Aldosterone Induces Angiotensin Converting Enzyme Gene Expression via a JAK2-Dependent Pathway in Rat Endothelial Cells
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《内分泌学杂志》
Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Tokyo 113-8519, Japan
Abstract
Aldosterone is currently recognized as a risk hormone for cardiovascular disease. However, the cellular mechanism by which aldosterone acts on vasculature has not been well understood. In the present study, we investigated whether aldosterone affects angiotensin-converting enzyme (ACE) gene expression in rat endothelial cells. Cultured rat aortic endothelial cells (RAECs) from Sprague-Dawley rats were used in the study. ACE mRNA levels and its enzyme activities in RAECs were examined by real-time RT-PCR and enzyme assay using hippuryl-His-Leu as substrates, respectively. Aldosterone significantly increased steady-state ACE mRNA levels and its enzymatic activities. This effect was dose dependent and time dependent and abolished by mineralocorticoid receptor antagonist spironolactone or transcription inhibitor actinomycin D. Dexamethasone also increased steady-state ACE mRNA levels, whose effect was completely blocked by glucocorticoid receptor antagonist RU486, but not by spironolactone. By contrast, the aldosterone-induced ACE mRNA expression was only partially blocked by RU486. The stimulatory effect of aldosterone on ACE mRNA expression was completely blocked by a protein tyrosine kinase inhibitor (genistein) and JAK2 inhibitor (AG490), partially by Src kinase inhibitor (PP2) and epidermal growth factor receptor kinase inhibitor (AG1478), but not by platelet-derived growth factor receptor kinase inhibitor (AG1296). Transfection of dominant-negative JAK2 construct, but not wild-type construct, significantly blocked the aldosterone-induced ACE mRNA up-regulation. Furthermore, aldosterone induced phosphorylation of JAK2, whose effect was blocked by spironolactone and actinomycin D. In conclusion, the present study demonstrates for the first time that aldosterone induces ACE gene expression and its enzyme activity mainly via a mineralocorticoid receptor-mediated and JAK2-dependent pathway in rat endothelial cells. This may constitute a positive feedback loop for a local renin-angiotensin system, possibly involved in the development of aldosterone-induced endothelial dysfunction and vascular injury.
Introduction
TWO RECENT CLINICAL studies, the Randomized Aldactone Evaluation Study (1) and Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (2), have demonstrated the pathophysiological importance of aldosterone in the development and/or progression of cardiovascular disease as a risk hormone. In addition, numerous experimental studies have shown that the pathophysiological role of aldosterone in cardiovascular disease is mediated not merely by its volume expansion/hypertensive effect but also by its direct action on cardiovascular tissue (3, 4, 5). However, the molecular and cellular mechanism by which aldosterone induces vascular injury has not been well understood.
It has been well recognized that both a systemic and local renin-angiotensin system (RAS) plays a pivotal role in the regulation of the cardiovascular function. Tissue RAS is enhanced in the vasculature of atherosclerosis and hypertension as well as the hypertrophied and the failing heart, where local angiotensin II (Ang II) exerts its major effects via Ang II type 1 (AT1) receptor (6, 7, 8, 9). However, recent accumulating lines of evidence suggest the importance of the pathophysiological roles of aldosterone in several extrarenal tissues, especially in the cardiovascular system (3, 5). For example, it has recently been reported that aldosterone up-regulates AT1 receptors in rat vascular smooth muscle cell (VSMCs) and potentiates Ang II-stimulated VSMC proliferation (10, 11). Furthermore, it has recently been shown that aldosterone induces angiotensin-converting enzyme (ACE) gene expression in cultured neonatal rat cardiocytes (12), which may be partly involved in cardiac hypertrophy. Taken together, these data suggest the pathophysiological role of aldosterone as a positive modulator of cardiovascular RAS. However, it remains elusive whether aldosterone has any direct effect on vascular endothelial cells.
These observations led us to examine whether aldosterone directly affects ACE gene expression and its enzymatic activity in rat vascular endothelial cells [rat aortic endothelial cells (RAECs)] and, if so, to elucidate the molecular mechanism by which ACE gene expression is regulated by aldosterone.
Materials and Methods
Materials
Aldosterone was purchased from Acros Organics (Geel, Belgium), spironolactone and genistein from Sigma Chemical Co. (St. Louis, Mo), actinomycin D from Biomol Research Laboratories Inc. (Plymouth, PA), PP2, AG1478, AG1296, and AG490 from Calbiochem (La Jolla, CA), and hippuryl-His-Leu from the Peptide Institute (Osaka, Japan). PCR primers were synthesized by JbioS Inc. (Saitama, Japan). The plasmid, JAK2 KE pRK5, a dominant-negative (DN) form of JAK2 (13), was kindly provided by Dr. James N. Ihle, St. Jude Children’s Research Hospital (Memphis, TN).
The appropriate concentrations of each kinase inhibitor used in the present study were chosen for its selective effect from previous publications: genistein (14), AG490 (15), PP2 (16, 17), AG1478 (11), and AG1296 (19).
Cell culture
RAECs were prepared from the thoracic aorta of 6-wk-old male Sprague-Dawley rats using the explant method (20) and cultured in Medium 199 containing 10% fetal bovine serum (Cell Culture Laboratories, Cleveland, OH) and 30 μg/ml endothelial cell growth supplement (BD Biosciences, Bedford, MA) on collagen-coated dishes (IWAKI, Chiba, Japan) at 37 C in an atmosphere of 5% CO2. The RAECs used in the present study showed typical cobblestone appearance, and more than 95% of cells incorporated diI-acetylated low-density lipoprotein (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) with negative immunostaining for -smooth muscle actin (data not shown). Subcultured RAECs (4–10 passages) were grown to confluence and then starved with Medium199 containing 0.5% calf serum for 24 h and used for subsequent experiments; the responses to aldosterone varied depending on the number of cell passages.
