz Biotechnology Inc. PTX was from List Biological Laboratories. [γ−32P]ATP was from New England Nuclear Corp.
Cell Culture and Preparation of Cell Extracts
CHO-AT1a cells were maintained in 100-mm dishes in HAM’s F-12 medium with 10% FBS as described.13 14 Cells were growth-arrested by incubation in HAM’s F-12 medium containing 1 mg/mL BSA and 20 mmol/L HEPES (pH 7.4) for 72 hours before use. Cells were treated with Ang II in serum-free BSA-containing medium. After washing, the harvested cells were lysed by WCE buffer containing 25 mmol/L HEPES (pH 7.7), 0.3 mol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.1% Triton X-100, 0.5 mmol/L dithiothreitol, 20 mmol/L β-glycerophosphate, 0.1 mmol/L sodium orthovanadate, 5 μg/mL leupeptin and aprotinin, and 0.1 mmol/L PMSF. The cell suspension was rotated at 4°C for 30 minutes, and lysate was centrifuged at 14 000g at 4°C for 10 minutes. Protein concentration was estimated by Bio-Rad protein assay.
Solid-Phase Kinase Assay
Cell extracts were diluted so that the final composition of the WCE buffer was 20 mmol/L HEPES (pH 7.7), 75 mmol/L NaCl, 2.5 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.05% Triton X-100, 0.5 mmol/L dithiothreitol, 20 mmol/L β-glycerophosphate, 0.1 mmol/L sodium orthovanadate, 5 μg/mL leupeptin and aprotinin, and 0.1 mmol/L PMSF. The extracts were mixed with 20 μL GST–c-Jun coupled to glutathione-Sepharose beads, which were prepared with the GST–c-Jun 1/79 plasmid.1 The mixture was rotated at 4°C overnight in a microcentrifuge tube and pelleted by centrifugation at 10 000g for 20 seconds. After four 1-mL washes in HEPES binding buffer (20 mmol/L HEPES [pH 7.7], 50 mmol/L NaCl, 2.5 mmol/L MgCl2, 0.1 mmol/L EDTA, and 0.05% Triton X-100), the pelleted beads were resuspended in 60 μL kinase buffer (20 mmol/L HEPES [pH 7.6], 20 mmol/L MgCl2, 20 mmol/L β-glycerophosphate, 20 mmol/L p-nitrophenyl phosphate, 0.1 mmol/L Na3VO4, and 2 mmol/L dithiothreitol) containing 20 μmol/L ATP and 5 μCi of [γ−32P]ATP. After 30 minutes at 30°C, the reaction was terminated by washing with HEPES binding buffer. Phosphorylated proteins were eluted with 1.5× Laemm-li sample buffer and resolved on 10% SDS-polyacrylamide gel, followed by autoradiography. Autoradiograms from the JNK activity studies were analyzed with an automated computerized densitometer (SCISCAN 5000, US Biochemical). Measurements were made in the linear range, and the values are expressed as arbitrary optical density units or fold over control.
Immunoprecipitation and Kinase Assay
Cell extracts in diluted WCE buffer (20 mmol/L HEPES [pH 7.7], 75 mmol/L NaCl, 2.5 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.05% Triton X-100, 0.5 mmol/L dithiothreitol, 20 mmol/L β-glycerophosphate, 0.1 mmol/L sodium orthovanadate, 5 μg/mL leupeptin and aprotinin, and 0.1 mmol/L PMSF) were mixed with 10 μL JNK-1 antibody (Santa Cruz Biotechnology), and the mixture was rotated at 4°C overnight and then was added to 50 μL protein A Sepharose. After 1 hour of incubation at 4°C, the beads were washed four times with diluted WCE buffer, and the pelleted beads were resuspended in 60 μL kinase buffer. The kinase buffer, the phosphorylation reaction, and the procedures were conducted as described in “Solid-Phase Kinase Assay.” The antibody used was specific for the JNK1 protein and does not react to any significant degree with JNK2.
The results are expressed as mean±SE from combined experiments, as noted in each legend. ANOVA with Dunnett’s test, the Tukey-Kramer multiple-comparison test, or Student’s t test was used to analyze the data.
Effect of Ang II on JNK Activity
JNK activity was assayed in quiescent CHO-AT1a cells to determine the time-response to Ang II treatment. Fig 1a⇓ is a representative autoradiogram of a gel showing that Ang II induces a time-dependent increase of JNK activity. The average JNK activity from several experiments using densitometric measurements of the GST–c-Jun1/79 band at different time periods is shown in Fig 1b⇓. The results show that JNK activation can been seen at 5 minutes (2.5-fold), with a peak at 30 minutes (>9-fold) and with the activity maintained until 3 hours (2.7-fold).
