Accepted Manuscript
Title: Angiotensin 1-7 suppresses Angiotensin II mediated aldosterone production via JAK/STAT signaling inhibition
Authors: Kiyotaka Itcho, Kenji Oki, Kazuhiro Kobuke, Haruya Ohno, Masayasu Yoneda, Noboru Hattori
The Journal of Steroid Biochemistry & Molecular Biology
| PII: | S0960-0760(18)30123-7 |
| DOI: | https://doi.org/10.1016/j.jsbmb.2018.08.007 |
| Reference: | SBMB 5197 |
| To appear in: | Journal of Steroid Biochemistry & Molecular Biology |
| Received date: | 26-2-2018 |
| Revised date: | 3-8-2018 |
| Accepted date: | 12-8-2018 |
Please cite this article as: Itcho K, Oki K, Kobuke K, Ohno H, Yoneda M, Hattori N, Angiotensin 1-7 suppresses Angiotensin II mediated aldosterone production via JAK/STAT signaling inhibition, Journal of Steroid Biochemistry and Molecular Biology (2018), https://doi.org/10.1016/j.jsbmb.2018.08.007
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Angiotensin 1-7 suppresses Angiotensin II mediated aldosterone production via JAK/STAT signaling inhibition
Short title: Aldosterone production via Ang1-7 and JAK/STAT
Kiyotaka Itcho, Kenji Oki, Kazuhiro Kobuke, Haruya Ohno, Masayasu Yoneda, Noboru Hattori Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan.
Precis: We demonstrated that angiotensin 1-7 negatively regulates angiotensin II mediated aldosterone production, and this inhibitory effect was associated with JAK/STAT signaling in human adrenal cells.
Correspondence: Kenji Oki Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan.
Email: kenjioki@hiroshima-u.ac.jp
Disclosure. The author reports no conflicts of interest in this work.
Graphical abstract
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A-II
A-II
Ang1-7
AT1R
M
MASR
AT1R
MASR
JAK
JAK
ERK
Ca2+
PKA
ERK
STAT
Ca2+
PKA
STAT
nucleus
nucleus
RIPT
Highlights
· Ang1-7 suppressed A-II-stimulated aldosterone production in HAC15 cells
· Ang1-7 antagonist abrogated Ang1-7-mediated inhibition of aldosterone production.
· JAK-STAT phosphorylation was inhibited by Ang1-7 in A-Il stimulated HAC15 cells.
· STAT signal inhibitors abrogated A-II-stimulated aldosterone production in HAC15.
Abstract:
Angiotensin 1-7 (Ang1-7), which is a protein cleaved from angiotensin II (A-II), binds to the MAS receptor. Ang1-7 has been demonstrated to exert protective effects against A-II-mediated cardiac, atherosclerotic, and renal damages. The aims of our study were to demonstrate the inhibitory role of Ang1-7 in A-II-mediated aldosterone production by interacting with the MAS receptor in human adrenocortical carcinoma (HAC15) cells, and clarify the intracellular signaling mechanisms underlying the inhibition of aldosterone production by Ang1-7. Ang1-7 significantly suppressed A-II-stimulated aldosterone production, and partially abrogated A-II- induced upregulation of CYP11B2 expression. Treatment with a selective Ang1-7 antagonist abrogated
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Ang1-7-mediated inhibition of aldosterone production in HAC15 cells. Incubation of A-II-treated HAC15 cells with conditioned medium containing Ang1-7 was demonstrated to suppress A-II-mediated aldosterone production and CYP11B2 expression. Proteomic analysis showed that Ang1-7 predominantly inhibited the phosphorylation of JAK-STAT proteins in A-II stimulated HAC15 cells. Treatment of HAC15 cells with a STAT3 inhibitor partially but significantly repressed A-II-mediated aldosterone production by 63.2%. Similarly, treatment with a STAT5 inhibitor significantly abrogated A-II-stimulated aldosterone production in HAC15 cells by 60.7%. In conclusion, we demonstrated that Ang1-7 negatively regulates A-II-mediated aldosterone production, and the observed inhibition of aldosterone production was associated with JAK/STAT signaling in human adrenal cells. Therefore, activation of Ang1-7 or stimulation of the MAS receptor, which inhibits aldosterone production, is a promising therapeutic approach for the prevention of cardiovascular events that can directly affect the target organs.