Quantification of ACE mRNA
Total RNA was extracted using RNA Bee (TEL-TEST, Friendswood, TX) according to the manufacturer’s protocol. Five micrograms of total RNA were reverse transcribed with the first-strand cDNA synthesis kit (Amersham Pharmacia Biotech, Piscataway, NJ), using random hexamers, according to the manufacturer’s instructions. Rat ACE mRNA levels were quantified with real-time RT-PCR using fluorescent SYBR green technology (LightCycler; Roche Molecular Biochemicals, Mannheim, Germany), as described (21). Rat acidic ribosomal phosphoprotein P0 (ARPP P0), a housekeeping gene, mRNA levels were quantitated by TaqMan fluorescence methods as described (22), except for the use of QuantiTect Probe PCR kit (QIAGEN, Chatsworth, CA) and LightCycler technology. The sequences of PCR primers and TaqMan probe are as follows: ACE forward, 5'-CAGAGGCCAACTGGCATTAT-3', and reverse, 5'-CTGGAAGTTGCTCACGTCAA-3' (product size, 137 bp); mineralocorticoid receptor (MR) forward, 5'-GCAGCGAAACAGATGATCCAG-3', and reverse, 5'-TCTCCAACTCAAAGCGAACGA-3' (product size, 134 bp); ARPP P0 forward, 5'-TAGAGGGTGTCCGCAATGTG-3', and reverse, 5'-GACAAAGCCAGGACCCTTTTGT-3', TaqMan probe, 5'-ACCCGACTGTTGCCTCAGTGCCTCACTCCA-3' (product size, 107 bp). In both the SYBR green and TaqMan real-time PCR methods, the fluorescence data were quantitatively analyzed using serial dilution of control samples included in each reaction to produce a standard curve. To compare the relative expression of ACE gene, ARPP P0 was used as an endogenous internal control; the relative levels of ACE mRNA to ARPP P0 were calculated and shown in each figure.
Measurement of ACE activity
RAECs were plated in 6-cm collagen-coated plates with Medium199 and allowed to grow to confluence. ACE enzymatic activity in RAECs was measured using a fluorometric assay as described (23). Briefly, cells were washed three times with cold PBS, harvested by scraping, and homogenized on ice by sonication. The homogenate containing 50 μg protein was incubated for 1 h at 37 C with hippuryl-His-Leu as substrates (5 mM). Hippuric acid formed by ACE is extracted with ethyl acetate. The ethyl acetate fractions were evaporated and redissolved in distilled water, and the amounts of hippuric acid in each fraction were determined by absorbance at 228 nm.
Transfection of JAK2 constructs
Transient transfection of plasmids JAK2 KE pRK5 and backbone vector (pRK5) into RAECs was performed using synthetic cationic liposome, (+)-N,N [bis (2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy) propyl] ammonium iodide with transferrin-receptor-operated transfer as described previously (24, 25). Briefly, 30 μl of liposome (1 mg/ml; Promega, Madison, WI) was added to 200 μl serum-free medium containing 192 μg of human holo-transferrin (Sigma), incubated for 20 min at room temperature, mixed with 200 μl serum-free medium containing 10 μg expression vector DNA, and further incubated for 20 min. The mixture was then overlaid on cells that had been covered with 1.2 ml of serum-free medium and incubated for 2 h. Cells were then stimulated with or without aldosterone for 18 h. To determine the transfection efficiency, JAK2 mRNA levels after transfection were quantified with real-time RT-PCR using primer pairs capable of amplifying both endogenous JAK2 and transfected DN-JAK2; forward primer 5'-GCAGCCCTAAGGACTTCAAC-3' and reverse primer 5'-CCGCTGAGGTTGTATTCTCC-3'. To remove the plasmid DNA contamination due to transfection, RNA samples were treated with RNase-free DNase (Promega), and then DNase was inactivated and removed by phenol/chloroform extraction and subsequent ethanol precipitation before RT. Transfection of RAECs with DN-JAK2 construct resulted in about 100-fold increase in JAK2 transcripts compared with that of empty vector (supplemental Fig. 2, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).
Immunoprecipitation and immunoblotting
Cells were harvested and lysed in immunoprecipitation buffer [50 mM HEPES (pH 7.6), 150 mM NaCl, 1 mM EGTA, 1% Nonidet P40, 10 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 5 mM NaF]. Cell lysates (300 μg) were incubated with 3 μg rabbit polyclonal anti-JAK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in a volume of 500 μl at 4 C overnight, followed by the addition of protein G beads (50% slurry) and additional incubation for 1 h. The beads were washed three times with immunoprecipitation buffer, solubilized in Laemmli sample buffer, and subjected to 10% SDS-PAGE. Immunoblot analysis was performed using mouse monoclonal anti-phosphotyrosine antibody (PY99: Santa Cruz Biotechnology), followed by reprobing with anti-JAK2 antibody as previously described (22).
Statistical analysis
Data were expressed as mean ± SEM. Differences between groups were examined for statistical significance using unpaired t test or ANOVA with Dunn’s post hoc test, if appropriate. P values less than 0.05 were considered statistically significant.
Results
Aldosterone increases ACE mRNA and its enzymatic activity
Aldosterone dose-dependently (3 x 10–9 to 10–7 M) increased steady-state ACE mRNA levels in RAECs (Fig. 1A); a significant (P < 0.05) increase was induced by as low as 3 x 10–9 M and a 17.5-fold increase by 10–7 M. Aldosterone time-dependently increased steady-state ACE mRNA levels in RAECs (Fig. 1C); about 5- and 10-fold increases were observed after 8 and 18 h, respectively. The ACE gene expression induced by aldosterone (5 x 10–7 M) was completely blocked by pretreatment with a selective MR antagonist, spironolactone (10–5 M), but partially inhibited by a selective glucocorticoid receptor (GR) antagonist, RU486 (10–6 M) (Fig. 2A). Dexamethasone (5 x 10–7 M) also increased steady-state ACE mRNA levels in RAECs, whose effect was completely blocked by RU486, but not by spironolactone (Fig. 2B). RAECs used in our study expressed both MR and GR mRNAs as revealed by RT-PCR (data not shown). These results suggest that ACE gene expression induced by aldosterone is mediated mainly via MR at lower concentrations but partly via GR at higher concentrations, whereas ACE gene expression is also regulated by glucocorticoids exclusively via GR.
