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【摘要】
Objective- Although accumulating evidences suggest that impaired thyroid function is a risk for ischemic heart disease, the molecular mechanism of anti-atherosclerotic effects of thyroid hormone is poorly defined. We examined whether thyroid hormone affects signaling pathway of angiotensin II (Ang II), which is critically involved in a broad aspect of cardiovascular disease process.
Methods and Results- 3,3',5-triiodo- L -thyronine (T3) did not show a significant effect on Ang II-induced activation of extracellular signal-regulated protein kinase or p38 mitogen-activated protein kinase in vascular smooth muscle cells (VSMCs), whereas T3 inhibited Ang II-induced activation of cAMP response element (CRE) binding protein (CREB), a nuclear transcription factor involved in the vascular remodeling process. Coimmunoprecipitaion assay revealed the protein-protein interaction between thyroid hormone receptor and CREB. T3 reduced an expression level of interleukin (IL)-6 mRNA, CRE-dependent promoter activity, and protein synthesis induced by Ang II. Administration of T3 (100 µg/100 g for 14 days) to rats attenuated neointimal formation after balloon injury of carotid artery with reduced CREB activation and BrdU incorporation.
Conclusion- These results suggested that T3 inhibits CREB/CRE signaling pathway and suppresses cytokine expression and VSMCs proliferation, which may account for, at least in part, an anti-atherosclerotic effect of thyroid hormone.
The molecular mechanism of anti-atherosclerotic effects of thyroid hormone is poorly defined. We showed in the present study that thyroid hormone inhibits cAMP response element binding protein activity and suppresses angiotensin II-induced cytokine expression and hypertrophy of vascular smooth muscle cells, which may be involved in anti-atherosclerotic effect.
【关键词】 angiotensin II cAMP response element binding protein thyroid hormone vascular remodeling
Introduction
Thyroid hormone has various effects on the cardiovascular system. Hypothyroidism is known to be associated with accelerated atherosclerosis and coronary artery disease. 1,2 The Rotterdam study showed that subclinical hypothyroidism is a risk factor for atherosclerosis and myocardial infarction independently of total cholesterol level. 3 It was shown that more angiographic progression of coronary atherosclerosis was documented in patients with the lower serum thyroid hormone level after 2 years of observation. 2,4 These results suggest that thyroid hormone is protective against atherosclerosis; however, the molecular mechanism of anti-atherosclerotic effects has remained to be elucidated.
3,3',5-triiodo- L -thyronine (T3) is an active form of thyroid hormone, which binds to thyroid hormone receptor (TR). The activated TR recruits transcriptional co-activators and induces gene expression through binding to thyroid hormone response element (TRE) in the promoter region of thyroid hormone responsive genes. 5
Angiotensin II (Ang II) has multiple biological functions and is involved in a broad aspect of cardiovascular disease process. There are 2 isoforms for Ang II receptor designated type 1 receptor (AT 1 R) and type 2 receptor (AT 2 R). AT 1 R mediates most of the traditional biological effects of Ang II, including vasoconstriction, water and sodium retention, and hypertrophy and hyperplasia of vascular smooth muscle cells (VSMCs). We previously reported that Ang II induced IL-6 expression and vascular hypertrophy through cAMP response element-binding protein (CREB). 6,7 CREB is a 43-kDa nuclear transcription factor bound to cAMP response element (CRE). 8,9 The functional state of CREB is regulated by phosphorylation of serine residue at 133 (Ser133), which promotes association with transcriptional co-activator proteins, CREB-binding protein (CBP) and p300. Overexpression of dominant negative CREB, of which serine 133 is replaced with alanine, attenuated neointimal formation after balloon injury of rat carotid artery. 10
A recent study showed a physical interaction between TR and CREB 11 in fibroblasts. However, it is not clear whether this novel signaling cross talk affects the vascular remodeling process. We examined the effect of T3 on Ang II signaling pathway and the vascular remodeling process.