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Abbreviations
A-Il, angiotensin II; AT1R, angiotensin type 1 receptor; MAPK, mitogen-activated protein kinase; ACE2, Angiotensin-converting enzyme 2; HAC15, human adrenocortical carcinoma; qPCR, quantitative polymerase chain reaction; angiotensin IV, AngIV;
Keywords: angiotensin II; aldosterone; angiotensin 1-7; JAK/STAT signaling
1. Introduction
Aldosterone secretion is primarily controlled by the renin-angiotensin-aldosterone system in the zona glomerulosa of the adrenal gland. Binding of angiotensin II (A-II) with angiotensin type 1 receptor (AT1R) stimulates aldosterone production through inositol trisphosphate/Ca2+ signaling by activation of voltage- gated calcium channel and release from endoplasmic reticulum [1, 2]. Additionally, A-II-mediated intracellular signaling pathways, including diacylglycerol/protein kinase C, mitogen-activated protein kinase (MAPK) pathway, and tyrosine kinase cascade also stimulate aldosterone production [3-5]. The secreted aldosterone promotes salt and water reabsorption by binding to mineralocorticoid receptors, which are predominantly found in the distal tubules of the kidneys. Excess aldosterone secretion leads to hypertension
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and causes cardiac, vascular, and renal disorders [6, 7]. Thus, there is urgent need to elucidate the mechanisms that regulate aldosterone production for the development of new therapeutic agents.
Angiotensin-converting enzyme removes two amino acids from angiotensin I (A-I) (1-10) and converts to A-II, which contains eight amino acids (1-8). Angiotensin-converting enzyme 2 (ACE2) catalyzes the conversion of A-II to angiotensin 1-7 (Ang1-7). In a previous study, we demonstrated that A-Il is primarily metabolized to Ang1-7 and slightly metabolized to angiotensin IV (AngIV, 3-8) in human adrenocortical carcinoma (HAC15) cells [8]. Importantly, the effects of Ang1-7 are mediated by the MAS receptor.
Basic studies suggested that Ang1-7 exerts the protective effects against A-II-mediated cardiac, atherosclerotic, and renal damages [9, 10]. ACE2-knockout rodent models presented cardiac dysfunction [11], atherosclerosis [12], and severe glomerulosclerosis and proteinuria [13]. Recently, Ang1-7 infusion in rats fed with low-sodium diet showed lower aldosterone levels and higher plasma renin activity [14]. Furthermore, same group reported the suppression of A-II-mediated aldosterone production by Ang1-7 in isolated rat glomerulosa cells [14]. Therefore, Ang1-7 is useful for the development of novel therapeutic agents. However, the exact molecular mechanisms underlying the suppression of A-II-mediated aldosterone production by Ang1-7 have not been elucidated in adrenal cells.
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Taken together, we first aimed to demonstrate that Ang1-7 abrogates A-II-mediated aldosterone production via the MAS receptor in HAC15 cells. Considering our previous results on A-II metabolites, we hypothesized that A-II mediated aldosterone production had negative feedback mechanism via its conversion to Ang1-7. Thus, we next aimed to demonstrate the inhibition of A-II-mediated aldosterone production by Ang1-7 and conditioned medium after A-Il is metabolized in HAC15 cells. Third, we validated the intracellular signaling mechanisms that mediate the inhibition of aldosterone production by Ang1-7.