The aldosterone-induced ACE gene expression was blocked by pretreatment with a transcription inhibitor, actinomycin D (5 x 10–6 M), but not with AT1 receptor blocker (valsartan, 10–6 M) (data not shown). Furthermore, aldosterone dose-dependently (10–9 to 10–7 M) increased ACE activity (Fig. 3A); about 2.7-fold increase was induced by as low as 10–9 M and 10.3-fold increase by 10–7 M. The aldosterone-stimulated ACE activity was also blocked by spironolactone (Fig. 3B). Thus, our data indicate that aldosterone induces ACE gene expression and increases its enzyme activity mainly via MR in RAECs.
Protein tyrosine kinase (PTK) inhibitors blocks aldosterone-induced ACE gene expression
To further characterize the molecular mechanism(s) underlying the aldosterone-induced ACE mRNA expression, we tested the effects of various PTK inhibitors on aldosterone-induced ACE gene up-regulation (Fig. 4).
The aldosterone-induced ACE mRNA expression was completely inhibited by a nonselective PTK inhibitor, genistein (10–4 M) (Fig. 4A) and JAK2 inhibitor, AG490 (10–4 M) (Fig. 4B). As shown in Fig. 5, the inhibitory effects of genistein (10–5 to 10–4 M) and AG490 (10–6 to 10–4 M) were concentration dependent. In contrast, a selective inhibitor of the Src kinase family (PP2, 10–5 M) (Fig. 4C) and an epidermal growth factor receptor (EGFR) kinase inhibitor (AG1478, 3 x 10–7 M) (Fig. 4D) only partially inhibited the aldosterone-induced ACE mRNA expression, and platelet-derived growth factor receptor kinase inhibitor (AG1296, 10–5 M) (Fig. 4E) was without effect.
DN-JAK2 construct suppressed aldosterone-induced ACE gene expression
To determine whether JAK2 is actually involved in the aldosterone-induced ACE mRNA expression, we examined the effects of overexpression of DN-JAK2 (JAK2 KE pRK5) or empty vector (pRK5). Transfection of RAECs with DN-JAK2 construct resulted in a significant (P < 0.05) decrease in the aldosterone-induced ACE expression, whereas transfection of empty vector did not affect aldosterone-induced ACE mRNA expression; basal ACE mRNA levels without aldosterone treatment were unaffected by transfection of either vectors (Fig. 6). Thus, these results suggest that JAK2 among several PTKs is mainly involved in the aldosterone-stimulated ACE gene expression in endothelial cells.
Aldosterone stimulated JAK2 phosphorylation
To ascertain whether aldosterone activates JAK2, we assessed tyrosine phosphorylation of JAK2 by immunoprecipitation with anti-JAK2 antibody coupled with immunoblotting with anti-phosphotyrosine antibody. Aldosterone stimulated phosphorylation of JAK2 in a time-dependent manner, which peaked after 2 h and then declined thereafter; no distinct band was noted when nonimmune IgG was used for immunoprecipitation (Fig. 7A). JAK2 phosphorylation was induced by aldosterone in a dose-dependent manner (10–8 to 10–7 M), whose effect was blocked by actinomycin D (Fig. 7B) and spironolactone (data not shown). These results indicate that the aldosterone-induced JAK2 phosphorylation response is transcription-dependent genomic action.
Discussion
The present study demonstrates for the first time that aldosterone directly up-regulates ACE gene expression and its enzymatic activity in rat endothelial cells, whose effect was completely blocked by a selective MR antagonist (spironolactone). Our data are consistent with a recent study showing that aldosterone up-regulates ACE gene expression in rat neonatal cardiomyocytes (12).
It has been recently recognized that aldosterone plays a pivotal role in the development and/or progression of cardiovascular disease as a critical and final component of RAS (1, 2, 3, 4, 5). In experimental Ang II-induced hypertension models, administration of MR antagonist or adrenalectomy significantly attenuated the vascular injury of coronary, renal, and cerebral arteries without lowering of blood pressure (3, 5). In mineralocorticoids/high-salt hypertensive rats, cardiovascular injury develops in a similar manner to the Ang II-induced hypertension models (3, 5). Vascular injury in these hypertensive models is preceded by the development of a vascular inflammatory phenotype associated with expression of a variety of proinflammatory genes, including COX-2, cytokines, osteopontin, and MCP-1 (4, 26). However, the underlying cellular mechanism of vascular injury by aldosterone has not been well understood.
It has been reported that MR mRNA is expressed not only in epithelial cells but also in nonepithelial tissue including cardiovascular tissue (27, 28, 29). We confirmed that RAECs used in the present study also expressed MR mRNA. The minimum effective concentrations of aldosterone required for ACE gene induction and increase in ACE activity (1–3 x 10–9 M) in RAECs appeared to be almost comparable to its physiological concentrations (10–9 M) in the circulation (30). Furthermore, the aldosterone-induced ACE gene expression and enzyme activity in RAECs were inhibited by a selective MR antagonist (spironolactone) at the concentration (10–6 M) almost equivalent to those in plasma concentrations (10–7 to 10–6 M) in the clinical setting (31). Thus, it is possible to speculate that spironolactone may be clinically effective in blocking the aldosterone-induced ACE gene expression in endothelium.
