1Department of Molecular and Internal Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan; and 2Department of Nephrology and Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria 3168, Australia
Submitted 14 April 2003 ; accepted in final form 19 October 2003
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ABSTRACT |
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angiotensin II type 1 and type 2 receptor; extracellular signal-regulated kinase 1/2; thrombospondin-1 blocking peptide
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.
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METHODS |
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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-cm2 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% CO2 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% CO2 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 105 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 MgCl2. -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 AT1 or AT2 receptor, HMC and HUVEC were seeded in 75-cm2 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 Na3VO4, 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-cm2 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.
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RESULTS |
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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. 2AI). 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. 2AV), whereas no staining was seen with an isotype, irrelevant control antibody (data not shown).
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For quantification of the cell-associated TSP-1 induced by different concentrations of ANG II, we performed Western blot analysis (Fig. 2B). 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. 3A). 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. 3B).
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These results demonstrate that ANG II upregulates TSP-1 mRNA and protein levels in cultured HMC.
HMC express AT1 and AT2 receptors. Western blot analysis identified the presence of both AT1 and AT2 receptors in quiescent, starved HMC (Fig. 4, A and B). As a positive control, HUVEC were shown to strongly express both AT1 and AT2 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).
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ANG II induces TSP-1 production via the AT1 receptor. Because both AT1 and AT2 receptor protein existed in HMC, we examined which receptor was involved in ANG II-induced TSP-1 production.
Incubation of HMC with the AT1-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 AT2-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).
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These results indicate that ANG II-induced TSP-1 production operates via the AT1 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).
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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.
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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. 8E).
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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. 9E).
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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. 10E).
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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.
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DISCUSSION |
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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 AT1 receptor, as demonstrated by inhibition with valsartan. Western blotting identified the presence of both AT1 and AT2 receptor subtypes in HMC, although AT2 levels were markedly lower than in HUVEC. Blockade of the AT2 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 AT1 receptor (7). However, a second study of cultured endothelial cells found that ANG II signaling via the AT1 receptor inhibited TSP-1 mRNA expression, whereas signaling via the AT2 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 AT1 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 AT1 receptor blockers seen in human and experimental kidney disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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