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【摘要】 ANG II induces secretion and activation of transforming growth factor- (TGF- ) by glomerular mesangial cells. However, the mechanisms that operate this are unclear. Thrombospondin-1 (TSP-1), which is produced by mesangial cells in damaged glomeruli, is one of several molecules known to activate the latent TGF- 1 complex. Therefore, we examined whether the ANG II-induced activation of latent TGF- 1 in human mesangial cells (HMC) operates via TSP-1. The addition of ANG II (1-100 nM) to HMC significantly increased TSP-1 mRNA within 6 h, followed by an increase in TSP-1 protein production as shown by Western blot analysis of cells and immunoassay of the culture supernatant. Production of ANG II-induced TSP-1 mRNA and protein was completely inhibited by an ANG II type 1 (AT 1 )-receptor antagonist but was unaffected by an AT 2 -receptor antagonist. Use of a TSP-1-specific blocking peptide demonstrated that the ANG II-induced activation of latent TGF- 1 operates via TSP-1. Next, we investigated the role of ERK1/2, p38 MAPK, and JNK in ANG II-induced TSP-1 production in HMC. The addition of the upstream ERK1/2 inhibitor PD-98059 did not affect ANG II-induced TSP-1 production, whereas addition of either the p38 MAPK inhibitor SB-203580 or the JNK inhibitor SP-600125 significantly reduced TSP-1 production. In conclusion, this study has demonstrated that ANG II-induced activation of latent TGF- 1 in HMC operates via TSP-1. Furthermore, ANG II-induced TSP-1 production is dependent on p38 MAPK and JNK signaling.
【关键词】 angiotensin II type and type receptor extracellular signalregulated kinase / thrombospondin blocking peptide
ANG II plays an important role in the development of glomerulosclerosis ( 39 ). In both clinical and experimental studies, angiotensin-converting enzyme inhibitors and ANG II type 1 (AT 1 )-receptor antagonists have shown their renoprotective effects, which cannot be explained entirely by their hemodynamic effects ( 6, 16, 21, 32 ). In addition to mediating contraction, ANG II can induce a variety of responses in glomerular mesangial cells, such as cell growth ( 39 ), hypertrophy ( 2 ), and production of ECM ( 38 ).
ANG II stimulates ECM protein synthesis in rat mesangial cells through the induction of transforming growth factor- (TGF- ) expression ( 3, 14 ). TGF- is secreted as a biologically inactive complex. These complexes require cleavage to an active form, which is then able to bind to the TGF- receptors on the cell surface and exert biological effects ( 22 ). ANG II stimulation of mesangial cells results in an increase in both total TGF- production and in levels of active TGF- ( 3, 14 ). While many studies have examined the molecular mechanisms of ANG II-induced TGF- 1 gene transcription, very little is known about how ANG II promotes activation of the latent TGF- complex.
Thrombospondin-1 (TSP-1) is a multifunctional matrix protein consisting of a trimer of three disulfide-linked 180-kDa subunits ( 1 ). Originally identified as a constituent of the -granules of platelets, TSP-1 plays an important role in wound healing and activates the latent TGF- 1 complex via a protease- and cell-independent mechanism in vitro ( 28 ) and in vivo ( 9 ). Upregulation of TSP-1 production by glomerular mesangial cells has been described in several experimental renal diseases and in vitro ( 5, 12, 17 ), suggesting that TSP-1 may play an important role in TGF- 1 -driven glomerulosclerosis. However, it is unknown whether TSP-1 is the mechanism by which ANG II induces TGF- 1 activation in glomerular mesangial cells.
METHODS
Reagents. Reagents for these studies were prepared as follows. ANG II and ITS (insulin, transferrin, sodium selenite) were from Sigma (St. Louis, MO); RPMI-1640 medium without D -glucose, HEPES, penicillin, streptomycin, and L -glutamine were from Life Technologies (Grand Island, NY); D -glucose was from Kanto Chemica (Tokyo, Japan); and FCS was from Mitsubishi Kasei (Tokyo, Japan). Valsartan, a selective AT 1 receptor blocker, was kindly provided by Novartis Pharma (Basel, Switzerland). PD-123319, a selective AT 2 receptor blocker, and anti- -tubulin antibody were from Sigma; PD-98059 (MAPK/ERK inhibitor) and SB-203580 (p38 MAPK inhibitor) were from Calbiochem (San Diego, CA); SP-600125 (JNK inhibitor) was from Biomol Research Labs (Plymouth Meeting, PA); a human TGF- 1 immunoassay kit (DB100) was from R&D Systems (Minneapolis, MN); a human TSP-1 immunoassay kit and rabbit anti-human TSP-1 polyclonal antibody were from Cytimmune Sciences, (College Park, MD); and rabbit anti-AT 1 receptor polyclonal antibody and goat anti-AT 2 receptor polyclonal antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-JNK (Thr183/Tyr185), and JNK were from Cell Signaling Technology (Beverly, MA); horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG antibody was from DakoCytomation (Carpinteria, CA); FITC-conjugated goat anti-rabbit IgG and 4,6-diamidino-2-phenylindole were from Molecular Probes (Leiden, The Netherlands); HRP-conjugated donkey anti-rabbit IgG, PVDF membranes, and the enhanced chemiluminescence (ECL) Western blotting detection system were from Amersham Pharmacia Biotech (Buckinghamshire, UK); and the TSP-1-blocking peptide GGWSHW (W-peptide) and the negative control peptide GGYSHW (Y-peptide) were synthesized by Kurabo (Osaka, Japan).