Aldosterone and glucocorticoids interact with MR with equal affinity, and aldosterone in higher concentration causes crossover activation of GR (32). We therefore examined the effect of dexamethasone on ACE gene expression and the specificities of aldosterone and dexamethasone responses by their selective MR and GR antagonist, respectively. The present results suggest that aldosterone-induced ACE response in the higher concentration (greater than 5 x 10–7 M), although partly via GR, is mainly mediated via MR, whereas dexamethasone-induced ACE response is exclusively mediated via GR. Furthermore, it should be noted that 11-hydroxysteroid dehydrogenase-2 (11-HSD2), which converts glucocorticoids into inactive 11-dehydro compounds to protect MR from the abundant glucocorticoids in renal epithelial cells, has recently been demonstrated in human aorta and rat endothelial cells (33, 34). We have also confirmed that our RAECs express not only MR mRNA but also 11-HSD2 mRNA by the quantitative RT-PCR experiments (unpublished observations). Taken together, it is suggested that the glucocorticoid-inactivating enzyme 11-HSD2 could play a functional role in the maintenance of ligand selectivity for MR in endothelial cells. However, the molecular interaction between MR and GR activation in the regulation of endothelial ACE gene expression remains to be determined.
Aldosterone synthase (CYP11B2) mRNA and its enzyme activity have been recently reported to be expressed in cardiovascular tissue, including endothelial cells and cardiomyocytes (35, 36, 37, 38). However, the presence of aldosterone synthase in human endothelial cells (39) and rat heart (40) has very recently been challenged. Assuming that aldosterone may be synthesized by cardiovascular tissue, it is likely that the approximate aldosterone concentration at the local level should be high enough to exert its paracrine action. An alternative possibility is that circulating aldosterone may be readily bound and concentrated by and released from cardiovascular tissue in a sufficient amount locally, as recently suggested by Funder (41). Thus, the critical issue as to whether cardiovascular tissue synthesizes and secretes aldosterone in an autocrine/paracrine fashion remains to be addressed.
The endothelial ACE up-regulation by aldosterone as demonstrated in this study may partly contribute to the development and/or progression of vascular damage resulting from activation of tissue RAS via a positive feedback mechanism as has been suggested in rat cardiomyocytes (12). It has very recently been reported that aldosterone up-regulates AT1 receptor in rat VSMCs and potentiates Ang II-stimulated VSMC proliferation (10, 11). Taken together, it is strongly suggested that a positive feedback loop of local RAS in the vasculature is partly responsible for the development and/or progression of aldosterone-induced endothelial dysfunction and vascular injury.
The present study clearly shows that aldosterone-induced up-regulation of the ACE gene in endothelial cells is blocked by a nonselective PTK inhibitor (genistein) as well as a specific inhibitor of JAK2 (AG490) in a dose-dependent manner. Furthermore, overexpression of DN-JAK2 mutant in endothelial cells decreased the aldosterone-induced ACE gene expression. Thus, our data suggest that aldosterone preferentially activates JAK2 among various PTKs, which constitutes an essential signal for endothelial ACE gene expression. Our contention is supported by the data that aldosterone induced JAK2 phosphorylation via a MR-mediated pathway.
The exact molecular mechanism by which aldosterone activates JAK2 remains unknown. Although it has been shown that the JAK family is activated via cytokine receptors or G protein-coupled receptors (42, 43), there has been no study thus far reported showing that a nuclear receptor activates the JAK family. In the present study, phosphorylation of JAK2 by aldosterone was completely blocked by actinomycin D, indicating that aldosterone-induced JAK2 phosphorylation is via its genomic action. Aldosterone has been shown to potentiate Ang II-induced ERK1/2 and JNK activation and oxygen radical production in rat VSMCs (11). Thus, aldosterone may potentiate Ang II-induced JAK activation. However, failure of AT1 receptor blocker (valsartan) to block the aldosterone-induced ACE gene expression in this study excluded the potential involvement of endogenous Ang II in the mechanism of aldosterone-induced ACE up-regulation.
In the present study, both c-Src inhibitor (PP2) and EGFR inhibitor (AG1478) partially suppressed the aldosterone-induced ACE gene expression. It has been shown that c-Src is associated with and activated by JAK2 (44). We have previously shown that c-Src is an upstream signal for Ang II-induced EGFR transactivation in rat VSMCs (18). Thus, aldosterone may activate c-Src and subsequent EGFR transactivation as a downstream signal resulting from JAK2 activation, which may be responsible, at least in part, for the aldosterone-induced ACE gene expression. It has been shown that EGFR inhibitor (AG1478) reduced the aldosterone-induced ERK1/2 phosphorylation in rat VSMCs (11). However, the question as to how aldosterone-induced activation of several PTKs, including c-Src, EGFR, and JAK2, leads to ACE gene expression remains open to question.
In conclusion, our present study demonstrates for the first time that aldosterone directly increases ACE gene expression and its enzyme activity mainly via a MR-mediated and JAK2-dependent pathway in rat endothelial cells. The aldosterone-induced JAK2 pathway gives a new insight into the cellular signaling mechanism of aldosterone’s action on cardiovascular tissue. Thus, regulation of the endothelial ACE gene by aldosterone may constitute a positive feedback loop for local RAS, possibly involved in the development and/or progression of aldosterone-induced endothelial dysfunction and vascular injury.
Footnotes
This study was supported in part by Grant-in-aid from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health, Welfare and Labor, of Japan.
Abbreviations: ACE, Angiotensin-converting enzyme; Ang II, angiotensin II; ARPP P0, acidic ribosomal phosphoprotein P0; AT1, Ang II type 1; DN, dominant negative; EGFR, epithelial growth factor receptor; GR, glucocorticoid receptor; 11-HSD2, 11-hydroxysteroid dehydrogenase-2; MR, mineralocorticoid receptor; PTK, protein tyrosine kinase; RAEC, rat aortic endothelial cell; RAS, renin-angiotensin system; VSMC, vascular smooth muscle cell.
