c-Jun NH2-terminal kinase mediation of angiotensin II-induced proliferation of human mesangial cells
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《美国生理学杂志》
Department of Nephrology, Nanjing Children's Hospital, Center of Pediatric Nephrology, Nanjing Medical University, and Department of Medical Oncology, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing, China
Division of Nephrology, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, Utah
ABSTRACT
Angiotensin II (ANG II) has been shown to activate c-Jun NH2-terminal kinase (JNK) in cultured mesangial cells, but the functional implication of this phenomenon remains to be determined, largely due to the lack of an effective approach to block JNK. Therefore, the present study was carried out to examine whether JNK is involved in ANG II-induced cell proliferation in cultured human mesangial cells (HMCs) with the use of a newly developed JNK-selective blocker, SP-600125. Within minutes, treatment with 100 nM ANG II activated all three members of MAP kinase family, including extracellular signal-regulated protein kinase (Erk) 1/2, JNK, and p38 in cultured HMCs, as assessed by immunoblotting detection of phosphorylation of MAP kinases. ANG II-dependent activation of JNK was further confirmed by detection of increased phosphorylation and transcription activity of c-Jun after the ANG II treatment. SP-600125 ranging from 5 to 10 μM almost completely abolished the activation of JNK by ANG II without affecting the activities of Erk1/2 and p38. After treatment with 100 ng ANG II, there was a steady increase in [3H]thymidine incorporation that was blocked by SP-60025 in a dose- and time-dependent manner. Similarly, SP-600125 dose dependently reduced the ANG II-induced increase in cell number. The antiproliferative effect of SP-60025 was further determined by cell-cycle analysis with flow cytometry. Twenty-four hours after ANG II treatment, 50% of the quiescent HMCs (G0/G1) progressed into the S phase, and the cell cycle progression was almost completely prevented in the presence of SP-60025. Our data suggest that JNK mediates the proliferative effect of ANG II in cultured HMCs and thus represents a novel therapeutic target for treatment of chronic renal diseases.
cell cycle
MESANGIAL CELLS (MCS) ARE a prominent cell type located inside the glomeruli or in the extraglomerular mesangium of the juxtaglomerular apparatus and play an important role in maintenance of structural and functional integrity of the glomeruli (6, 26). MCs are considered to be derived mesenchymal cells and share similar properties with vascular smooth muscle cells (VSMCs). Like VSMCs, MCs can exhibit contractile and proliferative responses to vasoactive substances such as angiotensin II (ANG II) (39). Proliferation of MCs is almost invariably associated with various forms of glomerular renal diseases. Understanding the mechanisms governing MC proliferation may lead to the development of new therapeutic interventions for the devastating diseases.
ANG II is best known for its role in the control of extracellular volume and blood pressure (12, 13, 28). Emerging evidence suggests that ANG II plays a role in the pathogenesis of various forms of chronic renal diseases through mediating the inflammatory responses. In this regard, angiotensin-converting enzyme inhibitors and AT1-receptor antagonists exhibit renal beneficial effects in both human and animals that cannot be entirely attributed to their homodynamic effects (19, 21). In particular, these maneuvers have been shown to be effective in the attenuation of glomerulosclerosis (27). A large body of experimental data demonstrates that ANG II stimulates proliferation of MCs (2, 16, 18, 30–32, 40) as well as VSMCs (11, 35, 38, 41). The growth-promoting effect of ANG II is mediated by the AT1 receptor, which is a G protein-coupled, seven-transmembrane receptor.
The signaling pathway modulating the growth-promoting effects of ANG II in MCs is poorly characterized. Emerging evidence suggests a potential role of mitogen-activated protein kinases (MAPKs) in mediating the growth-promoting effect of ANG II in MCs. MAPKs are a family of serine/threonine protein kinases that are an important part of intracellular signaling pathways, connecting extracellular signals to intracellular regulatory proteins. This family consists of three major members, Erk1/2, JNK, and p38. Erk1/2 is typically stimulated by growth factor acting via a tyrosine kinase receptor and is widely associated with cell proliferation, whereas JNK and p38 kinase are more preferably activated by cellular stress and implicated in regulation of survival and apoptosis. Indeed, in MCs, blockade of Erk1/2 with PD-098059 completely abolished the proliferative effects of basic fibroblast growth factor (bFGF), one of the major of players in MC proliferation (18). Like bFGF, ANG II can activate Erk1/2 in MCs, whereas the mitogenic effects of ANG II in these cells are not significantly affected by PD-098059 (18). These findings reveal distinct roles of Erk1/2 in the mitogenic response induced by activation of a tyrosine kinase receptor and G protein-coupled receptors. It seams reasonable to speculate that other members of MAPK family or distinct signaling pathways may mediate the mitogenic effects of ANG II in MCs. In the present study, we present compelling evidence that mitogenic effects of ANG II in human MCs (HMCs) are mediated by JNK. This information may be helpful in developing more effective therapeutic regimens for chronic renal diseases by targeting JNK.