Materials and Methods
Materials
Dulbecco?s modified Eagle?s medium (DMEM) and fetal bovine serum was purchased from GIBCO BRL. Bovine serum albumin, T3 and an anti- tubulin antibody were purchased from Sigma-Aldrich, Inc. Ang II was purchased from Peninsula Laboratories, Inc. [ 3 H]-leucine and [ 32 P] -dCTP were purchased from PerkinElmer Life Sciences. Horseradish peroxidase-conjugated secondary antibodies (anti-rabbit and anti-mouse IgG) were purchased from VECTOR Laboratories Inc. An anti thyroid hormone receptor (anti-TR) antibody that recognizes both TR and TRßwere purchased from Santa Cruz Biotechnology, Inc. Other antibodies used in the experiments were obtained from Cell Signaling Technology. Other chemical reagents were purchased from Wako Pure chemicals unless specifically mentioned.
Cell Culture
VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats (Kyudo Co; Kamamoto, Japan). Passages between 5 and 13 were used for the experiments as described previously. 6
Animals
All procedures and care were approved by the Committee on Ethics of Animal Experiments, Kyushu University, and were conducted according to animal care guidelines of the American Physiological Society. Adult male, 12 to 13-week-old Sprague-Dawley rats (350 to 400 g) were anesthetized by intraperitoneal injection of pentobarbital sodium. The left common carotid artery was denuded of the endothelium with a 2-Fr Fogarty balloon catheter (Baxter) that was introduced through the external carotid artery. Inflation and retraction of the balloon catheter were repeated 3 times. Then rats received intraperitoneal injection of T3 (100 µg/100 g body weight suspended in 0.02 N NaOH) every other day for 2 weeks (Hyperthyroid group). Control group received injection of 0.02 N NaOH. Systolic blood pressure and heart rate were measured using tail-cuff method (UR-5000, UEDA). After 2 weeks, rats were euthanized under pentobarbital anesthesia. Carotid arteries were quickly removed and blood samples were collected. Serum concentrations of T3, T4 and thyroid-stimulating hormone (TSH) were measured by radioimmunoassay.
Common carotid artery was ligated just proximal of the bifurcation. The extent of neointimal formation in the just proximal portion of the ligation was examined histologically.
Morphometry and Immunohistochemistry
Serial cross-sections of the carotid rings were stained with hematoxylin and eosin and subjected to morphometry for assessing the intima-media area ratio (I/M ratio) and to immunohistochemistry with the use of the denoted primary antibody and a commercially available detection system as described previously. 10
Detection of Apoptosis and DNA Synthesis In Vivo
Apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method with an apoptosis in situ detection kit (Wako Pure Chemicals) as described previously. 10 In vivo labeling with BrdU (0.5 mg/kg), a thymidine analogue that was injected intraperitoneally 3 hours before preparation of the artery, was performed to identify replicating cells by detection of DNA synthesis. The incorporated BrdU was detected immunohistochemically with an anti-BrdU antibody (Cell proliferation kit, Amersham Pharmacia Biotech) as described previously. 10
Western Blot Analysis
VSMCs were lysed and Western blot analyses of CREB, extracellular signal-regulated protein kinase (extracellular signal regulated kinase ) and p38 mitogen-activated protein kinase (p38-MAPK) were performed as described previously. 7
Immunoprecipitations
VSMCs were lysed in NP-40 lysis buffer 12 and immunoprecipitated with a primary antibody (specific for TR or CREB). Immunoprecipitated proteins were electrophoresed on 12% SDS-PAGE as described previously. 12 An antibody against CREB or TR was used for immunoblotting.
Northern Blot Analysis
Total RNA was prepared according to an acid guanidinium-thiocyanate-phenol-chloroform extraction method. Northern blot analysis for IL-6 and 18S rRNA were performed as described previously. 13
Measurement of CRE-Dependent Promoter Activity
VSMCs (5 x 10 5 ) were prepared in a 6-cm tissue culture dish; 5 µg of CRE (3 copies) /luciferase fusion DNA construct with thymidine kinase (TK) promoter and 2 µg of LacZ gene (driven by simian virus 40 promoter-enhancer sequence) were introduced to VSMCs with the DEAE-dextran method according to the manufacturer?s instruction (Promega Corporation). VSMCs were cultured in DMEM with 10% fetal bovine serum for 24 hours, and then incubated with T3 for 30 minutes and stimulated with Ang II (1µmol/L) for 24 hours in DMEM containing 0.1% bovine serum albumin. The luciferase activity was measured and normalized by ß-galactosidase activity as described previously. 13
Measurement of Protein Synthesis
VSMCs were preincubated with T3 for 30 minutes and stimulated with Ang II for 1 hour. Then the medium was changed to a fresh DMEM with 0.1% bovine serum albumin and incubated for additional 23 hours. The cells were labeled with [ 3 H]-leucine during the last 12 hours. Incorporation of [ 3 H]-leucine was measured by a liquid scintillation counter as described previously. 14
Statistical Analysis
Statistical analysis was performed with 1-way ANOVA and Fisher test if appropriate. Data are shown as mean±SEM. P <0.05 was considered to be statistically significant.
