Vascular Biology Center, Medical College of Georgia, Augusta, Georgia
Submitted 9 August 2004 ; accepted in final form 10 December 2004
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ABSTRACT |
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reactive oxygen species; 5-hydroxytryptamine
Investigators at our laboratory (2) previously reported that inhibition of ANG II-induced JAK2 activation reduced proteinuria in the STZ-induced diabetes rat model. Because 5-HT is a known mitogen and because inhibition of the 5-HT2A receptor in vivo demonstrates some protective effects in diabetes, we examined the ability of 5-HT to activate the JAK/STAT pathway. While studies have shown the presence of 5-HT2A, 5-HT2B, and 5-HT1B receptors in VSMCs (26), there are currently no data on the ability of the 5-HT2B and 5-HT1B receptors to activate the JAK/STAT pathway in this cell type. In skeletal muscle, 5-HT via the 5-HT2A receptor activates both JAK2 and STAT3 (11). Furthermore, in VSMCs, 5-HT, again through the 5-HT2A receptor, is linked to increased phosphorylation of JAK2 and activation of Na+/H+ exchanger type 1 (8). Furthermore, our laboratory (2) previously discovered a significant increase in the level of JAK2 activation under conditions of STZ-induced diabetes. The JAK/STAT pathway has been implicated in VSMC proliferation (19) as well as in high glucose-induced TGF- and fibronectin synthesis in glomerular mesangial cells (10). In addition, recent studies have shown that 5-HT, via the 5-HT2A receptor, induces TGF-
1 expression via reactive oxygen species (ROS) (10). Levels of ROS in diabetes are elevated and have been linked to increased levels of endothelin-converting enzyme expression and synthesis (12, 14). Prior work has shown that Src homology-1 domain phosphatase, an inhibitory protein for prolonged activation of the JAK/STAT pathway, is sensitive to inhibition by ROS in the model of STZ-induced diabetes (2). These data suggest that levels of ROS in the diabetic condition are an important consideration in studying signaling mechanisms. Therefore, we investigated both the mechanisms by which 5-HT activates the JAK/STAT pathway in VSMCs under the conditions of normal and high glucose as well as the potential alterations in 5-HT signaling observed in in vitro and in vivo models. To specifically address this topic, we tested the hypothesis that 5-HT activates the JAK/STAT pathway through the generation of ROS in VSMCs.
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MATERIALS AND METHODS |
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Antagonist and agonist studies. All antagonists were incubated for 30 min before the addition of agonists, except for catalase (50 U/ml) and superoxide dismutase (SOD; 100 U/ml), which were incubated for 18 h before the addition of agonists. All agonists were incubated for 5 min for the JAK2 and JAK1 studies and 10 min for activation in the STAT studies.
STZ-induced diabetes. All studies were conducted with the approval of the Medical College of Georgia Animal Care Committee and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (225250 g), purchased from Harlan Laboratories (Indianapolis, IN), were rendered diabetic by administration of a single intravenous injection of STZ (60 mg/kg) made in fresh 0.1 M citrate buffer, pH 4.5. Age-matched control rats were administered buffer only. The diabetic state was confirmed 48 h later by measurement of tail blood glucose level using the Accu-Chek glucometer (Roche Diagnostics, Indianapolis, IN). All rats administered STZ had a blood glucose concentration exceeding 15 mM and thus were considered diabetic. All diabetic rats were treated with up to 2 U/day insulin via insulin pellets (LinShin Canada, Scarborough, ON, Canada) to prevent ketoacidosis. Insulin treatment did not result in normalization of blood glucose. All animals were fed standard Purina rat chow (Ralston Purina, Richmond, IN), had free access to tap water ad libitum, and were maintained on a 12:12-h light-dark cycle.
Isolation of rat thoracic aorta. Sham tissues or tissues obtained from rats treated with STZ and STZ with ketanserin were quick frozen with liquid nitrogen, pulverized with a liquid nitrogen-cooled mortar and pestle, and solubilized in lysis buffer [0.5 mol/l Tris·HCl (pH 6.8), 10% sodium dodecyl sulfate (SDS), 10% glycerol] with protease inhibitors (0.5 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and a tyrosine phosphatase inhibitor (1 mM sodium orthovanadate). Homogenates were sonicated for 1 min at setting 7 in a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) and then centrifuged (11,000 g for 10 min at 4°C) and the protein concentration for each sample was assessed using a modification of Bradford's method (27).