MATERIALS AND METHODS
Plasmids and materials. The PathDetectk (Stratagene, La Jolla, CA) system used was the c-Jun-trans-Reporting System (219000) and consists of the following components: 1) a luciferase reporter plasmid containing five tandem repeats of the yeast GAL4-binding site upstream of the luciferase gene, 2) an expression construct encoding either the DNA-binding domain of GAL4 only or the activation domain of c-Jun fused to the GAL4 DNA-binding domain. Reagents were obtained as follows: SP-600125, N1-methyl-1,9-pyrazoloanthrone (N1-methyl-substituted pyrazolanthrone), PD-98059, and SB-203580 were from Calbiochem (San Diego, CA). These compounds were poorly soluble in water, and stock solutions were made by using 100% dimethylsulfoxide. Rabbit polyclonal phospho-stress-activated protein kinase/JNK (Thr183/Tyr185), phospho-ERK1/2, phospho-p38 MAPK, and phospho-c-Jun (Ser63) antibodies were from Cell Signaling Technology (Beverly, MA). All other reagents were from Sigma (St. Louis, MO).
HMC isolation and culture. Normal-appearing portions of human kidneys that were surgically removed for renal carcinoma were used to culture MCs from outgrowths of collagenase-treated glomeruli. HMCs were established and characterized as previously reported (9, 25). Briefly, cells were grown until confluent in RPMI 1640 buffered with 10 mmol/l HEPES to pH 7.4 and supplemented with 20% FCS, 5 μg/ml insulin and transferrin, 100 U/ml penicillin, and 100 mg/ml streptomycin. For passage, confluent cells were washed with PBS, removed with 0.025% trypsin/0.5 mM EDTA in PBS, and plated in RPMI. Experiments included in this study were performed in cells between passages 5 and 10.
Western blot analysis. At indicated time points, HMCs were rapidly washed with ice-cold PBS and lysed for 10 min on ice in lysis buffer (50 mM Tris, pH 7.5, 40 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 μg/ml leupeptin, 2 mM DTT, and 1 mM PMSF). Lysates were cleared by centrifugation at 14,000 g (4°C) for 10 min. Total protein was quantified by the Bradford assay. Equal amounts of lysates were fractionated by 10% SDS-PAGE and electrotransferred to Bio-Blot nitrocellulose membranes (Bio-Rad). The membranes were blocked in TBST (20 mM Tris-base, pH 7.6, 150 mM NaCl, 0.1% Tween 20) containing 5% bovine serum albumin for 1 h at room temperature and incubated with primary antibody in the blocking solution at 4°C overnight. The membranes were incubated with 1:1,000 diluted HRP-conjugated secondary antibody at room temperature for 1 h and visualized by an enhanced chemiluminescence kit (Amersham). The quantitation of the chemiluminescent signal was carried out with the use of UVP software.
DNA synthesis and cell count. To estimate DNA synthesis, HMCs were stimulated by ANG II with or without SP-600125 for 19 h and pulsed with 1 μCi/ml [3H]thymidine for 5 h. Cells were then washed twice with ice-cold PBS, incubated for 5 min in 5% TCA, washed by methanol, and dissolved in 99% formic acid. The incorporation of [3H]thymidine into TCA-insoluble material was measured by a liquid scintillation spectrophotometer. For the assay of cell growth, HMCs in six-well plates were stimulated by ANG II with or without SP-600125, and the cell number was counted with a Coulter counter.
Flow cytometry. At indicated time points, HMCs were harvested by trypsinization and fixed in iced 70% ethanol and suspended in 1 ml of propidium iodide staining solution (50 μg/ml propidium iodide, 30 U/ml RNase A, 0.1% Triton X-100, 4 mM sodium citrate) and incubated at 37°C for 10 min before addition of NaCl at a final concentration of 138 mM NaCl. Typically, 10,000 gated events were collected on a FACscan (Beckton Dickinson, Franklin Lakes, NJ) and analyzed by using CELLQUEST software (Beckton Dickinson). Cell-cycle analyses were performed with Flowjo Software (Tree Star, San Carlos, CA).
Cell viability. MC viability was assessed using a lactate dehydrogenase (LDH) cytotoxicity detection kit that measures LDH release into the culture medium. The manufacturer's protocols were followed.
Statistical analysis. All experiments were repeated at least three times. Results are presented as the means ± SE from three separate experiments where indicated. Statistical comparisons between multiple groups were performed by ANOVA, and Bonferroni's method was applied to control for multiple testing. A minimum value of P < 0.05 was considered to represent statistical significance.
RESULTS
Effect of ANG II on JNK activity. We investigated the activation kinetics of JNK after treatment of HMCs with 100 nM ANG II. JNK activities were determined by immunoblotting detection of phosphorylations of JNK and its target transcription factor c-Jun using antibodies against Thr183/Tyr185-phosphorylated JNK and Ser63-phosphorylated c-Jun, respectively. Within minutes of ANG II treatment, phosphorylation of both JNK and c-Jun started to increase and peaked at 30 min and was reduced thereafter (Fig. 1A). The relative abundance of the Ser63-phosphorylated c-Jun band was evaluated by densitometry. Then, the 30-min time point was used to determine the dose response of ANG II activation of JNK. As shown in Fig. 1B, ANG II increased phosphorylations of both JNK and c-Jun in a dose-dependent manner, with a maximal stimulation at 100 nM. This dose was therefore used in subsequent experiments.
SP-600125 inhibits ANG II-induced phosphorylation of c-Jun. To test the ability of SP-600125 to inhibit JNK activity in HMCs, we measured the phosphorylation of c-Jun residue Ser63 by Western blotting. As shown in Fig. 2, SP-600125 reduced the ANG II-induced Ser63 phosphorylation of c-Jun in a dose-dependent manner with 75% reduction at 10 μM and 90% reduction at 20 μM. The level of LDH in the medium in the presence or absence of 20 μM SP-600125 was determined to examine toxicities of the compound. The treatment with 20 μM SP-600125 had no significant effect on LDH release. LDH release, expressed as a percentage of the total LDH activity of the corresponding culture dish, was 8.23 ± 1.65% in control and 8.86 ± 1.91% in the SP-600125 group (n = 6/group, P > 0.05).