Results
T3 Suppressed Ang II-Induced Phosphorylation of CREB at Ser133
We previously reported that Ang II induced CREB activation with a peak at 5 minutes. 7 VSMCs were preincubated with T3 for 30 minutes and stimulated with Ang II for 5 minutes. Western blot analysis using an antibody against phosphorylated form of CREB at Ser133 (P-CREB) was performed. T3 suppressed Ang II-induced phosphorylation of CREB in a dose-dependent manner ( Figure 1 A). Expression level of total CREB (lower panel) was not affected either by Ang II or T3.
Figure 1. T3 suppressed Ang II-induced phosphorylation of CREB at Ser133. A, VSMCs were preincubated with T3 for 30 minutes at concentrations varying from 10 -9 to 10 -6 mol/L and stimulated with Ang II (10 -7 mol/L) for 5 minutes (n=10). Phosphorylation of CREB was detected by Western blot analysis using a phospho-specific CREB antibody. Density of the specific band was scanned and quantified with an imaging analyzer. The ratio of phosphorylated CREB to total CREB is shown in the right panel. The values (mean±SEM) are expressed as a percent of control (100%). ** P <0.01 vs control, ## P <0.01 vs Ang II-stimulated cell, # P <0.5 vs Ang II-stimulated cell. B, CRE (3 copies) /luciferase fusion DNA construct with thymidine kinase promoter (TK) is shown in left panel. The construct and LacZ gene were introduced to VSMCs with the DEAE-dextran method. VSMCs were preincubated with T3 (10 -6 mol/L) for 30 minutes and stimulated with Ang II (10 -6 mol/L) for 24 hours (n=4). The luciferase activity was normalized by the ß-galactosidase activity. Relative luciferase activity of unstimulated VSMCs (control) was set as 100%. Mean±SEM, ** P <0.01, * P <0.05, ## P <0.01 vs Ang II-stimulated cell.
T3 Suppressed CRE-Dependent Transcription Induced by Ang II
A luciferase reporter construct driven by 3 copies of CRE and TK promoter ( Figure 1 B, left) was introduced VSMCs and luciferase activity was examined. Ang II increased CRE-dependent promoter activity after 24 hours of stimulation, and T3 suppressed the effect of Ang II ( Figure 1 B).
T3 Had Minimal Effect on Ang II-Induced MAPKs Activation
Ang II induced CREB activation is dependent on ERK and p38MAPK. 7 T3 slightly inhibited the Ang II-induced ERK or p38MAPK phosphorylation as shown in figure 2A and 2 B. However, densitometric analysis revealed that inhibition of MAPK phosphorylation by T3 is statistically insignificant (Ang II versus T3+Ang II, pERK; P =0.182, p38MAPK; P =0.135). Therefore, it was suggested that MAPK does not play a pivotal role in the T3-induced inhibition of CREB phosphorylation.