Cell culture. Primary normal human mesangial cells (HMC; CC-2559, lot 8F1507) were purchased from BioWhittaker (Walkersville, MD) and originated from a 62-yr-old Caucasian woman. HMC were seeded in 75-cm 2 tissue culture flasks and routinely cultured in modified MCDB medium (BioWhittaker) supplemented with 5% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in an atmosphere of 5% CO 2 in air in a humidified incubator. The medium was replaced every 48 h. HMC were used for experiments at the passages 5-7. For all experiments, cells were made quiescent in RPMI-1640 medium containing 4 mM D -glucose, 20 mM HEPES, 2 mM L -glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, ITS (5 µg/ml, 5 µg/ml, 5 ng/ml, respectively), and 0.01% FCS for 48 h before administration of agents and during the experimental period. The same culture medium was used as the control medium. Additions of valsartan or PD-123319 were made 30 min before ANG II stimulation, whereas additions of PD-98059, SB-203580, and SP-600125 (or DMSO vehicle) were made 60 min before ANG II stimulation.
Human umbilical vein endothelial cells (HUVEC) were purchased from American Type Culture Collection (Rockville, MD) and grown in M199 medium supplemented with 10% FCS, 2 mM L -glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin 37°C in an atmosphere of 5% CO 2 in air in a humidified incubator. The medium was also replaced every 48 h. HUVEC were starved in M199 medium without FCS for 48 h before analysis of ANG II receptor subtypes.
Quantitative RT-PCR. HMC (1 x 10 5 cells/well) were seeded on six-well plates and grown until cells were 80% confluent. Cells then were made quiescent and incubated with various concentrations of ANG II for the specified time. Total RNA was extracted using the TRIzol reagent (Life Technologies). Equal amounts (2 µg) of total RNA from each sample were converted to cDNA by M-MLV reverse transcriptase RNaseH - (ReverTra Ace; Toyobo, Osaka, Japan) with oligo dT20 primer in a 20-µl reaction volume. We performed real-time PCR using the LightCycler quick system 350S (Roche Diagnostics, Tokyo, Japan). The RT reaction was subjected to PCR amplification using LightCycler Fast Start DNA Master SYBR Green I (Roche Diagnostics) in a 20-µl reaction volume with 0.3 µM of each primer and 3 mM MgCl 2. -Actin was used as the internal control. The primer sequences are as follows: TSP-1 (235 bp), sense 5'-CCTATGCTGGTGGTAGACTA-3' and antisense 5'-ACGTTCTAGGAGTCCACACT-3'; and -actin (260 bp), sense 5'-GCAAAGACCTGTACGCCAAC-3' and antisense 5'-CTAGAAGCATTTGCGGTGGA-3'. The amplification program was 95°C for 10 min and then 40 cycles consisting of 95°C for 10 s, 62°C for 10 s, and 72°C for 10 s. Amplification products were analyzed by a melting curve, which confirmed the presence of a single PCR product in all reactions (apart from negative controls). Quantification of PCR products was measured by fit-point analysis, and melting curve analysis was performed in all measurement. The results of TSP-1 were normalized by -actin. For visualization of PCR products, we amplified each cDNA using the same methods described above and terminated the reaction at the optimal cycle, which was in the range of threshold amplification. The PCR products were removed from each capillary, run on a 1.5% agarose gel, and visualized by ethidium bromide staining.