Immunoblotting protocol.
VSMC and tissue lysates were resolved by performing 7.5% SDS-PAGE, transferred to nitrocellulose membranes, blocked for 60 min at 22°C in Tris-buffered saline with 0.05% Tween 20 (TTBS), pH 7.4, with 5% skimmed milk powder. Membranes were incubated overnight at 4°C with affinity-purified anti-phosphotyrosine-specific and total JAK and STAT antibodies (JAK1, JAK2, STAT1, and STAT3). Membranes were washed with TTBS and then incubated with the appropriate secondary antibodies, either a goat anti-rabbit IgG or a goat anti-mouse horseradish peroxidase conjugate (60 min, 22°C). After being washed with TTBS, the bands were visualized using SuperSignal substrate chemiluminescence (Pierce Biotechnology, Rockford, IL) and Kodak Biomax film. Membranes were also incubated with smooth muscle -actin antibody (Oncogene, Boston, MA) and the appropriate secondary antibody to ensure equal loading of total protein. Molecular mass markers assessed specificity of the bands.
Adenoviral infection of cells. The recombinant adenoviruses expressing control green fluorescent protein and Cu/Zn-SOD were obtained from D. Fulton (Medical College of Georgia). Monolayers were incubated with recombinant adenovirus at a multiplicity of infection of 100. After infection, normal 10% FBS supplemented DMEM was added for the cell recovery period, followed by serum starvation in serum-free normal and high glucose DMEM. Experiments were performed as described previously.
DHE staining. Aortas were placed into freezing mold with Tissue-Tek OCT (Sakura Finetek, Torrance, CA) and snap frozen with liquid nitrogen. Samples were then sectioned on a cryostat, place on slides and in situ superoxide generation was evaluated in the cryosections with the oxidative fluorescent dye dihydroethidium (DHE). Cryosections (12 µm) were incubated with DHE (2 µmol/l) in PBS, with or without polyethylene glycol (PEG)-SOD (150 U/ml), which was added 30 min before staining. Fluorescence images were obtained with a Bio-Rad MRC 1024 scanning confocal microscope. For each slide, at least five images from different sections of the slide were captured and average staining intensity was calculated with MetaMorph software (Universal Imaging, Downingtown, PA).
Cell proliferation assay and Coulter counting. Proliferation was measured using the Cell Titer 96 aqueous nonradioactive cell proliferation assay (Promega, Madison, WI) as described previously (16). Briefly, VSMCs were grown to confluence in a 75-mm2 flask and detached with trypsin-EDTA (0.05% trypsin, 0.53 M EDTA; Life Technologies, Carlsbad, CA). Cells (n = 20,000) were plated into 96-well plates and allowed to settle for 4 h in DMEM supplemented with 10% FBS. Before experiments, cells were growth arrested in serum-deprived DMEM for 24 h (time 0). Cells were then stimulated with 106 M 5-HT. After timed 5-HT exposure, the phenazine methosulfate/MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt] mix was added to each well (final volume of 20 µl/100 µl of medium) and then incubated for an additional 60 min in 5% CO2 at 37°C. A 10% SDS solution was then added to stop the reaction, and the absorbance of formazan was measured at 490 nm. The JAK2 inhibitor AG490 (10 µM) was added 16 h before experimentation.
[3H]thymidine incorporation. VSMCs were plated in 96-well plates and maintained in DMEM supplemented with 10% FBS as described for the cell proliferation assay above. After (24 and 48 h) 5-HT exposure, cells were pulsed with 1 mCi/ml [3H]thymidine (New England Nuclear, Boston, MA) and then harvested into trichloroacetic acid-precipitable material. Cells were washed with PBS, incubated in 10% trichloroacetic acid at 4°C, dissolved at room temperature in 0.1 M tris(hydroxymethyl)aminomethane, and dried on filter paper. The filter paper was washed three times with PBS, and then samples were placed in scintillation liquid and counted with a scintillation counter (Beckman, Palo Alto, CA). Data were plotted as cpm/well. Each experimental data point represents duplicate wells from at least 10 different experiments. The JAK2 inhibitor AG490 (10 µM) was added 16 h before experimentation.