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Abstract
Aldosterone is currently recognized as a risk hormone for cardiovascular disease. However, the cellular mechanism by which aldosterone acts on vasculature has not been well understood. In the present study, we investigated whether aldosterone affects angiotensin-converting enzyme (ACE) gene expression in rat endothelial cells. Cultured rat aortic endothelial cells (RAECs) from Sprague-Dawley rats were used in the study. ACE mRNA levels and its enzyme activities in RAECs were examined by real-time RT-PCR and enzyme assay using hippuryl-His-Leu as substrates, respectively. Aldosterone significantly increased steady-state ACE mRNA levels and its enzymatic activities. This effect was dose dependent and time dependent and abolished by mineralocorticoid receptor antagonist spironolactone or transcription inhibitor actinomycin D. Dexamethasone also increased steady-state ACE mRNA levels, whose effect was completely blocked by glucocorticoid receptor antagonist RU486, but not by spironolactone. By contrast, the aldosterone-induced ACE mRNA expression was only partially blocked by RU486. The stimulatory effect of aldosterone on ACE mRNA expression was completely blocked by a protein tyrosine kinase inhibitor (genistein) and JAK2 inhibitor (AG490), partially by Src kinase inhibitor (PP2) and epidermal growth factor receptor kinase inhibitor (AG1478), but not by platelet-derived growth factor receptor kinase inhibitor (AG1296). Transfection of dominant-negative JAK2 construct, but not wild-type construct, significantly blocked the aldosterone-induced ACE mRNA up-regulation. Furthermore, aldosterone induced phosphorylation of JAK2, whose effect was blocked by spironolactone and actinomycin D. In conclusion, the present study demonstrates for the first time that aldosterone induces ACE gene expression and its enzyme activity mainly via a mineralocorticoid receptor-mediated and JAK2-dependent pathway in rat endothelial cells. This may constitute a positive feedback loop for a local renin-angiotensin system, possibly involved in the development of aldosterone-induced endothelial dysfunction and vascular injury.
Introduction
TWO RECENT CLINICAL studies, the Randomized Aldactone Evaluation Study (1) and Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (2), have demonstrated the pathophysiological importance of aldosterone in the development and/or progression of cardiovascular disease as a risk hormone. In addition, numerous experimental studies have shown that the pathophysiological role of aldosterone in cardiovascular disease is mediated not merely by its volume expansion/hypertensive effect but also by its direct action on cardiovascular tissue (3, 4, 5). However, the molecular and cellular mechanism by which aldosterone induces vascular injury has not been well understood.
It has been well recognized that both a systemic and local renin-angiotensin system (RAS) plays a pivotal role in the regulation of the cardiovascular function. Tissue RAS is enhanced in the vasculature of atherosclerosis and hypertension as well as the hypertrophied and the failing heart, where local angiotensin II (Ang II) exerts its major effects via Ang II type 1 (AT1) receptor (6, 7, 8, 9). However, recent accumulating lines of evidence suggest the importance of the pathophysiological roles of aldosterone in several extrarenal tissues, especially in the cardiovascular system (3, 5). For example, it has recently been reported that aldosterone up-regulates AT1 receptors in rat vascular smooth muscle cell (VSMCs) and potentiates Ang II-stimulated VSMC proliferation (10, 11). Furthermore, it has recently been shown that aldosterone induces angiotensin-converting enzyme (ACE) gene expression in cultured neonatal rat cardiocytes (12), which may be partly involved in cardiac hypertrophy. Taken together, these data suggest the pathophysiological role of aldosterone as a positive modulator of cardiovascular RAS. However, it remains elusive whether aldosterone has any direct effect on vascular endothelial cells.
These observations led us to examine whether aldosterone directly affects ACE gene expression and its enzymatic activity in rat vascular endothelial cells [rat aortic endothelial cells (RAECs)] and, if so, to elucidate the molecular mechanism by which ACE gene expression is regulated by aldosterone.
Materials and Methods
Materials
Aldosterone was purchased from Acros Organics (Geel, Belgium), spironolactone and genistein from Sigma Chemical Co. (St. Louis, Mo), actinomycin D from Biomol Research Laboratories Inc. (Plymouth, PA), PP2, AG1478, AG1296, and AG490 from Calbiochem (La Jolla, CA), and hippuryl-His-Leu from the Peptide Institute (Osaka, Japan). PCR primers were synthesized by JbioS Inc. (Saitama, Japan). The plasmid, JAK2 KE pRK5, a dominant-negative (DN) form of JAK2 (13), was kindly provided by Dr. James N. Ihle, St. Jude Children’s Research Hospital (Memphis, TN).
The appropriate concentrations of each kinase inhibitor used in the present study were chosen for its selective effect from previous publications: genistein (14), AG490 (15), PP2 (16, 17), AG1478 (11), and AG1296 (19).
Cell culture
RAECs were prepared from the thoracic aorta of 6-wk-old male Sprague-Dawley rats using the explant method (20) and cultured in Medium 199 containing 10% fetal bovine serum (Cell Culture Laboratories, Cleveland, OH) and 30 μg/ml endothelial cell growth supplement (BD Biosciences, Bedford, MA) on collagen-coated dishes (IWAKI, Chiba, Japan) at 37 C in an atmosphere of 5% CO2. The RAECs used in the present study showed typical cobblestone appearance, and more than 95% of cells incorporated diI-acetylated low-density lipoprotein (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) with negative immunostaining for -smooth muscle actin (data not shown). Subcultured RAECs (4–10 passages) were grown to confluence and then starved with Medium199 containing 0.5% calf serum for 24 h and used for subsequent experiments; the responses to aldosterone varied depending on the number of cell passages.