2. Material and Methods
2.1. Cell culture and materials
HAC15 cells derived from human adrenocortical carcinoma cells were kindly provided by Dr. William Rainey (University of Michigan, USA). HAC15 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM):F12 (1:1) supplemented with 10% Cosmic Calf serum (GE Healthcare-HyClone, Logan, UT, USA) at 37 ℃ in the presence of 5% CO2. Cells were serum-deprived in DMEM:F12 containing 0.1% Cosmic Calf
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serum for 24 h before measurement of aldosterone levels and mRNA expression. Cells were incubated in DMEM:F12 containing 0.1% Cosmic Calf serum with or without stimulants for 24 h and 3 h, for determination of aldosterone levels and mRNA expression, respectively.
A-II, Ang1-7, and forskolin were purchased from Sigma Aldrich Co. Ltd. (St. Louis, MO, USA). AngIV was obtained from American Peptide Co. Inc. (Sunnyvale, CA, USA). A779, a selective Ang1-7 antagonist, was obtained from Bachem (Bubendorf, Switzerland). The JAK/STAT signaling inhibitors which are Stattic (#ab120952) and IQDMA (#ab141192) were purchased from Abcam (Cambridge, UK).
2.2. RNA extraction and quantitative polymerase chain reaction (qPCR) assays
RNA extraction and qPCR assays were performed as previously reported [15]. Briefly, total RNA was extracted from cell cultures using RNeasy Mini kit (Qiagen, Hilden, Germany). For reverse transcription, 300 ng of total RNA was incubated with Takara PrimeScript RT Master Mix (Takara Bio Inc., Shiga, Japan) following the manufacturer’s protocol. The mRNA expression levels of CYP11B2 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were determined using a Taqman® Gene Expression Assay kit (Applied Biosystems, Waltham, MA, USA). mRNA levels were determined as arbitrary units normalized against GAPDH expression levels.
2.3. Phospho-kinase array
The Proteome Profiler™ Array Human Phospho-Kinase Array Kit (R&D Systems, Minneapolis, MN, USA) was used for the phospho-kinase assay following the manufacture’s protocol. The assay is based on ELISA method using antibodies for phosphorylated protein. HAC15 cells were grown to confluence, and were serum-deprived in DMEM:F12 containing 0.1% Cosmic Calf serum for 24 h. Then, cells were pretreated with or without Ang1-7 for 30 min and subsequently incubated with A-II (n=3). Cells were harvested with the provided buffer on ice. The absorbance was measured using ImageQuant LAS 500 (GE Healthcare, Chicago, IL, USA).
2.4. Western blot analysis and PKA kinase activity assay
Cell lysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transfer, and blot processing were performed as previously reported [16]. Briefly, cell lysates were collected using RIPA buffer supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Total proteins were separated via SDS-PAGE, and the bands were transferred onto a
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polyvinylidene difluoride membrane. Blots were probed using antibodies of ERK (1:5000 dilution) and phosphorylated ERK (1:5000 dilution), namely p44/42 MAPK (Cell Signaling, #4695, Danvers, MA, USA) and phospho-p44/42 MAPK (Cell Signaling, #4370, Danvers, MA, USA), respectively. Additionally, cell lysates were analyzed for PKA activity using a PKA kinase activity kit (Enzo Biochem Inc., Farmingdale, NY, USA).
2.5. Aldosterone and protein assay
Aldosterone levels in cell culture were measured using an ELISA kit as previously described [2]. Cellular protein levels were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).
2.6. Statistical analysis
Results were expressed as mean + S.E. of at least three independent experiments. In each experiment, samples were assayed in triplicate or quadruplicate. Statistically significant differences between two groups were analyzed using t-test. Multiple groups were analyzed by one-way ANOVA followed by Bonferroni comparisons. P<0.05 was considered statistically significant. Analyses were performed using SPSS for Windows (release 24.0; SPSS Inc., Chicago, IL, USA).