Data analysis and statistics. Quantitation of band density was performed using NIH Image software (Scion). Band density is reported in arbitrary densitometric units and was examined using two-way ANOVA and the Student-Newman-Keuls post hoc test. Samples were compared within normal and high glucose treatment groups and across all treatments to determine whether glucose alone had any effect. In vivo data were studied using one-way ANOVA and the Student-Newman-Keuls post hoc test. Data are reported as means ± SE for the number of animals and samples indicated. Values were considered significant at P < 0.05.
Chemicals. Molecular mass standards, acrylamide, SDS, N-N'-methylene-bisacrylamide, N,N,N',N'-tetramethylenediamine, protein assay reagents, and nitrocellulose membranes were purchased from Bio-Rad Laboratories. STZ, BW723C86, RU24969, CGS12066A, ketanserin tartrate, and GR55562 were purchased from Tocris (Ballwin, MO). Monoclonal antibodies to JAK1 and JAK2 were procured from Transduction Laboratories (Lexington, KY). Anti-phosphotyrosine antibodies for STAT1 and STAT3 were purchased from Cell Signaling Technology (Beverly, MA). The anti-phosphotyrosine antibodies for JAK1 and JAK2 were obtained from Biosource International (Camarillo, CA). DHE was purchased from Molecular Probes (Eugene, OR). The SuperSignal substrate chemiluminescence detection kit was obtained from Pierce Biotechnology. Goat anti-mouse IgG and anti-rabbit IgG were acquired from Amersham Biosciences (Piscataway, NJ). PEG-SOD, catalase-PEG, apocynin (4'-hydroxy-3'-methoxyacetophenone), diphenyleneiodonium chloride (DPI), 5-HT, Tween 20, and all other chemicals were purchased from the Sigma Chemical (St. Louis, MO).
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RESULTS |
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Role of ROS in 5-HT-induced activation of the JAK/STAT pathway. To further investigate the mechanisms of 5-HT activation of the JAK/STAT pathway in VSMCs, we studied the potential role of ROS. Under the normal glucose conditions, we found that neither the NAD(P)H oxidase inhibitor apocynin (100 nM) nor the flavoprotein inhibitor DPI (1 µM) had any effect on the 5-HT-induced phosphorylation of JAK2 (Fig. 3). Under conditions of high glucose, we found that only DPI significantly inhibited 5-HT-induced activation of JAK2 (Fig. 3). We next treated VSMCs with PEG-conjugated catalase to inhibit the formation of H2O2 and found that it had no effect on 5-HT-induced activation of JAK2 under conditions of normal or high glucose (Fig. 3). Treating the cells with PEG-SOD also had no effect under the conditions of normal and high glucose (Fig. 3). To determine whether the lack of effect with PEG-SOD treatment was due to inadequate cell permeability, we transfected VSMCs with a Cu/Zn-SOD adenovirus. Overexpression of the SOD virus still had no effect on the 5-HT-induced activation of JAK2 (Fig. 4A). The lack of effect was not due to an expression problem, because we observed significant expression of the SOD protein (Fig. 4B). In the high-glucose treatment groups, we again observed the expected increase in basal JAK phosphorylation levels (Fig. 4A). However, this increase was not affected by the expression of the SOD virus.
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DISCUSSION |
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In addition, previous work (19) showed an increase in the basal level of JAK2 phosphorylation in VSMCs under high-glucose conditions. We also observed this change but found it to be specific to JAK2, because we observed no increased basal phosphorylation levels of JAK1, STAT1, or STAT3. The increased basal phosphorylation of JAK2 was not observed in cells treated with inhibitors of ROS or NAD(P)H oxidase. High glucose alone in endothelial cells is known to active PKC via the polyol pathway, and this leads to the generation of NAD(P)H-dependent ROS and enhanced basal JAK phosphorylation (19). Therefore, the observed lack of increased basal phosphorylation of JAK2 in VSMCs under high-glucose conditions with these inhibitors is not surprising. Furthermore, unlike the results obtained with ANG II (19), another G protein-coupled receptor agonist that uses similar signaling pathways, we did not observe an enhanced activation of JAK2 by 5-HT under high-glucose conditions. These findings suggest that while there are many similarities between ANG II and 5-HT signaling, they use different mechanisms to activate the JAK/STAT pathway.