To determine the specificity of SP-600125 in the inhibition of JNK, we examined whether SP-600125 affected the activity of other members of the MAPK family, including Erk and p38. Phosphorylation of Erk and p38 was determined by immunoblotting. As shown in Fig. 3, ANG II significantly induced phosphorylation of both Erk1/2 and p38 MAPK. The activations of Erk1/2 and p38 after ANG II treatment were blocked by PD-98059, an inhibitor of ERK1/2, and SB-203580, an inhibitor of p38, respectively. In contrast, neither Erk1/2 nor p38 activity was affected by 20 μM SP-600125.
SP-600125 reduces ANG II-induced transcriptional activity of c-Jun. JNK increases AP-1-dependent gene expression by stimulating transcriptional activity of c-Jun through phosphorylation at its the NH2-terminus trans-activation domain. Therefore, we examined the possibility that SP-600125 might affect the transcriptional activity of c-Jun. To address this possibility, HMCs were transiently transfected with an expression construct encoding a chimeric protein consisting of the trans-activation domain of c-Jun fused to the GAL4 DNA-binding domain and a luciferase reporter driven by GAL4-binding sites. Cells were subsequently treated with ANG II (100 nM) in the presence or absence of various concentrations of SP-600125 (1–20 μM). As shown in Fig. 4, the activity of a GAL4-driven reporter was markedly stimulated by ANG II that was blocked by SP-600125 in a dose-dependent manner.
SP-600125 inhibits ANG II-induced cell proliferation. We determined the effects of SP-600125 on ANG II-induced proliferation of HMCs. Increases in DNA synthesis must precede cell proliferation. Therefore, the capability of SP-600125 to inhibit the ANG II-dependent incorporation of [3H]thymidine into DNA was first examined. As shown in Fig. 5A, ANG II linearly increased [3H]thymidine incorporation into HMCs over a 3-day incubation period, and the stimulation was markedly attenuated in the presence of 10 μM SP-600125. The inhibitory effect of SP-600125 on ANG II-induced 3H incorporation was seen at 24 h and persisted until 72 h. Subsequently, the 48-h time point was chosen to obtain a dose-response curve for SP-600125 inhibition of 3H incorporation. As shown in Fig. 5B, SP-600125 ranging from 1 to 20 μM significantly inhibited ANG II-dependent [3H]thymidine incorporation in a dose-dependent manner.
The inhibitory effects of SP-600125 on ANG II-induced HMC proliferation, as assessed by cell number, are shown in Fig. 6. Quiescent HMCs were treated for 48 h with 100 nM ANG II in the presence or absence of SP-600125. SP-600125 ranging from 1 to 20 μM dose dependently reduced cell number. The specific inhibition of HMC proliferation by SP-600125 was further confirmed by detection of [3H]thymidine incorporation and cell number after treatment of ANG II with ERK 1/2 inhibitor PD-98059 or p38 inhibitor SB-203580. As shown in Figs. 5C and 6B, PD-98059 and SB-203580 have no effect on the HMCs proliferation.
The antiproliferative effect of SP-600125 was also assessed by flow cytometry. As shown in Fig. 7A, in unstimulated quiescent HMCs, 95% of the cells were in the growth-arrested (G0/G1) phase of the cell cycle (Fig. 7A), whereas after 24-h stimulation with ANG II, 50% of the HMCs entered the S phase of the cell cycle (Fig. 7B). When various of concentrations of SP-600125 (1, 10, and 20 μM) were present, the effect of ANG II was almost completely abolished and the cells stayed in the G0/G1 phase (Fig. 7, C–F).
DISCUSSION
ANG II stimulates MC proliferation, which in part underlies the pathogenesis of chronic renal diseases. Despite extensive investigation, the mechanism of ANG II-induced mitogenic effects in MCs is poorly understood. The present study examined the potential role of JNK in mediating the ANG II-induced mitogenic effect in cultured HMCs with the use of a newly developed JNK-specific inhibitor, SP-600125. We demonstrated that ANG II significantly induced proliferation of HMCs associated with activation of JNK and that inhibition of JNK with SP-600125 blocked the mitogenic effect of ANG II. To our knowledge, this is the first report addressing the role of JNK in mediating the ANG II-induced mitogenic effects in cultured MCs.
Benette et al. (3) characterized SP-600125 as a selective inhibitor of JNK in 2001 (3) and since then SP-600125 has been widely used to investigate the function of JNK in a variety of model systems. Recently, Heo et al. (15) determined the ternary structure of JNK1 complexed with the scaffolding protein (JNK-interacting protein-1) and SP-600125, providing the basis for the JNK specificity of the compound. Therefore, we chose to use SP-600125 to examine the potential role of JNK in mediating the mitogenic effect of ANG II in cultured HMCs. SP-600125 in a dose range of 1–20 μM significantly blocked the ANG II-induced phosphorylation of c-Jun. In the same dose range, SP-600125 blocked the ANG II-induced transcriptional activity of c-Jun, as determined using a GAL4 reporter system. In contrast, at the maximal dose of 20 μM, SP-600125 had no effect on ANG II-induced phosphorylation of Erk1/2 and p38. SP-600125 inhibited ANG II-induced c-Jun phosphorylation in HMCs with an IC50 of 5–10 μM, similar to the IC50 value previously reported in Jurkat T cells (3). In the same report, partial inhibition of other MAPK pathways was observed only when SP-600125 was used at concentrations >25 μM.