Western blot analysis. For detection of AT 1 or AT 2 receptor, HMC and HUVEC were seeded in 75-cm 2 tissue culture flasks and cultured until 80% confluent. After being made quiescent, cells were washed twice with ice-cold PBS and then lysed in 500 µl of ice-cold lysis buffer (in mM: 10 Tris·HCl, pH 7.4, 100 NaCl, 1 EDTA, 1 EGTA, 1 NaF, 2 Na 3 VO 4, and 1 PMSF, as well as 1% Triton X-100, 10% glycerol, 0.5% deoxycholate, and 10% protease inhibitor cocktail for mammalian tissues; Sigma). The lysates were put on ice and vortexed every 2 min for 10 min. Lysates were then centrifuged at 15,000 g for 20 min at 4°C, and the supernatants were aliquoted and stored at -80°C. The protein content of cell lysates was determined by a BCA protein assay (Pierce, Rockford, IL). Lysates (15 µg of protein) were separated on 10% polyacrylamide gels using SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were blocked overnight at 4°C with 20 mM Tris·HCl, pH 7.4, and 140 mM NaCl with 0.05% Tween 20 (TBST buffer) containing 5% nonfat dry milk, incubated for 2 h at 4°C with each primary antibody (1:200 dilution), washed three times in TBST buffer, incubated with secondary antibody (HRP-conjugated donkey anti-rabbit IgG at 1:5,000 dilution or HRP-conjugated rabbit anti-goat IgG at 1:2,000 dilution) for 2 h at room temperature, and the reaction products were then detected by the ECL Western blotting detection system.
For detection of phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-JNK (Thr183/Tyr185), and JNK, HMC were seeded in 25-cm 2 tissue culture flasks and grown to 80% confluence. After HMC were made quiescent, cells were incubated with ANG II for the specified time, lysed with 100 µl of ice-cold lysis buffer, and stored as described above. Lysates (20 µg of protein) were separated on 10% polyacrylamide gels using SDS-PAGE and transferred to PVDF membranes. For detection of cell-associated TSP-1 and -tubulin, HMC were seeded on six-well plates and grown to 80% confluence. After HMC were made quiescent, cells were incubated with ANG II for 24 h, lysed with 100 µl of ice-cold lysis buffer, and stored as described above. Lysates (15 µg of protein) were separated on 7.5% polyacrylamide gels using SDS-PAGE and transferred to PVDF membranes. The blots were blocked for 2 h at room temperature with TBST containing 5% nonfat dry milk, then incubated overnight at 4°C with each primary antibody (1:1,000 dilution), washed three times in TBST buffer, incubated with HRP-conjugated secondary antibody (1:5,000 dilution) for 2 h, and the reaction products were detected by the ECL Western blotting detection system using X-ray film.
The intensity of each band was estimated using National Institutes of Health Image software (version 1.6).
Immunoassays. HMC were seeded on 24-well plates, grown to 80% confluency, and then made quiescent. Cells then were incubated in the presence or absence of ANG II for the specified time. The culture medium was collected and centrifuged, and the supernatant was stored at -30°C until assayed. The concentration of TSP-1 in the media was measured with a competitive enzyme immunoassay kit according to the manufacturer's instructions.
TGF- 1 in culture media was determined by ELISA ( 18 ). The total amount of TGF- 1 was determined by acidification of samples before assay, whereas activated TGF- 1 in culture media was determined without acidification according to the manufacturer's instructions. The TSP-1 blocking peptide (W-peptide) and negative control peptide (Y-peptide) were added to HMC at the same time as ANG II.
Quantification of TSP-1 and TGF- 1 in the culture media was normalized by the total cell protein contents determined by the BCA protein assay.
Immunostaining of TSP-1. HMC were cultured on two-well chamber slides (Nalge Nunc), made quiescent, and then incubated in the test media for 24 h. Then cells were fixed in cold acetone/methanol at -20°C for 5 min, rehydrated in PBS, and incubated for 2 h in PBS containing 20% Block Ace (Dainippon Seiyaku, Tokyo, Japan). This was followed by overnight incubation with a primary antibody (rabbit anti-human TSP-1 polyclonal antibody, 1:100 dilution) at 4°C. After being washed, cells were incubated with FITC-conjugated goat anti-rabbit IgG (5 µg/ml) for 1 h at room temperature and nuclei were counterstained with 300 nM 4,6-diamidino-2-phenylindole for 3 min. Stained specimens were examined under a laser scanning microscope (AX-80, Olympus, Tokyo, Japan).
Statistical analysis. Results are expressed as means ± SE of at least three experiments. Statistical analysis was performed with ANOVA followed by Tukey's post hoc test. Differences were taken as statistically significant at P < 0.05.
RESULTS
ANG II induces TSP-1 gene and protein expression in HMC. Constitutive expression of TSP-1 mRNA was detected in quiescent HMC by RT-PCR ( Fig. 1, A and B ). The addition of 100 nM ANG II induced a significant increase in TSP-1 mRNA levels (1.8-fold) within 3 h, which was maintained for the 12-h study period ( Fig. 1, A and B ). The effect of different doses of ANG II on TSP-1 mRNA was investigated at a 6-h time point. Incubation with 1 nM ANG II induced a significant increase (1.25-fold) in TSP-1 mRNA levels, with greater increases in TSP-1 mRNA evident with 10 and 100 nM ANG II (1.45- and 1.65-fold; Fig. 1, C and D ).