Figure 2. An effect of T3 on Ang II-induced MAP kinase activation. VSMCs were preincubated with T3 (10 -6 mol/L) for 30 minutes and stimulated with Ang II (10 -7 mol/L) for 5 minutes. Western blot analysis of (A) phosphorylated ERK (P-ERK) and ERK (n=6), (B) phosphorylated p38-MAPK (P-p38), and p38-MAPK (n=7) were performed as described in the legend to Figure 1. Density of the specific band was scanned and quantified with an imaging analyzer. The ratio of phosphorylated ERK to total ERK and phosphorylated p38-MAPK to total p38-MAPK are shown. ** P <0.05, NS=not significant. C, VSMCs were stimulated with T3 (10 -6 mol/L) for 30 minutes, lysed and immunoprecipitated (IP) with an antibody specific to TR (left panel) or an antibody specific to CREB (right panel). Both TR and CREB were detected by immunoblot analysis (IB) as described in the legend to Figure 1. A representative autoradiograph is shown. (n=6)
CREB Interacts With Thyroid Hormone Receptor
Recently, it has been suggested that T3 induced a direct protein-protein interaction between TR and CREB. 11 Immunoprecipitation analysis revealed that CREB was coimmunoprecipitated with TR and vice versa ( Figure 2 C). However, the amount of associated protein was almost the same between T3-stimulated cells (T3) and nonstimulated cells (C), suggesting that CREB and TR are constitutively interacting.
T3 Suppressed Angiotensin II-Induced IL-6 Expression
It was examined whether T3-induced inhibition of CREB results in the suppression of Ang II-induced gene expression. Preincubation with T3 for 30 minutes reduced the expression level of Ang II-induced IL-6 mRNA, which is dependent on CRE and CREB 6 ( Figure 3 A).
Figure 3. Effects of T3 on Ang II-induced IL-6 mRNA expression and protein synthesis. A, VSMCs were preincubated with T3 (10 -6 mol/L) for 30 minutes and stimulated with Ang II (10 -6 mol/L) for another 30 minutes. IL-6 mRNA expression was detected by Northern blot analysis. The radioactivity of the band of IL-6 mRNA was counted by a Bioimage Analyzer and was normalized by that of rRNA. The ratio of radioactivity of IL-6 mRNA to that of rRNA is shown (n=6). The values are expressed as mean±SEM. ** P <0.01, * P <0.05. B, VSMCs were preincubated with T3 (10 -6 mol/L) for 30 minutes and stimulated with Ang II (10 -7 mol/L) for 1 hour. The medium was changed to fresh serum free medium and incubated for additional 23 hours. The protocol is shown in the upper panel. Incorporation of [ 3 H]-leucine was measured by a liquid scintillation counter. Results are expressed as mean±SEM. [ 3 H]-leucine incorporation of unstimulated cells was set as 100%. n=9, ** P <0.01.
T3 Inhibited Angiotensin II-Induced Protein Synthesis in VSMCs
The effect of T3 on Ang II-induced incorporation of [ 3 H]-leucine 3 hours) to T3 reduced AT 1 R expression level in VSMCs. 15 To exclude the possible effect of T3 inhibition of AT 1 R expression on Ang II-induced leucine incorporation, the medium was changed to a fresh serum free medium after VSMCs were preincubated with T3 for 30 minutes and stimulated with Ang II for additional 1 hour. Ang II weakly but significantly increased protein synthesis with 1 hour of stimulation and T3 inhibited Ang II-induced protein synthesis ( Figure 3 B).
Neointimal Formation of Balloon-Injured Artery Was Suppressed in Hyperthyroid Rats
We previously showed that inhibition of CREB function attenuated neointimal formation after vascular injury. 10 We assumed that T3 might show the same effect if T3 inhibits CREB function in vivo. We treated rats with T3 after balloon injury ( Table ). After 14 days of balloon injury of rat carotid artery, the cross-section of carotid artery showed a substantial neointimal formation ( Figure 4 A, left). Administration of T3 significantly suppressed the neointimal formation ( Figure 4 A, right and, 4 B). CREB-positive and phosphorylated CREB-positive cells were detected in the neointima of the carotid arteries ( Figure 4 C). T3 reduced the ratio of phosphorylated CREB positive cells to total cells ( Figure 4 C upper right panel), whereas the ratio of CREB positive cells to total cells was not changed ( Figure 4 C lower right panel).