Quantification of ACE mRNA
Total RNA was extracted using RNA Bee (TEL-TEST, Friendswood, TX) according to the manufacturer’s protocol. Five micrograms of total RNA were reverse transcribed with the first-strand cDNA synthesis kit (Amersham Pharmacia Biotech, Piscataway, NJ), using random hexamers, according to the manufacturer’s instructions. Rat ACE mRNA levels were quantified with real-time RT-PCR using fluorescent SYBR green technology (LightCycler; Roche Molecular Biochemicals, Mannheim, Germany), as described (21). Rat acidic ribosomal phosphoprotein P0 (ARPP P0), a housekeeping gene, mRNA levels were quantitated by TaqMan fluorescence methods as described (22), except for the use of QuantiTect Probe PCR kit (QIAGEN, Chatsworth, CA) and LightCycler technology. The sequences of PCR primers and TaqMan probe are as follows: ACE forward, 5'-CAGAGGCCAACTGGCATTAT-3', and reverse, 5'-CTGGAAGTTGCTCACGTCAA-3' (product size, 137 bp); mineralocorticoid receptor (MR) forward, 5'-GCAGCGAAACAGATGATCCAG-3', and reverse, 5'-TCTCCAACTCAAAGCGAACGA-3' (product size, 134 bp); ARPP P0 forward, 5'-TAGAGGGTGTCCGCAATGTG-3', and reverse, 5'-GACAAAGCCAGGACCCTTTTGT-3', TaqMan probe, 5'-ACCCGACTGTTGCCTCAGTGCCTCACTCCA-3' (product size, 107 bp). In both the SYBR green and TaqMan real-time PCR methods, the fluorescence data were quantitatively analyzed using serial dilution of control samples included in each reaction to produce a standard curve. To compare the relative expression of ACE gene, ARPP P0 was used as an endogenous internal control; the relative levels of ACE mRNA to ARPP P0 were calculated and shown in each figure.
Measurement of ACE activity
RAECs were plated in 6-cm collagen-coated plates with Medium199 and allowed to grow to confluence. ACE enzymatic activity in RAECs was measured using a fluorometric assay as described (23). Briefly, cells were washed three times with cold PBS, harvested by scraping, and homogenized on ice by sonication. The homogenate containing 50 μg protein was incubated for 1 h at 37 C with hippuryl-His-Leu as substrates (5 mM). Hippuric acid formed by ACE is extracted with ethyl acetate. The ethyl acetate fractions were evaporated and redissolved in distilled water, and the amounts of hippuric acid in each fraction were determined by absorbance at 228 nm.
Transfection of JAK2 constructs
Transient transfection of plasmids JAK2 KE pRK5 and backbone vector (pRK5) into RAECs was performed using synthetic cationic liposome, (+)-N,N [bis (2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy) propyl] ammonium iodide with transferrin-receptor-operated transfer as described previously (24, 25). Briefly, 30 μl of liposome (1 mg/ml; Promega, Madison, WI) was added to 200 μl serum-free medium containing 192 μg of human holo-transferrin (Sigma), incubated for 20 min at room temperature, mixed with 200 μl serum-free medium containing 10 μg expression vector DNA, and further incubated for 20 min. The mixture was then overlaid on cells that had been covered with 1.2 ml of serum-free medium and incubated for 2 h. Cells were then stimulated with or without aldosterone for 18 h. To determine the transfection efficiency, JAK2 mRNA levels after transfection were quantified with real-time RT-PCR using primer pairs capable of amplifying both endogenous JAK2 and transfected DN-JAK2; forward primer 5'-GCAGCCCTAAGGACTTCAAC-3' and reverse primer 5'-CCGCTGAGGTTGTATTCTCC-3'. To remove the plasmid DNA contamination due to transfection, RNA samples were treated with RNase-free DNase (Promega), and then DNase was inactivated and removed by phenol/chloroform extraction and subsequent ethanol precipitation before RT. Transfection of RAECs with DN-JAK2 construct resulted in about 100-fold increase in JAK2 transcripts compared with that of empty vector (supplemental Fig. 2, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).
Immunoprecipitation and immunoblotting
Cells were harvested and lysed in immunoprecipitation buffer [50 mM HEPES (pH 7.6), 150 mM NaCl, 1 mM EGTA, 1% Nonidet P40, 10 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 5 mM NaF]. Cell lysates (300 μg) were incubated with 3 μg rabbit polyclonal anti-JAK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in a volume of 500 μl at 4 C overnight, followed by the addition of protein G beads (50% slurry) and additional incubation for 1 h. The beads were washed three times with immunoprecipitation buffer, solubilized in Laemmli sample buffer, and subjected to 10% SDS-PAGE. Immunoblot analysis was performed using mouse monoclonal anti-phosphotyrosine antibody (PY99: Santa Cruz Biotechnology), followed by reprobing with anti-JAK2 antibody as previously described (22).
Statistical analysis
Data were expressed as mean ± SEM. Differences between groups were examined for statistical significance using unpaired t test or ANOVA with Dunn’s post hoc test, if appropriate. P values less than 0.05 were considered statistically significant.
Results
Aldosterone increases ACE mRNA and its enzymatic activity
Aldosterone dose-dependently (3 x 10–9 to 10–7 M) increased steady-state ACE mRNA levels in RAECs (Fig. 1A); a significant (P < 0.05) increase was induced by as low as 3 x 10–9 M and a 17.5-fold increase by 10–7 M. Aldosterone time-dependently increased steady-state ACE mRNA levels in RAECs (Fig. 1C); about 5- and 10-fold increases were observed after 8 and 18 h, respectively. The ACE gene expression induced by aldosterone (5 x 10–7 M) was completely blocked by pretreatment with a selective MR antagonist, spironolactone (10–5 M), but partially inhibited by a selective glucocorticoid receptor (GR) antagonist, RU486 (10–6 M) (Fig. 2A). Dexamethasone (5 x 10–7 M) also increased steady-state ACE mRNA levels in RAECs, whose effect was completely blocked by RU486, but not by spironolactone (Fig. 2B). RAECs used in our study expressed both MR and GR mRNAs as revealed by RT-PCR (data not shown). These results suggest that ACE gene expression induced by aldosterone is mediated mainly via MR at lower concentrations but partly via GR at higher concentrations, whereas ACE gene expression is also regulated by glucocorticoids exclusively via GR.