Fig. 1. ANG II induces thrombospondin-1 (TSP-1) gene expression in human mesangial cells (HMC). A : HMC were incubated in the absence or presence of 100 nM ANG II for indicated times, and real-time RT-PCR was performed. A representative gel shows TSP-1 ( top ) and -actin ( bottom ). B : graph showing the relative mRNA levels of TSP-1 normalized to -actin in the absence (open bars) or presence (filled bars) of 100 nM ANG II for indicated times relative to 0-h control levels. C : HMC were incubated for 6 h in the absence (open bars) or presence (filled bars) of various concentrations of ANG II, and real-time PCR was performed. A representative gel shows TSP-1 ( top ) and -actin ( bottom ). D : graph showing the relative mRNA levels in various concentrations of ANG II normalized to -actin relative to control levels. Values are means ± SE of 6 individual experiments. * P < 0.05 by ANOVA with Tukey's post hoc test.
Immunostaining showed the production of cell-associated TSP-1 protein by ANG II-stimulated HMC. In control cells, faint immunostaining for TSP-1 was observed ( Fig. 2 AI ). There was an increase in the intensity of TSP-1 immunostaining in HMC stimulated for 24 h with 1-100 nM ANG II ( Fig. 2, AII-IV ). As a positive control, TSP-1 immunostaining was increased by stimulation with 5% FCS ( Fig. 2 AV ), whereas no staining was seen with an isotype, irrelevant control antibody (data not shown).
Fig. 2. ANG II induces an increase in cell-associated TSP-1 protein in HMC. A : immunostaining was used to detect cell-associated TSP-1 in cultured HMC, with nuclei counterstained with 4,6-diamidino-2-phenylindole. HMC were cultured on glass slides for 24 h with medium alone as a negative control ( I ); 1 ( II ), 10 ( III ), and 100 nM ANG II ( IV ); and 5% FCS as a positive control ( V ). Photomicrographs are representative of similar results obtained from 3 separate experiments. Magnification x 200. B : HMC were incubated in various concentrations of ANG II for 24 h. Representative blots show TSP-1 ( top ) and -tubulin ( bottom ). C : graph showing the relative cell-associated TSP protein levels in various concentrations of ANG II normalized to -tubulin relative to control levels. Values are means ± SE of 4 individual experiments. * P < 0.05 by ANOVA with Tukey's post hoc test.
For quantification of the cell-associated TSP-1 induced by different concentrations of ANG II, we performed Western blot analysis ( Fig. 2 B ). Incubation with 1, 10, and 100 nM ANG II for 24 h significantly increased that cell-associated TSP-1/ -tubulin ratio (1.53-, 1.98-, and 2.72-fold, respectively).
Stimulation of HMC with 100 nM ANG II also caused a significant increase in the secretion of TSP-1 into the culture medium. This was evident within 12 h of ANG II addition [12-h control: 9.2 ± 1.1, 12-h ANG II: 18.9 ± 6.3, 24-h control: 16.4 ± 2.1, 24-h ANG II: 35.2 ± 1.5 (SE) µg/mg cell protein, respectively] ( Fig. 3 A ). Stimulation of HMC with 10 or 100 nM ANG II for 24 h increased TSP-1 secretion by HMC, whereas concentrations of 1 nM had no significant effect on TSP-1 secretion (control: 17.4 ± 1.7, 1 nM ANG II: 19.7 ± 3.5, 10 nM ANG II: 22.2 ± 4.6, 100 nM ANG II: 35.7 ± 2.2 µg/mg cell protein, respectively) ( Fig. 3 B ).
Fig. 3. ANG II induces secretion of TSP-1 protein by HMC. A : HMC were incubated in the absence (open bars) or presence (filled bars) of 100 nM ANG II for the indicated times. Secreted TSP-1 protein in the culture supernatant was measured by competitive enzyme immunoassay. B : secreted TSP-1 protein in the culture supernatant after incubation for 24 h in the presence of various concentrations of ANG II. Values are means ± SE of 6 individual experiments. * P <0.05 by ANOVA with Tukey's post hoc test.
These results demonstrate that ANG II upregulates TSP-1 mRNA and protein levels in cultured HMC.
HMC express AT 1 and AT 2 receptors. Western blot analysis identified the presence of both AT 1 and AT 2 receptors in quiescent, starved HMC ( Fig. 4, A and B ). As a positive control, HUVEC were shown to strongly express both AT 1 and AT 2 receptors. The specificity of the Western blotting results was confirmed by incubation of the primary antibodies with their respective blocking peptides, which prevented detection of the bands (data not shown).