Physiological Characteristics and Serum Hormone Concentration
Figure 4. Neointimal formation of balloon-injured artery was suppressed in hyperthyroid rats. A, Representative microphotographs of hematoxylin-eosin staining of carotid arteries after 14 days of balloon injury (BI) are shown. In the right panel, rats received intraperitoneal injection of T3 (100 µg/100 g body weight suspended in 0.02 N NaOH) every other day for 2 weeks after BI (BI+T3). In the left panel, BI group received injection of 0.02 N NaOH only. B, A bar graph shows I/M ratio in the BI and BI+T3 groups. C, Representative microphotographs of immunohistochemistry for CREB and phosphorylated CREB in injured carotid artery at 14 days after injury are shown at left panel. The ratio of phosphorylated CREB-positive or CREB-positive cell number to total cell number is shown in the right panel (n=7).
To examine whether T3 induces apoptosis in neointima of balloon-injured artery or not, we detected apoptotic cells with TUNEL method. Although TUNEL index (the ratio of TUNEL positive cells to total cells) were slightly increased in T3-treated rats compared with control, the increase was not statistically significant ( Figure 5 A). BrdU positive cells were increased in the neointima after 7 days of vascular injury. In T3-treated rats, BrdU labeling index was lower than control group (5.27±1.46% versus 9.73±1.89% in control, P <0.05), which suggests that T3 decreased cell proliferation in the neointima of balloon-injured artery ( Figure 5 B). To exclude the possible effect of high blood flow on the neointimal formation in hyperthyroid state, we examined the extent of neointimal formation in a carotid artery ligation model. The I/M ratio of hyperthyroid rats was also decreased compared with that of control rats in this model (Figure I, available online at http://atvb.ahajournals.org), suggesting that thyroid hormone inhibits neointimal formation, at least in part, through a direct effect on the blood vessel.
Figure 5. Effect of T3 on apoptosis and cell proliferation after balloon injury. A, TUNEL was performed in the cross-section of carotid arteries after 14 days of balloon injury with (BI+T3) or without (BI) T3 treatment (n=7). Representative microphotographs are shown in left panel and TUNEL index of intima or media is indicated in right panel. B, Representative microphotographs of BrdU labeling in the cross sections of carotid arteries after 7 days of balloon injury with (BI+T3) or without (BI) T3 treatment are shown in left panel. BrdU labeling index of intima or media is indicated in right panel. Results are expressed as mean±SEM (n=6). * P <0.05. NS=not significant.
Discussion
Ang II plays an important role in atherosclerotic cardiovascular disease and is known to accelerate atherogenesis through vascular hypertrophy, cytokine production, and cell growth. 16-18 Many reports have shown that MAPKs are critically involved in these processes. 7,19 We previously demonstrated that Ang II induced CREB activation through ERK and p38-MAPK pathways. 7 In this study, we reported an inhibitory effect of T3 on Ang II-induced CREB phosphorylation ( Figure 1 ). T3, however, showed insignificant effects on ERK or p38-MAPK activation. Although we could not exclude the possibility that the T3-induced mild attenuation of MAPK phosphorylation affect CREB phosphorylation, the coimmunoprecipitation assay suggests that protein-protein interaction between CREB and TR may play a role ( Figure 3 ). To our knowledge, this is the first report to show an existence of crosstalk between Ang II signaling and T3 signaling in VSMC.
Contradicting results are reported in terms of the effect of thyroid hormone on intracellular cAMP level. Marchal et al 20 reported that T3 increased cAMP production in myoblasts. In contrast, it was reported that T3 or T4 inhibited basal and corticotropin (ACTH)-stimulated levels of intracellular cAMP in adrenal cells. 21 Incubation with T3 alone did not affect the phosphorylation level of CREB (data not shown) or CRE-dependent gene transcription ( Figure 1 B) in our VSMCs. These data may suggest that cAMP level is not affected by T3 in VSMCs and the effect of thyroid hormone on cAMP level may be cell type-dependent.
Ang II is involved in vascular remodeling after balloon injury. It is reported that AT 1 R mediates the progression of neointimal thickening after balloon-injured artery in rat. 22,23 We showed that hyperthyroidism downregulated AT 1 R in the aorta in the previous study. 15 It was also demonstrated that T3 suppressed CREB phosphorylation and decreased cell proliferation in the neointima of balloon-injured artery. Therefore the decreased AT 1 R level may be responsible for the decreased CREB phosphorylation and neointimal formation. However, in vitro study clearly demonstrated that T3 inhibited CREB phosphorylation with 30 minutes? of preincubation, which is insufficient to downregulate AT 1 R. It may be, therefore, plausible to assume that both downregulation of AT 1 R and inhibition of CREB phosphorylation are responsible for the reduced neointimal formation in T3-treated rats. However, it is difficult to examine these two effects separately in vivo.