3. Results
3.1. Effects of A-Il metabolites on A-II- and forskolin-induced aldosterone production
Consistent with our previous findings, Ang1-7 or AngIV did not significantly affect aldosterone production at baseline (Figure 1A and 1B) [8]. Ang1-7 was found to inhibit A-II-stimulated aldosterone production by 23.3% (P<0.05, Figure 1A), whereas AngIV did not inhibit A-II-induced aldosterone production (Figure 1B). Upregulation of CYP11B2 expression by A-II was partially inhibited by Ang1-7 (P<0.05, Figure 1C). Ang1-7 did not affect forskolin-mediated aldosterone production (Figure 1D).
3.2. Effects of Ang1-7 antagonist on A-II mediated aldosterone production
A779, a selective Ang1-7 antagonist, did not significantly affect aldosterone production at baseline (Figure 2). By contrast, A779 significantly abrogated Ang1-7 mediated inhibition of aldosterone production. In addition, A-II-stimulated HAC15 cells treated with Ang1-7 and A779 showed significantly higher aldosterone levels than those of HAC15 cells treated with or without Ang1-7 (Figure 2).
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3.3. Effects of A-II depleted conditioned medium on aldosterone production
We previously demonstrated that A-II is completely cleaved within 6 h by HAC15 cells, whereas Ang1-7 was detected up to 6 h after stimulation [8]. Therefore, HAC15 cells were treated with the conditioned medium after 6 h of incubation with A-II. As shown in Figure 3A and 3B, treatment with the conditioned medium did not stimulate aldosterone production or induce CYP11B2 expression, consistent with our previous results indicating that the conditioned medium did not contain A-II. On the other hand, the conditioned medium significantly suppressed A-II-mediated aldosterone production and inhibited CYP11B2 expression (Figure 3A and 3B).
3.4. The intracellular signaling via MAS receptor
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Previous studies suggested that Ang1-7 attenuated A-II-mediated ERK phosphorylation and PKA activity [17-19]. However, we observed no significant differences in ERK phosphorylation or PKA activity between A-Il stimulated cells with and without Ang1-7 pretreatment (Figure S1 and S2). Therefore, we conducted proteomic analysis of phospho-protein to evaluate the mechanisms by which Ang1-7 inhibits A-II- induced aldosterone production via the MAS receptor. We analyzed the phospho-proteins which showed more than 20% reduction following Ang1-7 treatment, and the proteins were annotated using the KEGG pathway database (http://www.genome.jp/kegg/). Ang1-7 predominantly inhibited the phosphorylation of the proteins involved in JAK-STAT signaling in A-II-stimulated HAC15 cells (Figure 4).
3.5. Effects of JAK/STAT inhibition on aldosterone production
Based on the proteomic analysis and the previous report which JAK/STAT signaling was activated by A-Il stimulation [20], we focused on the effects of JAK/STAT inhibition on aldosterone production. HAC15 cells were treated with Stattic and IQDMA to inhibit STAT3 and STAT5, respectively. 30 uM Stattic or 10 UM IQDMA did not influence aldosterone concentration in media of HAC15 cells (Figure S3). Since cytotoxicity were visible in HAC15 cells using more than 30 uM Stattic or more than 10 UM IQDMA, we applied 10 µM Stattic or 3 µM IQDMA in this study. Stattic (10uM) and IQDMA (3uM) partially but significantly repressed A- Il-mediated aldosterone production by 63.2% and by 60.7%, respectively (Figure 5A and 5B).
4. Discussion
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Our results demonstrated that Ang1-7 suppressed A-II-mediated aldosterone production in HAC15 cells. Treatment with A779, a selective Ang1-7 antagonist, was found to enhance A-II-mediated aldosterone production in HAC15 cells. A-II stimulated aldosterone production in HAC15 cells was significantly reduced after treatment with conditioned medium, in which A-II might be converted to Ang1-7. Proteomic analysis revealed that Ang1-7 inhibited STAT phosphorylation, which in turn decreased aldosterone production in HAC15 cells.