In the present study, ANG II-induced HMC proliferation was confirmed by three different approaches: [3H]thymidine incorporation, cell number, and flow cytometry. The mitogenic effect of ANG II was consistently blocked by SP-600125, with a noticeable effect at 1 μM and maximal effect at 20 μM. As discussed above, SP-600125 in the dose range of 1–20 μM selectively blocked JNK but not Erk or p38 activity. Therefore, the attenuation of ANG II-induced proliferation of HMCs by SP-600125 is due to specific inhibition of JNK. This finding agrees with the report that in cultured HMCs, transfection with the dominant-negative JNK construct inhibited PDGF-induced proliferation (22). Using SP-600125, increasing numbers of recent studies document important roles of JNK in mediation of a wide variety of cellular responses to ANG II. JNK in conjunction with other members of MAPK pathway is involved in ANG II stimulation of thrombospondin-1 and the latent transforming growth factor-1 complex in HMCs (29), cell migration in smooth muscle cells (24), and osteopontin expression in rat cardiac fibroblasts (42). We believe that the increasing use of SP-600125 will facilitate our understanding of the previously unidentified functions of JNK in ANG II-elicited signaling transductions or in other types of cellular responses.
To address the possibility that the antiproliferative effect of SP-600125 might be due to its cytotoxicity, we compared LDH release in HMCs treated with or without the compound. We found that SP-600125 at a dose of up to 20 μM had no significant effect on LDH release, thus ruling out the cytotoxicities of the compound under current experimental conditions.
It is well recognized that MC proliferation can be stimulated by activation of tyrosine kinase receptors for epidermal growth factor (EGF) (7, 36), platelet derived growth factor (PDGF) (1, 7), and bFGF (8, 17), and of G protein-coupled receptors for ANG II (2, 16) and endothelin-1 (33, 34). The signaling transductions elicited by activation of tyrosine kinase receptors and G protein-coupled receptors converge at Erk1/2. The role of Erk1/2 in mediation of the mitogenic effect of growth factors is well documented. In MCs, blockade of Erk1/2 with PD-098059 completely abolished the proliferative effects of bFGF, one of the major of players in MC proliferation (18). In contrast, it has been observed that the mitogenic effects of ANG II in MCs are not affected by PD-098059 (18). These findings reveal distinct roles of Erk1/2 in the mitogenic responses induced by activation of tyrosine kinase receptors and G protein-coupled receptors. It has been speculated that other members of MAPK family or distinct signaling pathways may mediate the mitogenic effect of ANG II in MCs (18). In support of this speculation, the present study demonstrated the mitogenic effect of ANG II in MCs is mediated by JNK.
We also provide evidence indicating the mechanism of JNK mediation of ANG II action in cultured MCs. Using a GAL4 reporter system, we demonstrated that ANG II induced transcriptional activity of c-Jun that was inhibited by JNK blockade. Given the observation that SP-600125 blocked ANG II-induced phosphorylation of c-Jun, it is evident that phosphorylation is required for the ANG II activation of transcriptional activity of c-Jun. However, this does not exclude the possibility that JNK regulation of c-Jun may occur at other levels. In a separate study, we observe that JNK is involved in ANG II regulation of monocyte chemoattractant protein 1 (MCP-1) mRNA stability (data not shown). We speculate that ANG II regulation of c-Jun may occur at multiple levels involving phosphorylation, posttranscriptional regulation, etc. Additionally, despite the emphasis on the JNK/c-Jun pathway, other coactivators may also be involved. In particular, c-fos, a well-known partner of c-Jun, may act in concert with c-Jun to mediate the full proliferative response of MCs to ANG II.
The proliferation of MCs is the common pathological feature of glomerular diseases, including immunoglobulin A nephropathy, lupus nephritis, and diabetes (5, 23). In experimental animal models of nephritis, MC proliferation frequently precedes and is linked to the increase in extracellular matrix in the mesangium and to glomerulosclerosis. Inhibition of MC proliferation by complement depletion (4), blocking of PDGF (10, 20) and basic fibroblast growth factor (5), and inhibiting of their intracellular signaling pathways with phosphodiesterase inhibitors (37) reduce extracellular matrix expansion and glomerulosclerosis. Thus elucidation of mechanisms regulating MC proliferation is of critical importance and may provide fundamental knowledge for the development of new therapies for glomerular diseases. Given the effectiveness of SP-600125 in the attenuation of ANG II-induced proliferation in cultured HMCs, it is reasonable to speculate that this compound may hold promise for the treatment of glomerular diseases, especially those associated with an activated renin-angiotensin system. In line with this notion, recently the therapeutic effect of SP-600125 in inflammatory arthritis has been demonstrated in a rat adjuvant arthritis model (14).
In summary, the present study, for the first time, demonstrated that SP-600125, an inhibitor of JNK, reduced ANG II-induced HMC proliferation, as assessed by [3H]thymidine incorporation, cell count, and flow cytometry. The dose range used in the present study effectively blocked JNK without an effect on Erk or p38 activity. The observation not only provides insight into mechanisms of MC proliferation but also supports the therapeutic potential of SP-600125 regarding glomerular diseases and glomerulosclerosis.