The aldosterone-induced ACE gene expression was blocked by pretreatment with a transcription inhibitor, actinomycin D (5 x 10–6 M), but not with AT1 receptor blocker (valsartan, 10–6 M) (data not shown). Furthermore, aldosterone dose-dependently (10–9 to 10–7 M) increased ACE activity (Fig. 3A); about 2.7-fold increase was induced by as low as 10–9 M and 10.3-fold increase by 10–7 M. The aldosterone-stimulated ACE activity was also blocked by spironolactone (Fig. 3B). Thus, our data indicate that aldosterone induces ACE gene expression and increases its enzyme activity mainly via MR in RAECs.
Protein tyrosine kinase (PTK) inhibitors blocks aldosterone-induced ACE gene expression
To further characterize the molecular mechanism(s) underlying the aldosterone-induced ACE mRNA expression, we tested the effects of various PTK inhibitors on aldosterone-induced ACE gene up-regulation (Fig. 4).
The aldosterone-induced ACE mRNA expression was completely inhibited by a nonselective PTK inhibitor, genistein (10–4 M) (Fig. 4A) and JAK2 inhibitor, AG490 (10–4 M) (Fig. 4B). As shown in Fig. 5, the inhibitory effects of genistein (10–5 to 10–4 M) and AG490 (10–6 to 10–4 M) were concentration dependent. In contrast, a selective inhibitor of the Src kinase family (PP2, 10–5 M) (Fig. 4C) and an epidermal growth factor receptor (EGFR) kinase inhibitor (AG1478, 3 x 10–7 M) (Fig. 4D) only partially inhibited the aldosterone-induced ACE mRNA expression, and platelet-derived growth factor receptor kinase inhibitor (AG1296, 10–5 M) (Fig. 4E) was without effect.
DN-JAK2 construct suppressed aldosterone-induced ACE gene expression
To determine whether JAK2 is actually involved in the aldosterone-induced ACE mRNA expression, we examined the effects of overexpression of DN-JAK2 (JAK2 KE pRK5) or empty vector (pRK5). Transfection of RAECs with DN-JAK2 construct resulted in a significant (P < 0.05) decrease in the aldosterone-induced ACE expression, whereas transfection of empty vector did not affect aldosterone-induced ACE mRNA expression; basal ACE mRNA levels without aldosterone treatment were unaffected by transfection of either vectors (Fig. 6). Thus, these results suggest that JAK2 among several PTKs is mainly involved in the aldosterone-stimulated ACE gene expression in endothelial cells.
Aldosterone stimulated JAK2 phosphorylation
To ascertain whether aldosterone activates JAK2, we assessed tyrosine phosphorylation of JAK2 by immunoprecipitation with anti-JAK2 antibody coupled with immunoblotting with anti-phosphotyrosine antibody. Aldosterone stimulated phosphorylation of JAK2 in a time-dependent manner, which peaked after 2 h and then declined thereafter; no distinct band was noted when nonimmune IgG was used for immunoprecipitation (Fig. 7A). JAK2 phosphorylation was induced by aldosterone in a dose-dependent manner (10–8 to 10–7 M), whose effect was blocked by actinomycin D (Fig. 7B) and spironolactone (data not shown). These results indicate that the aldosterone-induced JAK2 phosphorylation response is transcription-dependent genomic action.
Discussion
The present study demonstrates for the first time that aldosterone directly up-regulates ACE gene expression and its enzymatic activity in rat endothelial cells, whose effect was completely blocked by a selective MR antagonist (spironolactone). Our data are consistent with a recent study showing that aldosterone up-regulates ACE gene expression in rat neonatal cardiomyocytes (12).
It has been recently recognized that aldosterone plays a pivotal role in the development and/or progression of cardiovascular disease as a critical and final component of RAS (1, 2, 3, 4, 5). In experimental Ang II-induced hypertension models, administration of MR antagonist or adrenalectomy significantly attenuated the vascular injury of coronary, renal, and cerebral arteries without lowering of blood pressure (3, 5). In mineralocorticoids/high-salt hypertensive rats, cardiovascular injury develops in a similar manner to the Ang II-induced hypertension models (3, 5). Vascular injury in these hypertensive models is preceded by the development of a vascular inflammatory phenotype associated with expression of a variety of proinflammatory genes, including COX-2, cytokines, osteopontin, and MCP-1 (4, 26). However, the underlying cellular mechanism of vascular injury by aldosterone has not been well understood.
It has been reported that MR mRNA is expressed not only in epithelial cells but also in nonepithelial tissue including cardiovascular tissue (27, 28, 29). We confirmed that RAECs used in the present study also expressed MR mRNA. The minimum effective concentrations of aldosterone required for ACE gene induction and increase in ACE activity (1–3 x 10–9 M) in RAECs appeared to be almost comparable to its physiological concentrations (10–9 M) in the circulation (30). Furthermore, the aldosterone-induced ACE gene expression and enzyme activity in RAECs were inhibited by a selective MR antagonist (spironolactone) at the concentration (10–6 M) almost equivalent to those in plasma concentrations (10–7 to 10–6 M) in the clinical setting (31). Thus, it is possible to speculate that spironolactone may be clinically effective in blocking the aldosterone-induced ACE gene expression in endothelium.