Although the JAK/STAT pathway is primarily regulated by cytokine receptor, AT1R stimulation has also been shown to activate JAK/STAT signaling [20]. A-Il stimulation induced rapid tyrosine phosphorylation of several proteins, and tyrosine phosphorylation of JAK2, STAT1, and STAT2 was observed within 5 min [3, 20]. AT1R, which belongs to a family of seven-transmembrane receptors, exerts its functions through a G protein-dependent signaling pathway. Tyrosine phosphorylation of JAK2 through AT1R requires G protein- dependent signaling, which is mediated by increased cytoplasmic Ca2+ concentrations and PKCo activation [21]. On the other hand, mutant AT1R, which cannot activate the G protein, was shown to activate the JAK/STAT pathway, indicating that the JAK/STAT pathway is stimulated by a G protein-independent mechanism [22]. Taken together, the above findings indicated that G protein-dependent and -independent effects by binding of A-II to AT1R induce JAK/STAT signaling.
We demonstrated that Ang1-7 suppressed A-II-mediated JAK/STAT signaling in HAC15 cells. Ang1- 7 was previously demonstrated to suppress JAK/STAT signaling to prevent pulmonary hypertension in a rat model [23]. Our results are consistent with a previous report showing that Ang1-7 inhibited the JAK/STAT pathway. On the other hand, Ang1-7 attenuated cardiac remodeling and ventricular dysfunction and was associated with reduced ERK activity following A-Il stimulation in rats [17, 18]. Furthermore, Ang1-7 was demonstrated to attenuate PKA signaling in a rat model [18, 19]. We investigated the effects of Ang1-7 on ERK phosphorylation and PKA signaling for the suppression of aldosterone production. Results of proteomic analysis, western blotting analysis, and ELISA consistently showed that Ang1-7 did not modulate ERK phosphorylation or PKA signaling (Figure 4, Figure S1, Figure S2). On the other hand, A-II mediated AMPK signaling were suppressed by Ang1-7 in HAC15 cells (Figure 4), however there were no evidence of the involvement of AMPK signaling on aldosterone production in adrenal cells. Further basic studies would be needed for the association between AMPK signaling and A-II mediated aldosterone production. It is
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suggested that suppression of JAK/STAT signaling is a pivotal role for the suppression of aldosterone production by Ang1-7 in adrenal cells (Figure 6).
We verified the novel findings that treatment with a MAS receptor antagonist further promoted aldosterone production under A-II stimulation. Ang1-7, which inhibits A-II-stimulated aldosterone production, is metabolized from A-Il in HAC15 cells [8]. The above results suggested a self-negative feedback mechanism in A-II-mediated aldosterone production. Furthermore, A-Il loading downregulated AT1R expression in adrenal cells [24], suggesting its additional role in the negative regulation of A-II-stimulated aldosterone production. Therefore, Ang1-7 or AT1R can serve as protective machinery from acute A-II stimulation to prevent A-II-induced excessive aldosterone production.
Ang1-7 exerted protective effects against A-II-mediated cardiac, atherosclerotic, and renal damage in rodent models [9, 10]. Our findings suggested that Ang1-7 suppresses aldosterone production in response to A-Il stimulation. Therefore, Ang1-7 can be used for the prevention of cardiovascular diseases by lowering aldosterone levels. Additionally, Ang1-7 and MAS receptor signaling have been shown to be involved in the inhibition of tumor cell growth and angiogenesis in colon cancer, lung cancer, and hepatocellular carcinoma [25-27]. Given that Ang1-7 exerts pleiotropic effects, such as the prevention of cardiovascular events and tumor progression, compounds targeting Ang1-7 or the MAS receptor could serve as effective therapeutic agents against cardiovascular diseases and tumors.
The present study demonstrated that Ang1-7, a metabolite of A-II, negatively regulates A-II- mediated aldosterone production. Moreover, inhibition of aldosterone production by Ang1-7 was associated with JAK/STAT signaling in human adrenal cells. Ang1-7 activation or MAS receptor stimulation serve as promising therapeutic strategies for suppressing aldosterone production as well as preventing cardiovascular events and tumor progression.