GRANTS
This work was supported by National Natural Science Foundation of China Grant 30100081, Natural Science Foundation of Jiangsu Province Grants 01KJB320009 and 01KJB320011 (A. Zhang), and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-066592 (T. Yang).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A. Zhang and G. Ding contributed equally to this work.
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Division of Nephrology, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, Utah
ABSTRACT
Angiotensin II (ANG II) has been shown to activate c-Jun NH2-terminal kinase (JNK) in cultured mesangial cells, but the functional implication of this phenomenon remains to be determined, largely due to the lack of an effective approach to block JNK. Therefore, the present study was carried out to examine whether JNK is involved in ANG II-induced cell proliferation in cultured human mesangial cells (HMCs) with the use of a newly developed JNK-selective blocker, SP-600125. Within minutes, treatment with 100 nM ANG II activated all three members of MAP kinase family, including extracellular signal-regulated protein kinase (Erk) 1/2, JNK, and p38 in cultured HMCs, as assessed by immunoblotting detection of phosphorylation of MAP kinases. ANG II-dependent activation of JNK was further confirmed by detection of increased phosphorylation and transcription activity of c-Jun after the ANG II treatment. SP-600125 ranging from 5 to 10 μM almost completely abolished the activation of JNK by ANG II without affecting the activities of Erk1/2 and p38. After treatment with 100 ng ANG II, there was a steady increase in [3H]thymidine incorporation that was blocked by SP-60025 in a dose- and time-dependent manner. Similarly, SP-600125 dose dependently reduced the ANG II-induced increase in cell number. The antiproliferative effect of SP-60025 was further determined by cell-cycle analysis with flow cytometry. Twenty-four hours after ANG II treatment, 50% of the quiescent HMCs (G0/G1) progressed into the S phase, and the cell cycle progression was almost completely prevented in the presence of SP-60025. Our data suggest that JNK mediates the proliferative effect of ANG II in cultured HMCs and thus represents a novel therapeutic target for treatment of chronic renal diseases.
cell cycle
MESANGIAL CELLS (MCS) ARE a prominent cell type located inside the glomeruli or in the extraglomerular mesangium of the juxtaglomerular apparatus and play an important role in maintenance of structural and functional integrity of the glomeruli (6, 26). MCs are considered to be derived mesenchymal cells and share similar properties with vascular smooth muscle cells (VSMCs). Like VSMCs, MCs can exhibit contractile and proliferative responses to vasoactive substances such as angiotensin II (ANG II) (39). Proliferation of MCs is almost invariably associated with various forms of glomerular renal diseases. Understanding the mechanisms governing MC proliferation may lead to the development of new therapeutic interventions for the devastating diseases.
ANG II is best known for its role in the control of extracellular volume and blood pressure (12, 13, 28). Emerging evidence suggests that ANG II plays a role in the pathogenesis of various forms of chronic renal diseases through mediating the inflammatory responses. In this regard, angiotensin-converting enzyme inhibitors and AT1-receptor antagonists exhibit renal beneficial effects in both human and animals that cannot be entirely attributed to their homodynamic effects (19, 21). In particular, these maneuvers have been shown to be effective in the attenuation of glomerulosclerosis (27). A large body of experimental data demonstrates that ANG II stimulates proliferation of MCs (2, 16, 18, 30–32, 40) as well as VSMCs (11, 35, 38, 41). The growth-promoting effect of ANG II is mediated by the AT1 receptor, which is a G protein-coupled, seven-transmembrane receptor.
The signaling pathway modulating the growth-promoting effects of ANG II in MCs is poorly characterized. Emerging evidence suggests a potential role of mitogen-activated protein kinases (MAPKs) in mediating the growth-promoting effect of ANG II in MCs. MAPKs are a family of serine/threonine protein kinases that are an important part of intracellular signaling pathways, connecting extracellular signals to intracellular regulatory proteins. This family consists of three major members, Erk1/2, JNK, and p38. Erk1/2 is typically stimulated by growth factor acting via a tyrosine kinase receptor and is widely associated with cell proliferation, whereas JNK and p38 kinase are more preferably activated by cellular stress and implicated in regulation of survival and apoptosis. Indeed, in MCs, blockade of Erk1/2 with PD-098059 completely abolished the proliferative effects of basic fibroblast growth factor (bFGF), one of the major of players in MC proliferation (18). Like bFGF, ANG II can activate Erk1/2 in MCs, whereas the mitogenic effects of ANG II in these cells are not significantly affected by PD-098059 (18). These findings reveal distinct roles of Erk1/2 in the mitogenic response induced by activation of a tyrosine kinase receptor and G protein-coupled receptors. It seams reasonable to speculate that other members of MAPK family or distinct signaling pathways may mediate the mitogenic effects of ANG II in MCs. In the present study, we present compelling evidence that mitogenic effects of ANG II in human MCs (HMCs) are mediated by JNK. This information may be helpful in developing more effective therapeutic regimens for chronic renal diseases by targeting JNK.
MATERIALS AND METHODS
Plasmids and materials. The PathDetectk (Stratagene, La Jolla, CA) system used was the c-Jun-trans-Reporting System (219000) and consists of the following components: 1) a luciferase reporter plasmid containing five tandem repeats of the yeast GAL4-binding site upstream of the luciferase gene, 2) an expression construct encoding either the DNA-binding domain of GAL4 only or the activation domain of c-Jun fused to the GAL4 DNA-binding domain. Reagents were obtained as follows: SP-600125, N1-methyl-1,9-pyrazoloanthrone (N1-methyl-substituted pyrazolanthrone), PD-98059, and SB-203580 were from Calbiochem (San Diego, CA). These compounds were poorly soluble in water, and stock solutions were made by using 100% dimethylsulfoxide. Rabbit polyclonal phospho-stress-activated protein kinase/JNK (Thr183/Tyr185), phospho-ERK1/2, phospho-p38 MAPK, and phospho-c-Jun (Ser63) antibodies were from Cell Signaling Technology (Beverly, MA). All other reagents were from Sigma (St. Louis, MO).