Aldosterone and glucocorticoids interact with MR with equal affinity, and aldosterone in higher concentration causes crossover activation of GR (32). We therefore examined the effect of dexamethasone on ACE gene expression and the specificities of aldosterone and dexamethasone responses by their selective MR and GR antagonist, respectively. The present results suggest that aldosterone-induced ACE response in the higher concentration (greater than 5 x 10–7 M), although partly via GR, is mainly mediated via MR, whereas dexamethasone-induced ACE response is exclusively mediated via GR. Furthermore, it should be noted that 11-hydroxysteroid dehydrogenase-2 (11-HSD2), which converts glucocorticoids into inactive 11-dehydro compounds to protect MR from the abundant glucocorticoids in renal epithelial cells, has recently been demonstrated in human aorta and rat endothelial cells (33, 34). We have also confirmed that our RAECs express not only MR mRNA but also 11-HSD2 mRNA by the quantitative RT-PCR experiments (unpublished observations). Taken together, it is suggested that the glucocorticoid-inactivating enzyme 11-HSD2 could play a functional role in the maintenance of ligand selectivity for MR in endothelial cells. However, the molecular interaction between MR and GR activation in the regulation of endothelial ACE gene expression remains to be determined.
Aldosterone synthase (CYP11B2) mRNA and its enzyme activity have been recently reported to be expressed in cardiovascular tissue, including endothelial cells and cardiomyocytes (35, 36, 37, 38). However, the presence of aldosterone synthase in human endothelial cells (39) and rat heart (40) has very recently been challenged. Assuming that aldosterone may be synthesized by cardiovascular tissue, it is likely that the approximate aldosterone concentration at the local level should be high enough to exert its paracrine action. An alternative possibility is that circulating aldosterone may be readily bound and concentrated by and released from cardiovascular tissue in a sufficient amount locally, as recently suggested by Funder (41). Thus, the critical issue as to whether cardiovascular tissue synthesizes and secretes aldosterone in an autocrine/paracrine fashion remains to be addressed.
The endothelial ACE up-regulation by aldosterone as demonstrated in this study may partly contribute to the development and/or progression of vascular damage resulting from activation of tissue RAS via a positive feedback mechanism as has been suggested in rat cardiomyocytes (12). It has very recently been reported that aldosterone up-regulates AT1 receptor in rat VSMCs and potentiates Ang II-stimulated VSMC proliferation (10, 11). Taken together, it is strongly suggested that a positive feedback loop of local RAS in the vasculature is partly responsible for the development and/or progression of aldosterone-induced endothelial dysfunction and vascular injury.
The present study clearly shows that aldosterone-induced up-regulation of the ACE gene in endothelial cells is blocked by a nonselective PTK inhibitor (genistein) as well as a specific inhibitor of JAK2 (AG490) in a dose-dependent manner. Furthermore, overexpression of DN-JAK2 mutant in endothelial cells decreased the aldosterone-induced ACE gene expression. Thus, our data suggest that aldosterone preferentially activates JAK2 among various PTKs, which constitutes an essential signal for endothelial ACE gene expression. Our contention is supported by the data that aldosterone induced JAK2 phosphorylation via a MR-mediated pathway.
The exact molecular mechanism by which aldosterone activates JAK2 remains unknown. Although it has been shown that the JAK family is activated via cytokine receptors or G protein-coupled receptors (42, 43), there has been no study thus far reported showing that a nuclear receptor activates the JAK family. In the present study, phosphorylation of JAK2 by aldosterone was completely blocked by actinomycin D, indicating that aldosterone-induced JAK2 phosphorylation is via its genomic action. Aldosterone has been shown to potentiate Ang II-induced ERK1/2 and JNK activation and oxygen radical production in rat VSMCs (11). Thus, aldosterone may potentiate Ang II-induced JAK activation. However, failure of AT1 receptor blocker (valsartan) to block the aldosterone-induced ACE gene expression in this study excluded the potential involvement of endogenous Ang II in the mechanism of aldosterone-induced ACE up-regulation.
In the present study, both c-Src inhibitor (PP2) and EGFR inhibitor (AG1478) partially suppressed the aldosterone-induced ACE gene expression. It has been shown that c-Src is associated with and activated by JAK2 (44). We have previously shown that c-Src is an upstream signal for Ang II-induced EGFR transactivation in rat VSMCs (18). Thus, aldosterone may activate c-Src and subsequent EGFR transactivation as a downstream signal resulting from JAK2 activation, which may be responsible, at least in part, for the aldosterone-induced ACE gene expression. It has been shown that EGFR inhibitor (AG1478) reduced the aldosterone-induced ERK1/2 phosphorylation in rat VSMCs (11). However, the question as to how aldosterone-induced activation of several PTKs, including c-Src, EGFR, and JAK2, leads to ACE gene expression remains open to question.
In conclusion, our present study demonstrates for the first time that aldosterone directly increases ACE gene expression and its enzyme activity mainly via a MR-mediated and JAK2-dependent pathway in rat endothelial cells. The aldosterone-induced JAK2 pathway gives a new insight into the cellular signaling mechanism of aldosterone’s action on cardiovascular tissue. Thus, regulation of the endothelial ACE gene by aldosterone may constitute a positive feedback loop for local RAS, possibly involved in the development and/or progression of aldosterone-induced endothelial dysfunction and vascular injury.
Footnotes
This study was supported in part by Grant-in-aid from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health, Welfare and Labor, of Japan.
Abbreviations: ACE, Angiotensin-converting enzyme; Ang II, angiotensin II; ARPP P0, acidic ribosomal phosphoprotein P0; AT1, Ang II type 1; DN, dominant negative; EGFR, epithelial growth factor receptor; GR, glucocorticoid receptor; 11-HSD2, 11-hydroxysteroid dehydrogenase-2; MR, mineralocorticoid receptor; PTK, protein tyrosine kinase; RAEC, rat aortic endothelial cell; RAS, renin-angiotensin system; VSMC, vascular smooth muscle cell.
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