Acknowledgments
None
Sources of Funding.
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This study was financially supported by JSPS KAKENHI Grant Number JP17H06893 (KI) and JP17K09883 (KO), Okinaka Memorial Institute for Medical Research Grant (KO), SENSHIN Medical Research Foundation (KO), and Japan Heart Foundation Dr. Hiroshi Irisawa & Dr.Aya Irisawa Memorial Research Grant (KO).
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Figure legends
Figure 1 Angiotensin II (A-II) metabolites including angiotensin 1-7 (Ang1-7) suppressed A-II- mediated aldosterone production and CYP11B2 mRNA expression in HAC15 cells. After reaching confluence, cells were serum-deprived in DMEM:F12 containing 0.1% Cosmic Calf serum for 24 h. After pretreatment with or without A-Il metabolites (10nM) for 30 min, cells then were incubated with or without stimulants. (A, B) Aldosterone levels were measured in cells incubated with or without A-II (10 nM) for 24 h. Results were expressed as fold change relative to aldosterone levels in unstimulated control cells. * , P<0.05 vs. control with A-Il stimulation by t-test (n=4). (C) After 3 h of incubation with or without A-II (10 nM), cells were collected for RNA extraction and real time RT-PCR analysis. Expression levels were normalized against GAPDH levels and expressed as fold change relative to unstimulated control cells. * , P<0.05 vs. control with A-Il stimulation by t-test (n=3). (D) Aldosterone levels were compared between cells incubated with or without forskolin (10 uM) for 24 h. Results were expressed as fold change relative to unstimulated control cells (n=4).
Figure 2 A779, a selective angiotensin 1-7 (Ang1-7) antagonist, potentiated angiotensin II (A-II)- stimulated aldosterone production in HAC15 cells. After reaching confluence, HAC15 cells were serum- deprived in DMEM:F12 containing 0.1% Cosmic Calf serum for 24 h. First, cells were pretreated with or without A779 (10 uM) for 30 min and subsequently pretreated with 10 nM Ang1-7 for 30 min. Finally, cells were incubated with or without 10 nM A-II. * , P<0.05 vs. A-Il stimulation by one-way ANOVA. #, P<0.05 vs. A-Il stimulation with Ang1-7 pretreatment by one-way ANOVA (n=3).
Figure 3 Conditioned medium suppressed angiotensin II (A-II)-mediated aldosterone production and CYP11B2 mRNA expression in HAC15 cells. Conditioned medium was obtained from the culture supernatant at 6 h after A-Il loading. After reaching confluence, cells were serum-deprived in DMEM:F12 containing 0.1% Cosmic Calf serum for 24 h. The conditioned medium or control medium (DMEM:F12 containing 0.1% Cosmic Calf serum) were incubated for 30 min before A-II (10 nM) loading. (A) For measurement of aldosterone levels, supernatants were collected after 24 h after stimulation with or without
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A-II (10 nM). Results were expressed as fold change relative to unstimulated control cells. * , P<0.05 vs. control with A-II stimulation by t-test (n=4). (B) After 3 h of incubation with or without A-II stimulation, the cells were collected for RNA extraction and real time RT-PCR performed. CYP11B2 mRNA expression levels were normalized against GAPDH levels and expressed as fold change versus unstimulated controls. *, P<0.05 vs. control with A-Il stimulation by t-test (n=3).
Figure 4 Results of proteomic analysis of phospho-proteins mediating angiotensin 1-7 (Ang1-7) inhibition. After reaching confluence, HAC15 cells were serum-deprived in DMEM:F12 containing 0.1% Cosmic Calf serum for 24 h. Then cells were incubated with A-II for 5 min after pretreatment with or without Ang1-7 metabolites for 30 min (n=3). Cells were harvested on ice, and phosphor-proteins were analyze using Proteome Profiler™ Array Human Phospho-Kinase Array Kit. Phospho-proteins that showed more than 20% downregulation by Ang1-7 were analyzed. The extent of down regulation of protein phosphorylation was shown in parentheses after protein name.