HMC isolation and culture. Normal-appearing portions of human kidneys that were surgically removed for renal carcinoma were used to culture MCs from outgrowths of collagenase-treated glomeruli. HMCs were established and characterized as previously reported (9, 25). Briefly, cells were grown until confluent in RPMI 1640 buffered with 10 mmol/l HEPES to pH 7.4 and supplemented with 20% FCS, 5 μg/ml insulin and transferrin, 100 U/ml penicillin, and 100 mg/ml streptomycin. For passage, confluent cells were washed with PBS, removed with 0.025% trypsin/0.5 mM EDTA in PBS, and plated in RPMI. Experiments included in this study were performed in cells between passages 5 and 10.
Western blot analysis. At indicated time points, HMCs were rapidly washed with ice-cold PBS and lysed for 10 min on ice in lysis buffer (50 mM Tris, pH 7.5, 40 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 μg/ml leupeptin, 2 mM DTT, and 1 mM PMSF). Lysates were cleared by centrifugation at 14,000 g (4°C) for 10 min. Total protein was quantified by the Bradford assay. Equal amounts of lysates were fractionated by 10% SDS-PAGE and electrotransferred to Bio-Blot nitrocellulose membranes (Bio-Rad). The membranes were blocked in TBST (20 mM Tris-base, pH 7.6, 150 mM NaCl, 0.1% Tween 20) containing 5% bovine serum albumin for 1 h at room temperature and incubated with primary antibody in the blocking solution at 4°C overnight. The membranes were incubated with 1:1,000 diluted HRP-conjugated secondary antibody at room temperature for 1 h and visualized by an enhanced chemiluminescence kit (Amersham). The quantitation of the chemiluminescent signal was carried out with the use of UVP software.
DNA synthesis and cell count. To estimate DNA synthesis, HMCs were stimulated by ANG II with or without SP-600125 for 19 h and pulsed with 1 μCi/ml [3H]thymidine for 5 h. Cells were then washed twice with ice-cold PBS, incubated for 5 min in 5% TCA, washed by methanol, and dissolved in 99% formic acid. The incorporation of [3H]thymidine into TCA-insoluble material was measured by a liquid scintillation spectrophotometer. For the assay of cell growth, HMCs in six-well plates were stimulated by ANG II with or without SP-600125, and the cell number was counted with a Coulter counter.
Flow cytometry. At indicated time points, HMCs were harvested by trypsinization and fixed in iced 70% ethanol and suspended in 1 ml of propidium iodide staining solution (50 μg/ml propidium iodide, 30 U/ml RNase A, 0.1% Triton X-100, 4 mM sodium citrate) and incubated at 37°C for 10 min before addition of NaCl at a final concentration of 138 mM NaCl. Typically, 10,000 gated events were collected on a FACscan (Beckton Dickinson, Franklin Lakes, NJ) and analyzed by using CELLQUEST software (Beckton Dickinson). Cell-cycle analyses were performed with Flowjo Software (Tree Star, San Carlos, CA).
Cell viability. MC viability was assessed using a lactate dehydrogenase (LDH) cytotoxicity detection kit that measures LDH release into the culture medium. The manufacturer's protocols were followed.
Statistical analysis. All experiments were repeated at least three times. Results are presented as the means ± SE from three separate experiments where indicated. Statistical comparisons between multiple groups were performed by ANOVA, and Bonferroni's method was applied to control for multiple testing. A minimum value of P < 0.05 was considered to represent statistical significance.
RESULTS
Effect of ANG II on JNK activity. We investigated the activation kinetics of JNK after treatment of HMCs with 100 nM ANG II. JNK activities were determined by immunoblotting detection of phosphorylations of JNK and its target transcription factor c-Jun using antibodies against Thr183/Tyr185-phosphorylated JNK and Ser63-phosphorylated c-Jun, respectively. Within minutes of ANG II treatment, phosphorylation of both JNK and c-Jun started to increase and peaked at 30 min and was reduced thereafter (Fig. 1A). The relative abundance of the Ser63-phosphorylated c-Jun band was evaluated by densitometry. Then, the 30-min time point was used to determine the dose response of ANG II activation of JNK. As shown in Fig. 1B, ANG II increased phosphorylations of both JNK and c-Jun in a dose-dependent manner, with a maximal stimulation at 100 nM. This dose was therefore used in subsequent experiments.
SP-600125 inhibits ANG II-induced phosphorylation of c-Jun. To test the ability of SP-600125 to inhibit JNK activity in HMCs, we measured the phosphorylation of c-Jun residue Ser63 by Western blotting. As shown in Fig. 2, SP-600125 reduced the ANG II-induced Ser63 phosphorylation of c-Jun in a dose-dependent manner with 75% reduction at 10 μM and 90% reduction at 20 μM. The level of LDH in the medium in the presence or absence of 20 μM SP-600125 was determined to examine toxicities of the compound. The treatment with 20 μM SP-600125 had no significant effect on LDH release. LDH release, expressed as a percentage of the total LDH activity of the corresponding culture dish, was 8.23 ± 1.65% in control and 8.86 ± 1.91% in the SP-600125 group (n = 6/group, P > 0.05).