We previously reported that overexpression of dominant negative CREB in injured rat carotid artery attenuated neointimal formation 10 with increased apoptosis and decreased proliferation. Although we expected that T3 has the same effect on injured artery, a significant increase in TUNEL index was not observed. This is probably because thyroid hormone has other effects than inhibiting CREB activity on blood vessel. And incubation of VSMCs with T3 for 24 hours failed to inhibit Ang II-induced CREB phosphorylation (data not shown). Further study is needed to clarify the mechanisms of these differences.
There are several reports describing that nuclear receptors can modulate gene expression by mechanism of protein-protein interaction with other transcription factors. Tagami et al reported an involvement of CREB in negative regulation of TSH promoter activity by T3. 24 They suggests that T3 induces release of the co-repressor/histone deacetylase (HDAC) complex from TR and recruits co-activators such as CBP to TR, which competes CBP away from the CREB on the promoter, causing repression of CREB-dependent transcription. It is also reported that the co-repressor complex containing HDAC released from TR binds to other transcription factors such as Octamer transcription factor-1 (Oct-1) 25 and nuclear factor- B (NF- B), 26 and inhibits the Oct-1-dependent or NF- B-dependent gene transcription. Tricostatin A (TSA), an inhibitor of HDAC, is reported to restore co-repressor-induced suppression of IL-2 gene expression that is activated by NF- B. We examined whether TSA abolishes T3-dependent suppression of Ang II-induced IL-6 expression. However, TSA did not affect T3 inhibition of Ang II-induced IL-6 mRNA expression (data not shown), suggesting that corepressor complex may not be involved. Recently, Mendez-Pertuz et al reported a transcriptional cross-talk between CREB and TR signaling pathways. 11 They showed that overexpression of CREB reduced T3-dependent transcriptional activation. To clarify the role of CREB, we took an advantage of overexpression of wild-type CREB by an adenovirus vector. Overexpression of CREB, however, did not restore an inhibitory effect of T3 on Ang II-induced IL-6 mRNA expression and Ang II-induced protein synthesis (data not shown). This may suggest that CREB is abundantly expressed in VSMCs. Actually, CREB is a ubiquitously expressed transcription factor and one report suggested that all the CRE sites in the genome are saturated by endogenous CREB. 27
In this report, we showed a ligand-independent interaction between CREB and TR. T3 did not change the amount of association of CREB with TR but inhibited Ang II-induced CREB phosphorylation, which suggests that T3 may cause conformational change of TR resulting in the inhibition of CREB phosphorylation.
In the present study, we used a relatively high concentration of T3 to stimulate VSMCs. However, Mizuma et al 28 have shown the presence of an iodothyronine deiodinase in human VSMCs. This suggests that VSMCs are able to convert T4 to T3 and that intracellular concentration of T3 in blood vessel may be higher than the serum concentration. In addition, plasma concentration of T3 was not so high and TSH was weakly suppressed in our hyperthyroid model ( Table ), indicating that neointimal formation was significantly reduced in the mild hyperthyroid state. We therefore believe that our results are clinically relevant.
In summary, T3 inhibited Ang II-induced CREB activation without affecting MAPK activation. T3 attenuated Ang II-induced cytokine expression and protein synthesis. Neointimal formation of balloon-injured artery was suppressed in T3-treated rats with reduced CREB activation and cell proliferation. These results may suggest that an anti-atherosclerotic effect of thyroid hormone is, at least in part, dependent on the inhibition of AT 1 R signaling and expression.
Acknowledgments
Sources of Funding
This study was supported in part by grants from Kimura Memorial Research Foundation, Takeda Science Foundation and a Grant-in-aid for Scientific Research from ministry of Education, Culture, Sports, Science, and Technology of Japan. (17590742).
Disclosures
None.
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作者单位：Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.