Furthermore, stimulation with the 5-HT2B receptor agonist BW723C86 did not activate JAK2 in VSMCs under conditions of normal or acute high glucose. These data suggest that although the receptor is present in VSMCs (26), it is not involved in 5-HT activation of the JAK/STAT pathway. This finding is consistent with previously published reports that although this receptor is present in VSMCs, it does not appear capable of activation (20, 25). The reasons for this are as yet unclear, but to date no one has shown measurable activation of this receptor in VSMCs in vitro. In addition, treatment with the 5-HT1B receptor agonist CGS12066A did not activate JAK2 and treatment with the 5-HT1B receptor agonist RU24969 activated JAK2 only at the highest concentration. However, the 5-HT1B receptor antagonist GR55562 did not affect the ability of 5-HT to activate JAK2. The discrepancy between these data suggests that RU24969 at high concentrations is not acting as a specific 5-HT1B receptor agonist. While the mechanism for this nonselectivity is unclear, it was previously reported (3). These data suggest that the 5-HT1B receptor also is not involved in 5-HT-induced activation of JAK2 in VSMCs.
Unlike the data reported for pulmonary VSMCs, in which the 5-HT1B receptor caused vasoconstriction and acted through NAD(P)H oxidase to increase O2 (13), activation of JAK2 in aortic VSMCs by 5-HT was not affected by treatment with catalase, SOD, and apocynin. This finding was not universal, because we observed significant inhibition of 5-HT-induced MAPK activation by both catalase and SOD. Previous work demonstrated that activation of the MAPK pathway in VSMCs occurs via the 5-HT2A receptor (1, 26). These findings suggest that 5-HT differentially uses ROS as a second messenger within the same cell and through the same receptor. While the mechanisms by which 5-HT activates the JAK/STAT pathway have yet to be elucidated, this finding is novel and very different from the mechanism used by ANG II. To date, no one has demonstrated that ANG II simultaneously activates ROS-dependent and -independent signaling mechanisms through the same receptor type.
These data also suggest that more investigation into peripheral 5-HT receptors and their functions is warranted. These future investigations should take into account the vascular bed and whether the arteries are in a diseased state, because other work has demonstrated that in hypertension, both the 5-HT2B and 5-HT1B receptors are upregulated and functional only in aorta and superior mesenteric arteries from DOCA-salt-hypertensive rats (4, 5). Changes in 5-HT receptor function in any other disease state have not been addressed to date, even though there are reports that the circulating level of this vasoactive substance are increased (14). Furthermore, the reduction in albuminuria in patients with diabetes using the 5-HT2A receptor antagonist sapogrelate (20), as well as the effects on insulin and blood glucose levels (22), suggests a role for 5-HT in diabetes.
In addition, we observed an increase in JAK2, STAT1 and STAT3 tyrosine phosphorylation levels in endothelium-denuded rat thoracic aorta in vivo. Furthermore, treatment of diabetic rats with ketanserin prevented the increase in pJAK2 and pSTAT1 levels observed in aortas from untreated diabetic rats. The present report is the first one involving diabetes in which STAT3 activation in VSMCs appears not to have occurred through 5-HT. These data correspond to recently published results showing that inhibition of JAK2 in STZ-induced diabetes had a protective effect in the kidney (2). This suggests that further investigation of the involvement of 5-HT in diabetic complications, particularly in the kidney, should be pursued.
In rat aortic VSMCs, 5-HT uses only the 5-HT2A receptor under the conditions of normal and acute high glucose to activate JAK2 and STAT1 in vitro. Furthermore, the activation of JAK2 by 5-HT appears to be independent of ROS and NAD(P)H oxidase. This finding is novel because other activators of the JAK/STAT pathway, such as ANG II, which is also a G protein-coupled receptor, appear to use ROS to activate this pathway. Furthermore, in vivo treatment with ketanserin reduced the activation of JAK2 and STAT1, suggesting that 5-HT via the 5-HT2A receptor maybe involved in complications of diabetes mellitus type 1.
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GRANTS |
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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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