To determine the specificity of SP-600125 in the inhibition of JNK, we examined whether SP-600125 affected the activity of other members of the MAPK family, including Erk and p38. Phosphorylation of Erk and p38 was determined by immunoblotting. As shown in Fig. 3, ANG II significantly induced phosphorylation of both Erk1/2 and p38 MAPK. The activations of Erk1/2 and p38 after ANG II treatment were blocked by PD-98059, an inhibitor of ERK1/2, and SB-203580, an inhibitor of p38, respectively. In contrast, neither Erk1/2 nor p38 activity was affected by 20 μM SP-600125.
SP-600125 reduces ANG II-induced transcriptional activity of c-Jun. JNK increases AP-1-dependent gene expression by stimulating transcriptional activity of c-Jun through phosphorylation at its the NH2-terminus trans-activation domain. Therefore, we examined the possibility that SP-600125 might affect the transcriptional activity of c-Jun. To address this possibility, HMCs were transiently transfected with an expression construct encoding a chimeric protein consisting of the trans-activation domain of c-Jun fused to the GAL4 DNA-binding domain and a luciferase reporter driven by GAL4-binding sites. Cells were subsequently treated with ANG II (100 nM) in the presence or absence of various concentrations of SP-600125 (1–20 μM). As shown in Fig. 4, the activity of a GAL4-driven reporter was markedly stimulated by ANG II that was blocked by SP-600125 in a dose-dependent manner.
SP-600125 inhibits ANG II-induced cell proliferation. We determined the effects of SP-600125 on ANG II-induced proliferation of HMCs. Increases in DNA synthesis must precede cell proliferation. Therefore, the capability of SP-600125 to inhibit the ANG II-dependent incorporation of [3H]thymidine into DNA was first examined. As shown in Fig. 5A, ANG II linearly increased [3H]thymidine incorporation into HMCs over a 3-day incubation period, and the stimulation was markedly attenuated in the presence of 10 μM SP-600125. The inhibitory effect of SP-600125 on ANG II-induced 3H incorporation was seen at 24 h and persisted until 72 h. Subsequently, the 48-h time point was chosen to obtain a dose-response curve for SP-600125 inhibition of 3H incorporation. As shown in Fig. 5B, SP-600125 ranging from 1 to 20 μM significantly inhibited ANG II-dependent [3H]thymidine incorporation in a dose-dependent manner.
The inhibitory effects of SP-600125 on ANG II-induced HMC proliferation, as assessed by cell number, are shown in Fig. 6. Quiescent HMCs were treated for 48 h with 100 nM ANG II in the presence or absence of SP-600125. SP-600125 ranging from 1 to 20 μM dose dependently reduced cell number. The specific inhibition of HMC proliferation by SP-600125 was further confirmed by detection of [3H]thymidine incorporation and cell number after treatment of ANG II with ERK 1/2 inhibitor PD-98059 or p38 inhibitor SB-203580. As shown in Figs. 5C and 6B, PD-98059 and SB-203580 have no effect on the HMCs proliferation.
The antiproliferative effect of SP-600125 was also assessed by flow cytometry. As shown in Fig. 7A, in unstimulated quiescent HMCs, 95% of the cells were in the growth-arrested (G0/G1) phase of the cell cycle (Fig. 7A), whereas after 24-h stimulation with ANG II, 50% of the HMCs entered the S phase of the cell cycle (Fig. 7B). When various of concentrations of SP-600125 (1, 10, and 20 μM) were present, the effect of ANG II was almost completely abolished and the cells stayed in the G0/G1 phase (Fig. 7, C–F).
DISCUSSION
ANG II stimulates MC proliferation, which in part underlies the pathogenesis of chronic renal diseases. Despite extensive investigation, the mechanism of ANG II-induced mitogenic effects in MCs is poorly understood. The present study examined the potential role of JNK in mediating the ANG II-induced mitogenic effect in cultured HMCs with the use of a newly developed JNK-specific inhibitor, SP-600125. We demonstrated that ANG II significantly induced proliferation of HMCs associated with activation of JNK and that inhibition of JNK with SP-600125 blocked the mitogenic effect of ANG II. To our knowledge, this is the first report addressing the role of JNK in mediating the ANG II-induced mitogenic effects in cultured MCs.
Benette et al. (3) characterized SP-600125 as a selective inhibitor of JNK in 2001 (3) and since then SP-600125 has been widely used to investigate the function of JNK in a variety of model systems. Recently, Heo et al. (15) determined the ternary structure of JNK1 complexed with the scaffolding protein (JNK-interacting protein-1) and SP-600125, providing the basis for the JNK specificity of the compound. Therefore, we chose to use SP-600125 to examine the potential role of JNK in mediating the mitogenic effect of ANG II in cultured HMCs. SP-600125 in a dose range of 1–20 μM significantly blocked the ANG II-induced phosphorylation of c-Jun. In the same dose range, SP-600125 blocked the ANG II-induced transcriptional activity of c-Jun, as determined using a GAL4 reporter system. In contrast, at the maximal dose of 20 μM, SP-600125 had no effect on ANG II-induced phosphorylation of Erk1/2 and p38. SP-600125 inhibited ANG II-induced c-Jun phosphorylation in HMCs with an IC50 of 5–10 μM, similar to the IC50 value previously reported in Jurkat T cells (3). In the same report, partial inhibition of other MAPK pathways was observed only when SP-600125 was used at concentrations >25 μM.