THE
Figure 5 JAK/STAT signaling inhibitors, Stattic and IQDMA, partially abrogated angiotensin II (A-II)- stimulated aldosterone production in HAC15 cells. After reaching confluence, HAC15 cells were serum- deprived in DMEM:F12 containing 0.1% Cosmic Calf serum for 24 h. Cells were pretreated with or without 10 uM Stattic (A) or 3 uM IQDMA (B) for 30 min, and subsequently incubation with or without 10 nM A-II for 24 h. * , P<0.05 vs. control with A-Il stimulation by t-test (n=3).
Figure 6 The considerable molecular mechanisms by which angiotensin 1-7 (Ang1-7) suppresses angiotensin II (A-II)-mediated aldosterone production. The binding of AT1R with A-Il induces JAK/STAT phosphorylation in adrenal cells, which in turn promotes the transcription of genes involve in aldosterone production. Ang1-7 abrogates A-II-induced phosphorylation of JAK/STAT proteins via the MAS receptor.
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A
none
A-II
6.0
Aldosterone levels (vs control w/o A-II)
5.0
T
*
1
4.0
3.0
2.0
1.0
0
control Ang1-7 control Ang1-7
C
none
A-II
CYP11B2 mRNA (vs control w/o A-II)
10.0
T
8.0
1
*
6.0
T
4.0
2.0
0
control Ang1-7 control Ang1-7
B
none
A-II
5.0
Aldosterone levels (vs control w/o A-II)
T
4.0
1
3.0
2.0
1.0
I
0
control AngIV control AngIV
D
Forskolin
aldosterone levels (vs control)
6.0
5.0
L
4.0
3.0
2.0
1.0
0
control control Ang1-7
ZEPTED MANUSCRIPT 0
15.0
* #
Relative aldosterone levels
12.0
*
9.0
6.0
3.0
E
E
A-II (10nM)
-
-
-
+
+
+
Ang1-7 (10nM)
-
-
-
+
A779 (10uM) - - +
-
-
+
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A
-
none
A-II
Aldosterone levels (vs control w/o A-II)
8.0
6.0
T
1
*
&
4.0
SCRIPT
2.0
0
control
conditioned medium
control
conditioned medium
B
10.0
none
A-II
CYP11B2 mRNA (vs control w/o A-II)
8.0
6.0
*
4.0
2.0
0
control
conditioned medium
control
conditioned medium
Others (n=5) WNK1 (37.3%) p70S6 kinase (35.0%) PDGFRØ (28.4%) PRAS40 (22.8%) p53 (20.1%)
JAK/STAT signaling (n=5)
STAT2 (22.1%) STAT3 (22.9%)
STAT5a (22.4%) STAT5b (24.4%) STAT6 (20.5%)
JNK and p38 MAP kinase pathway (n=2) p38a (21.0%) MSK1/2 (21.8%)
AMPK signaling (n=2) AMPKa1 (20.3%) AMPKa2 (30.1%)
ACCES
ACCEPTED MANUSCRIPT
A
none
A-II
B
none
A-II
12.0
25.0
aldosterone levels (vs control)
10.0
*
aldosterone levels (vs control)
20.0
8.0
15.0
6.0
10.0
4.0
2.0
5.0
0
8 88
0
control Stattic (10μM)
control Stattic (10μM)
control IQDMA control IQDMA (3ΜΜ) (3ΜΜ)
* ACCEPTED MAN SONRA MALESTAR
ACCEPTED MANUSCRIPT
A-II
AT1R
ÎN
MASR
JAK
ERK
Ca2+
PKA
STAT
nucleus
A-II
Ang1-7
AT1R
W
MASR
JAK
1
ERK
Ca2+
PKA
STAT
nucleus
ACCEPTED MANGE