Effect of the 12-LO Pathway on JNK Activity
To elucidate the effect of the LO pathway on JNK activity, we first evaluated whether the LO inhibitor CDC can affect Ang II–induced JNK activity. CDC was used at 10−6 mol/L, a concentration that is more specific for the 12-LO enzyme. As shown in Fig 2⇓, a 15-minute pretreatment of CHO-AT1a cells with 10−6 mol/L CDC caused a significant inhibition of Ang II–induced JNK activation at 30 minutes by 87.5% (n=4, P<.01). CDC alone did not significantly increase JNK activity (n=4) when all experiments were analyzed.
We next examined the direct effect of 12[S]-HETE addition on JNK activation. Direct addition of 12-HETE resulted in JNK activation in a time-dependent manner. The JNK activity at different time periods after 12-HETE addition is shown in Fig 3⇓. We also conducted experiments at other times showing that 12-HETE can increase JNK at 5 minutes (3.13±0.27-fold) and 30 minutes (2.80±0.40-fold). The data indicate that the JNK activity in response to 12-HETE can be seen at 5 minutes and reaches a peak at 10 minutes. The effect of 12-HETE on JNK activation may be sustained at least 1 hour. Direct addition of 15-HETE also activated JNK activity, as shown in Fig 4⇓. In contrast to 12-HETE action, the effect of 15-HETE was transient with a decline at 30 minutes and a complete absence of activity at 1 hour.
Involvement of ROIs and G Proteins in JNK Activation
To elucidate a possible mechanism of JNK activation, the cells were first treated with the antioxidant NAC for 1 hour before the addition of Ang II or 12-HETE. Cells were treated by Ang II or 12-HETE for 30 and 10 minutes, respectively. The data shown in Fig 5⇓ indicate that pretreatment of cells with 500 μmol/L NAC significantly inhibited the Ang II–induced JNK activity by ≈50% (P<.04). NAC alone had no effect on basal activity. NAC (500 μmol/L) also inhibited 12-HETE–induced JNK activity by ≈90% (P<.01). These results suggest that oxygen intermediates do play a role in JNK activation stimulated by Ang II and 12-HETE.
To explore whether a G protein–linked mechanism is involved in 12-HETE–induced JNK activation, we investigated the effect of PTX on 12-HETE action. The effect of PTX on 12-HETE–induced JNK activity is shown in Fig 6⇓. PTX (100 ng/mL) added 3 hours before the addition of 12-HETE completely eliminated 12-HETE–induced increases in JNK activity at 10 minutes, suggesting an important role of Gi protein signaling in 12-HETE action.
We have used the CHO-AT1a cell line, which has been stably overexpressed with the rat vascular AT1a receptor cDNA, as a model cell system to elucidate the possible role of Ang II in JNK regulation. The JNK antibody immunoprecipitation and kinase assay was mainly used in the present study with the JNK1 antibody, whereas the solid-phase kinase assay was used only in the experiment shown in Fig 1⇑. Our results show that Ang II can lead to a time-dependent activation of JNK, which demonstrates a peak effect at 30 minutes and can be maintained until 3 hours. These new results suggest that JNK pathway activation may be important for the sustained effects of Ang II type-1 receptor signaling.
The 12-LO pathway plays a key role in Ang II–induced growth and ERK activation. Therefore, we also evaluated the possible role of the LO pathway on JNK activation. First, we observed that Ang II–induced JNK activation could be inhibited by preincubation with CDC, a specific blocker of the LO pathway of arachidonate metabolism. This result indicates a potential role for LO metabolites in mediating the action of Ang II to stimulate JNK activity. Second, we observed that the direct addition of 12-HETE also induced a time-dependent activation on JNK. Ang II can increase 12-LO activity within 10 minutes in the CHO-AT1a cells, and these changes can be blocked by CDC.15 The present data clearly indicate a role of 12-LO activation in the later phases of Ang II–induced stimulation of JNK. The direct addition of 15-HETE also activated JNK activity, but the time course of activation was different from that of 12-HETE, suggesting a more specific role of the 12-LO pathway products in the sustained effects of Ang II activation. However, the results could be due to a more rapid loss of 15-HETE than of 12-HETE. More experiments are thus needed to characterize these differences.