In the present study, ANG II-induced HMC proliferation was confirmed by three different approaches: [3H]thymidine incorporation, cell number, and flow cytometry. The mitogenic effect of ANG II was consistently blocked by SP-600125, with a noticeable effect at 1 μM and maximal effect at 20 μM. As discussed above, SP-600125 in the dose range of 1–20 μM selectively blocked JNK but not Erk or p38 activity. Therefore, the attenuation of ANG II-induced proliferation of HMCs by SP-600125 is due to specific inhibition of JNK. This finding agrees with the report that in cultured HMCs, transfection with the dominant-negative JNK construct inhibited PDGF-induced proliferation (22). Using SP-600125, increasing numbers of recent studies document important roles of JNK in mediation of a wide variety of cellular responses to ANG II. JNK in conjunction with other members of MAPK pathway is involved in ANG II stimulation of thrombospondin-1 and the latent transforming growth factor-1 complex in HMCs (29), cell migration in smooth muscle cells (24), and osteopontin expression in rat cardiac fibroblasts (42). We believe that the increasing use of SP-600125 will facilitate our understanding of the previously unidentified functions of JNK in ANG II-elicited signaling transductions or in other types of cellular responses.
To address the possibility that the antiproliferative effect of SP-600125 might be due to its cytotoxicity, we compared LDH release in HMCs treated with or without the compound. We found that SP-600125 at a dose of up to 20 μM had no significant effect on LDH release, thus ruling out the cytotoxicities of the compound under current experimental conditions.
It is well recognized that MC proliferation can be stimulated by activation of tyrosine kinase receptors for epidermal growth factor (EGF) (7, 36), platelet derived growth factor (PDGF) (1, 7), and bFGF (8, 17), and of G protein-coupled receptors for ANG II (2, 16) and endothelin-1 (33, 34). The signaling transductions elicited by activation of tyrosine kinase receptors and G protein-coupled receptors converge at Erk1/2. The role of Erk1/2 in mediation of the mitogenic effect of growth factors is well documented. In MCs, blockade of Erk1/2 with PD-098059 completely abolished the proliferative effects of bFGF, one of the major of players in MC proliferation (18). In contrast, it has been observed that the mitogenic effects of ANG II in MCs are not affected by PD-098059 (18). These findings reveal distinct roles of Erk1/2 in the mitogenic responses induced by activation of tyrosine kinase receptors and G protein-coupled receptors. It has been speculated that other members of MAPK family or distinct signaling pathways may mediate the mitogenic effect of ANG II in MCs (18). In support of this speculation, the present study demonstrated the mitogenic effect of ANG II in MCs is mediated by JNK.
We also provide evidence indicating the mechanism of JNK mediation of ANG II action in cultured MCs. Using a GAL4 reporter system, we demonstrated that ANG II induced transcriptional activity of c-Jun that was inhibited by JNK blockade. Given the observation that SP-600125 blocked ANG II-induced phosphorylation of c-Jun, it is evident that phosphorylation is required for the ANG II activation of transcriptional activity of c-Jun. However, this does not exclude the possibility that JNK regulation of c-Jun may occur at other levels. In a separate study, we observe that JNK is involved in ANG II regulation of monocyte chemoattractant protein 1 (MCP-1) mRNA stability (data not shown). We speculate that ANG II regulation of c-Jun may occur at multiple levels involving phosphorylation, posttranscriptional regulation, etc. Additionally, despite the emphasis on the JNK/c-Jun pathway, other coactivators may also be involved. In particular, c-fos, a well-known partner of c-Jun, may act in concert with c-Jun to mediate the full proliferative response of MCs to ANG II.
The proliferation of MCs is the common pathological feature of glomerular diseases, including immunoglobulin A nephropathy, lupus nephritis, and diabetes (5, 23). In experimental animal models of nephritis, MC proliferation frequently precedes and is linked to the increase in extracellular matrix in the mesangium and to glomerulosclerosis. Inhibition of MC proliferation by complement depletion (4), blocking of PDGF (10, 20) and basic fibroblast growth factor (5), and inhibiting of their intracellular signaling pathways with phosphodiesterase inhibitors (37) reduce extracellular matrix expansion and glomerulosclerosis. Thus elucidation of mechanisms regulating MC proliferation is of critical importance and may provide fundamental knowledge for the development of new therapies for glomerular diseases. Given the effectiveness of SP-600125 in the attenuation of ANG II-induced proliferation in cultured HMCs, it is reasonable to speculate that this compound may hold promise for the treatment of glomerular diseases, especially those associated with an activated renin-angiotensin system. In line with this notion, recently the therapeutic effect of SP-600125 in inflammatory arthritis has been demonstrated in a rat adjuvant arthritis model (14).
In summary, the present study, for the first time, demonstrated that SP-600125, an inhibitor of JNK, reduced ANG II-induced HMC proliferation, as assessed by [3H]thymidine incorporation, cell count, and flow cytometry. The dose range used in the present study effectively blocked JNK without an effect on Erk or p38 activity. The observation not only provides insight into mechanisms of MC proliferation but also supports the therapeutic potential of SP-600125 regarding glomerular diseases and glomerulosclerosis.
GRANTS
This work was supported by National Natural Science Foundation of China Grant 30100081, Natural Science Foundation of Jiangsu Province Grants 01KJB320009 and 01KJB320011 (A. Zhang), and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-066592 (T. Yang).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A. Zhang and G. Ding contributed equally to this work.
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