Fig. 4. HMC express angiotensin type 1 and 2 receptors. Western blot analysis of quiescent HMC or human umbilical vein endothelial cells (HUVEC) was used to detect the presence of ANG II type 1 ( A ) and type 2 receptors ( B ). Data shown are representative of similar results obtained from 3 separate experiments.
ANG II induces TSP-1 production via the AT 1 receptor. Because both AT 1 and AT 2 receptor protein existed in HMC, we examined which receptor was involved in ANG II-induced TSP-1 production.
Incubation of HMC with the AT 1 -receptor antagonist valsartan (1 µM) abolished 100 nM ANG II-induced upregulation of TSP-1 mRNA levels assessed by RT-PCR ( Fig. 5, A and B ). Accordingly, the upregulation of TSP-1 protein was inhibited by varlsartan at the same extent ( Fig. 5, C-E ). In contrast, the AT 2 -receptor antagonist PD-123319 (1 µM) had no effect on ANG II-induced TSP-1 mRNA levels, cell-associated TSP-1 protein, or secreted TSP-1 protein ( Fig. 5, A-E ).
Fig. 5. ANG II induces TSP-1 production in HMC via the ANG II type 1 receptor. Quiescent HMC were incubated with 1 µM valsartan (selective type 1 receptor blocker), 1 µM PD-123319 (selective type 2 receptor blocker), or vehicle control for 30 min before stimulation with 100 nM ANG II. A : real-time RT-PCR was performed to assess the levels of TSP-1 mRNA expression. A representative gel shows TSP-1 ( top ) and -actin ( bottom ) for 6 h with or without 100 nM ANG II incubation. B : graph showing the relative TSP-1 mRNA levels normalized to -actin relative to control. Values are means ± SE of 3 individual experiments. * P < 0.05 by ANOVA with Tukey's post hoc test. C : representative blots show TSP-1 ( top ) and -tubulin ( bottom ) for 24 h with or without 100 nM ANG II incubation. D : graph showing the relative TSP-1 protein levels normalized to -tubulin relative to control levels. Values are means ± SE of 4 individual experiments. * P < 0.05 by ANOVA with Tukey's post hoc test. E : HMC cells were cultured with or without 100 nM ANG II for 24 h, and secreted TSP-1 protein was measured by competitive enzyme immunoassay. Values are means ± SE of 3 experiments. * P <0.05 by ANOVA with Tukey's post hoc test.
These results indicate that ANG II-induced TSP-1 production operates via the AT 1 receptor.
TSP-1 activates latent TGF- 1 in HMC. To determine whether TSP-1 actives latent TGF- 1, we measured the amount of total vs. activated TGF- 1 secreted into the culture media by ELISA using acidified or nonacidified samples, respectively. Stimulation of HMC with 100 nM ANG II for 24 h induced a significant increase in the total amount of secreted TGF- 1 (1.38-fold) and an increase in the level of active TGF- 1 (1.80-fold) ( Fig. 6, A and B ). Incubation of cells with the specific TSP-1 inhibitor W-peptide had no effect on ANG II-induced total TGF- 1 secretion but reduced the levels of activated TGF- 1 back to control levels ( Fig. 6, A and B ). The control Y-peptide had no effect on secretion of total or active TGF- 1 ( Fig. 6, A and B ).
Fig. 6. TSP-1 blocking peptide (GGWSHW; W-peptide) inhibits ANG II-induced activation of transforming growth factor (TGF)- 1. Quiescent HMC were cultured for 24 h with (filled bars) or without (open bars) 100 nM ANG II in the presence of 10 µM TSP-1 W-peptide, 10 µM negative control peptide (GGYSHW; Y-peptide), or medium alone (control). The culture medium was assayed by ELISA for total TGF- 1 content (acidified samples; A ) or active TGF- 1 content (no acidification; B ). Values are means ± SE of 4 experiments. * P <0.05 by ANOVA with Tukey's post hoc test.
These results demonstrate that ANG II-induced TSP-1 production is the major mechanism whereby ANG II induces activation of latent TGF- 1 in HMC.
ANG II induces ERK 1/2, p38 MAPK, and JNK activation in HMC. Having demonstrated that ANG II induces activation of latent TGF- 1 in HMC via TSP-1, we examined the mechanisms by which ANG II induces TSP-1 production. ANG II is known to induce a number of cellular responses via ERK1/2, p38 MAPK, and JNK in several types of cells ( 25, 34, 36 ). Therefore, we examined whether ANG II actually activates these three kinases in HMC.