There is little known about the mechanism whereby Ang II or 12-HETE can induce JNK activation. In our previous study, it was shown that Ang II caused a rapid and sustained activation of phospholipase D–mediated phosphatidylcholine hydrolysis, resulting in the formation of phosphatidic acid.14 Ang II in these cells also causes an activation of arachidonic acid release of >20-fold (authors’ unpublished data, 1996), indicating the activation of phospholipase A2. The activation of both phospholipase A2 and phospholipase D could stimulate the intracellular generation of ROIs through the formation of arachidonate and phosphatidic acid, respectively. In vascular smooth muscle cells, Ang II stimulates NADH and NADPH oxidase activity through phosphatidic acid generation, thus promoting superoxide formation.16 The LO products of arachidonate have also been demonstrated to lead to superoxide formation.17 Very recently, it was demonstrated that ROIs mediate cytokine activation of c-Jun NH2-terminal kinases.18 Taken together, these facts prompted us to investigate the role of ROIs in mediating JNK activation by Ang II and 12-HETE. It has been demonstrated that NAC is an effective free radical scavenger and increases the intracellular glutathione level, which in turn modulates the concentrations of ROIs via glutathione peroxidase.19 Therefore, the antioxidant NAC was used to perturb the intracellular ROI level to assess the role of endogenous ROIs in regulating JNK activity. Antioxidant treatment antagonized the stimulating effect of Ang II and 12-HETE on JNK activity (Fig 5⇑). These facts support a role of ROIs in modulating JNK activity. The exact pathway explaining how ROIs lead to JNK activation has yet to be identified. JNKs themselves do not appear to be the direct targets of ROIs because both H2O2 and S-nitroso-N-acetylpenicillamine could not activate JNKs immobilized onto GST–c-Jun glutathione beads.18 The direct modulation of p21ras activity by ROIs such as H2O2 and NO20 21 might produce candidate pathways that lead to the JNK activation.
PAK has been shown recently to be one upstream regulator of JNK.8 9 However, signaling from Rho proteins to JNK in 293T cells does not involve PAK.22 These findings demonstrate that Rac and/or Cdc42 might signal to JNK in a cell type–specific manner. Ang II has been demonstrated to activate PAK in CHO-AT1a cells in our laboratory (authors’ unpublished data, 1996). However, additional studies using wild-type, dominant-negative, and constitutively active PAK vectors will be needed for exploring the role of PAK as an upstream activator of JNK in the CHO-AT1a cells.
It is also possible that the effects of Ang II and 12-HETE on JNK may be via increases in Ca2+ concentration, since Ca2+ has been linked to JNK activation.11 It has recently been shown that Ang II–induced JNK activation in cardiac myocytes involves increases in Ca2+.23 12-HETE has been shown to increase intracellular Ca2+ in vascular and adrenal cells,24 supporting this hypothesis.
Recent evidence supports a role of βγ subunits of heterotrimeric Gi proteins in communicating G protein–coupled receptors with the JNK pathway, acting on a Ras- and Rac-dependent biochemical route.25 It is interesting that treatment with PTX almost completely inhibited 12-HETE–induced JNK activation, suggesting that 12-HETE may act via a Gi protein–linked receptor and that a βγ complex might be involved in 12-HETE–induced JNK activation.
The present study provides new evidence supporting a role of the 12-LO pathway in mediating Ang II–induced JNK activation. Furthermore, the results support the hypothesis that 12-LO products participate in JNK activation in part by altering redox tone and Gi-protein signaling.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|CHO-AT1a cells||=||Ang II receptor AT1a–transfected Chinese hamster ovary cells|
|ERK||=||extracellular-regulated protein kinase (MAP kinase and ERK are often used interchangeably)|
|JNK||=||c-Jun amino-terminal kinase (JNK and SAPK are often used interchangeably)|
|MAP kinase||=||mitogen-activated protein kinase|
|MEK||=||MAP kinase kinase|
|ROI||=||reactive oxygen intermediate|
|SAPK||=||stress-activated protein kinase|
This study was supported by National Institutes of Health grants R01 DK-39721 (Dr Nadler) and P01 HL-55798 (Dr Nadler). The authors would like to thank Dr Eric Clauser (College de France, Paris, France) for providing the CHO-AT1a cell line. We also thank Dr Michael Karin (University of California at San Diego) for providing GST–c-Jun1/79 plasmids, Dr Rama Natarajan for helpful suggestions, and Almira Fontanilla for secretarial assistance.
- Received February 11, 1997.
- Accepted July 22, 1997.
- © 1997 American Heart Association, Inc.
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