As shown in Fig. 7, stimulation of HMC with 100 nM ANG II induced a rapid activation (phosphorylation) of ERK1/2, p38 MAPK, and p46 JNK, which peaked at 5 min and then gradually decreased (2.89-, 2.40-, and 1.68-fold vs. control, respectively), whereas p54 JNK was not activated. Blots for total MAPKs remained constant throughout the duration of the experiments.
Fig. 7. ANG II activates ERK 1/2, p38 MAPK, and JNK in HMC. Quiescent HMC were stimulated with 100 nM ANG II for the times indicated, and then cells were lysed and analyzed by Western blotting for phosphorylated and total ERK1/2 ( A ) phosphorylated and total p38 MAPK ( C ), and phosphorylated and total JNK ( E ). B : ratio of phospho-ERK1/2 to total ERK1/2. D : ratio of phospho-p38 MAPK to total p38 MAPK. F : ratio of phospho-p46 JNK to total p46 JNK. Western blots shown are representative of similar results obtained from 3 separate experiments. Values are means ± SE of 3 experiments. * P < 0.05 by ANOVA with Tukey's post hoc test
ANG II induces TSP-1 production via p38 MAPK and JNK, but not ERK1/2. The addition of the MEK 1 inhibitor PD-98059 abolished ERK1/2 phosphorylation in control HMC ( Fig. 8, A, lane 2 ) and substantially inhibited ERK1/2 phosphorylation in ANG II-stimulated cells ( Fig. 8, A and B ). However, the addition of 0.5-50 µM PD-98059 did not affect cell-associated TSP-1 production ( Fig. 8, C and D ) or TSP-1 secretion into the medium ( Fig. 8 E ).
Fig. 8. ANG II-induced TSP-1 production in HMC does not operate via ERK1/2. A : quiescent HMC were stimulated with 100 nM ANG II for 5 min, and then cells were lysed and examined for phosphorylation of ERK1/2 by Western blotting. The blots shown are representative of similar results obtained from 3 separate experiments. The addition of 50 µM PD-98059 to HMC largely inhibited both basal and ANG II-induced ERK1/2 activation compared with DMSO vehicle control. B : graph showing the ratio of phospho- to total ERK1/2. Values are means ± SE of 3 experiments. * P < 0.05 by ANOVA with Tukey's post hoc test. C : quiescent HMC were stimulated for 24 h with (filled bars) or without (open bars) 100 nM ANG II plus PD-98059 or DMSO vehicle. The representative blots show cell-associated TSP-1 ( top ) and -tubulin ( bottom ) protein. D : graph showing the relative TSP-1 protein levels normalized to -tubulin relative to control levels. Values are means ± SE of 4 individual experiments. * P < 0.05 by ANOVA with Tukey's post hoc test. E : culture media were assayed for secreted TSP-1 by competitive enzyme immunoassay. Values are means ± SE of 6 experiments. * P < 0.05 by ANOVA with Tukey's post hoc test.
The addition of 0.2-20 µM SB-203580, a specific inhibitor of p38 MAPK, significantly reduced the phosphorylation of p38 MAPK ( Fig. 9, A and B ). Western blotting showed that 20 µM SB-203580 prevented the ANG II-induced increase in cell-associated TSP-1 ( Fig. 9, C and D ). Similarly, SB-203580 prevented the ANG II-induced increase in the secretion of TSP-1 into the culture medium in a dose-dependent fashion ( Fig. 9 E ).
Fig. 9. ANG II-induced TSP-1 production in HMC operates via p38 MAPK. A : quiescent HMC were stimulated with 100 nM ANG II for 5 min in the presence of SB-203580 or DMSO vehicle control. Cells were lysed and examined for phosphorylation of p38 MAPK by Western blotting. The blots shown are representative of similar results obtained from 3 separate experiments. B : graph showing the ratio of phospho- to total p38 MAPK relative to control cells. Values are means ± SE of 3 experiments. * P < 0.05 by ANOVA with Tukey's post hoc test. C : quiescent HMC were stimulated for 24 h with (filled bars) or without (open bars) 100 nM ANG II plus SB-203580 or DMSO vehicle. The representative blots showed cell-associated TSP-1 ( top ) and -tubulin ( bottom ) protein. D : graph showing the relative TSP-1 protein levels normalized to -tubulin relative to control levels. Values are means ± SE of 4 individual experiments. * P < 0.05 by ANOVA with Tukey's post hoc test. E : culture media were assayed for secreted TSP-1 by competitive enzyme immunoassay. Values are means ± SE of 6 experiments. * P < 0.05 by ANOVA with Tukey's post hoc test.
The addition of 20 µM SP-600125, a novel inhibitor of JNK, significantly reduced the phosphorylation of p46 JNK ( Fig. 10, A and B ). As shown in Fig. 10, C and D, 20 µM SP-600125 significantly reduced ANG II-induced cell-associated TSP-1 production. Similarly, SP-600125 substantially reduced the ANG II-induced increase in secreted TSP-1 ( Fig. 10 E ).
Fig. 10. ANG II-induced TSP-1 production in HMC also operates via p46 JNK. A : quiescent HMC were stimulated with 100 nM ANG II for 5 min in the presence of SP-600125 or DMSO vehicle control. Cells were lysed and examined for phosphorylation of JNK by Western blotting. Because p54 JNK was not activated by 100 nM ANG II, the blots present only p46 JNK. Data are representative of similar results obtained from 3 separate experiments. B : graph showing the ratio of phospho- to total p46 JNK relative to control cells. Values are means ± SE of 3 experiments. * P < 0.05 by ANOVA with Tukey's post hoc test. C : quiescent HMC were stimulated for 24 h with (filled bars) or without (open bars) 100 nM ANG II plus SP-600125 or DMSO vehicle. The representative blots show cell-associated TSP-1 ( top ) and -tubulin ( bottom ) protein. D : graph showing the relative TSP-1 protein levels normalized to -tubulin relative to control levels. Values are means ± SE of 4 individual experiments. * P < 0.05 by ANOVA with Tukey's post hoc test. E : culture media were assayed for secreted TSP-1 by competitive enzyme immunoassay. Values are means ± SE of 6 experiments. * P < 0.05 by ANOVA with Tukey's post hoc test.
In summary, these data show that p38 MAPK and p46 JNK play a major role in ANG II-induced TSP-1 production, whereas ERK1/2 is not involved.
DISCUSSION
This study identified that ANG II-induced TSP-1 mediated the activation of latent TGF- 1 in HMC. This was associated with an increase in TSP-1 mRNA and protein production, which was mediated by p38 MAPK and p46 JNK signaling after ANG II binding to the AT 1 receptor.
ANG II stimulation of quiescent HMC resulted in an increase in total TGF- 1 secreted into the culture media and in an increase in the amount of active TGF- 1. TSP-1 activation of latent TGF- 1 involves a domain consisting of three type I repeats ( 28 ). The amino acid sequence GGWSHW (W-peptide) in the first type 1 repeat, and the sequence KRFK between the first and the second type 1 repeats is responsible for TSP-1 activation of latent TGF- 1 ( 27 ). The W-peptide can be used as a specific inhibitor of TSP-1 activation, with the replacement of a single amino acid in the mutated Y-peptide serving as a control ( 24, 40 ). Using this strategy, we found two distinct mechanisms of activation of latent TGF- 1 by HMC. First, ANG II-induced activation of latent TGF- 1 operates via TSP-1. Second, the basal level of active TGF- 1 in quiescent HMC is produced by a TSP-1-independent mechanism. This is indicated by the finding that the W-peptide did not affect basal levels of active TGF- 1 in control cells, whereas the W-peptide reduced the levels of active TGF- 1 in ANG II-stimulated cells to those in control cells. The mechanism of latent TGF- 1 activation in control HMC is unknown but may involve a cell-associated plasminogen activator and subsequent generation of plasmin ( 14 ). However, such a mechanism would not be expected to operate with ANG II stimulation because ANG II rapidly induces synthesis of plasminogen activator inhibitor-1 ( 15 ).
ANG II-induced TSP-1-dependent activation of latent TGF- 1 was associated with an increase in TSP-1 mRNA levels and in cell-associated and secreted TSP-1 protein. This is the first demonstration that ANG II upregulates TSP-1 production in mesangial cells. Maximal induction of TSP-1 production was seen with 100 nM ANG II, but an increase in TSP-1 mRNA and cell-associated TSP-1 protein was observed with 1 and 10 nM ANG II. Although these levels of ANG II are high compared with those found in plasma, local ANG II levels in glomeruli are reported to be significantly higher than those found in the circulation ( 26, 29 ). In addition, ANG II-induced cell proliferation, collagen synthesis ( 38 ), TGF- production ( 3, 14 ), and fibronectin production ( 34 ) in mesangial cells have also been observed in the nanomolar range. Thus it is conceivable that concentrations of 1-100 nM ANG II may exist locally in the glomerulus in disorders involving local activation of the renin-angiotensin system.
ANG II-induced upregulation of TSP-1 mRNA and protein production operated via the AT 1 receptor, as demonstrated by inhibition with valsartan. Western blotting identified the presence of both AT 1 and AT 2 receptor subtypes in HMC, although AT 2 levels were markedly lower than in HUVEC. Blockade of the AT 2 receptor with PD-123319 was without effect. These findings in HMC are consistent with a previous study in cultured endothelial cells in which ANG II-induced TSP-1 mRNA operated via the AT 1 receptor ( 7 ). However, a second study of cultured endothelial cells found that ANG II signaling via the AT 1 receptor inhibited TSP-1 mRNA expression, whereas signaling via the AT 2 receptor increased TSP-1 gene expression ( 11 ).
The ability of ANG II to upregulate TSP-1 production by HMC raised the question of the signaling pathways by which it operates. ERK1/2, p38 MAPK, and JNK are cascades of serine/threonine kinases that transduce signals from the cell surface to the nucleus in response to growth factors and cellular stress ( 8, 20 ). ANG II is known to induce a variety of responses via these MAPK pathways. For example, ANG II activation of ERK1/2 induces hyperplasia and hypertrophy in vascular smooth muscle cells ( 30, 33, 37 ) and is involved in ECM production in mesangial cells ( 34 ). On the other hand, ANG II activation of p38 MAPK plays an important role in the hyper-trophic response of vascular smooth muscle cells and rat mesangial cells ( 19, 25, 36 ). However, very little is known about the role of ERK, p38 MAPK, and JNK signaling in TSP-1 production.
ANG II induction of TSP-1 production in HMC was dependent on signaling through p38 MAPK and p46 JNK, but not ERK 1/2. This is the first demonstration that ANG II-induced upregulation of TSP-1 operates via p38 MAPK and p46 JNK. This finding is consistent with studies in pancreatic tumor cells showing that TGF- 1 upregulation of TSP-1 mRNA also operates via p38 MAPK ( 31 ). Interestingly, TGF- 1 increased TSP-1 mRNA levels via prolonged mRNA stability in MG63 osteosarcoma cells ( 23 ). However, it is unclear whether ANG II upregulation of TSP-1 via p38 MAPK operates via prolonged mRNA stability.
The requirement of p38 MAPK signaling for ANG II-induced activation of latent TGF- 1 via TSP-1 suggests that activation of the p38 MAPK pathway may play an important role in ANG II-induced glomerulosclerosis. Currently, there is little information regarding the role of p38 MAPK signaling in renal fibrosis. Of interest, administration of a p38 MAPK inhibitor caused a significant reduction in bleomycin-induced lung fibrosis ( 35 ). However, it is unclear whether this results from a direct effect on the fibrotic process or simply from the inhibition of an inflammatory cascade that induces the fibrotic response.
The role of p38 MAPK signaling in ANG II-induced TGF- 1 activation is more complex than simply being required for upregulation of TSP-1 synthesis and secretion. Incubation of HMC with p38 MAPK inhibitor prevented the marked increase in the total amount of TGF- 1 secreted in response to ANG II stimulation (data not shown). Therefore, ANG II-induced p38 MAPK signaling is critical for both the production and activation of TGF- 1.
Many of the stimuli that activate the p38 MAPK pathway also activate the JNK pathway. Indeed, these pathways share common elements in their upstream signaling cascades ( 20 ). In addition to p38 MAPK, we identified the JNK signaling pathway as playing a major role in ANG II-induced TSP-1 production in HMC. Previous studies have identified an interaction between ANG II and JNK in cardiac organ gene expression and in the hypertrophic response in vitro and in vivo ( 4, 10 ); however, the role of ANG II-induced JNK activation in renal disease is not known. JNK activation induces phosphorylation of c-Jun, a component of the transcription factor complex AP-1 that binds to a specific DNA sequence called the "AP-1 binding site" ( 41 ). Because the TSP-1 gene promoter has three AP-1 binding sites ( 10 ), ANG II-induced JNK activation may contribute to increased TSP-1 gene transcription via the AP-1 complex. Further studies are required to clarify whether activation of JNK and p38 MAPK contributes solely to transcriptional regulation of the TSP-1 gene or if they have an additional role in the posttranscription events involved in TSP-1 protein production.
In summary, this study demonstrated that ANG II-induced activation of latent TGF- 1 in HMC operates via TSP-1. ANG II stimulation increased TSP-1 mRNA levels and increased cell-associated and -secreted TSP-1 protein. ANG II-induced TSP-1 production operates via the AT 1 receptor and signaling through the p38 MAPK and p46 JNK pathway. This mechanism may be important for the renoprotection afforded by angiotensin-converting enzyme inhibitors and AT 1 receptor blockers seen in human and experimental kidney disease.
ACKNOWLEDGMENTS
We thank Dr. Masayuki Kambe and Fujiko Kohno (Dept. of Clinical Laboratory Medicine, Div. of Medical Intelligence and Informatics, Programs for Applied Biomedicine, Hiroshima Univ. Graduate School of Biomedical Science) for technical assistance.
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作者单位：1 Department of Molecular and Internal Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-855 Japan; and 2 Department of Nephrology and Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria 316 Australia