Peroxisome proliferator-activated receptor-y suppresses CYP11B2 expression and aldosterone production
Akira Uruno1, Ken Matsuda2, Naoya Noguchi1, Takeo Yoshikawa1, Masataka Kudo2, Fumitoshi Satoh2, William E Rainey4, Xiao-Gang Hui3, Jun-ichi Akahira3,
Yasuhiro Nakamura3, Hironobu Sasano3, Hiroshi Okamoto1, Sadayoshi Ito2 and Akira Sugawara1
1Department of Advanced Biological Sciences for Regeneration, 2Division of Nephrology, Endocrinology and Vascular Medicine and 3Department of Pathology, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
4Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912, USA
(Correspondence should be addressed to A Sugawara; Email: akiras2i@med.tohoku.ac.jp)
Abstract
Peroxisome proliferator-activated receptor-Y (PPARY) is a nuclear receptor for the antidiabetic agent thiazolidinedione, which exerts various physiological activities, independent of lowering blood glucose. However, the role of PPARY in aldosterone production has not been clarified. The objective of this study was to investigate the effect of PPARY on aldosterone synthase gene (CYP11B2) expression and aldosterone production. Localization of PPARY expression in normal adrenal cortex was determined by immunohistochemistry. Aldosterone production and CYP11B2 expression levels were determined using human adrenocortical carcinoma H295R cells. Pioglitazone suppressed angiotensin II-induced aldosterone secretion and CYP11B2 expression. PPARY was expressed in zona glomerulosa in human normal adrenal gland. PPARY overexpression enhanced pioglitazone-mediated CYP11B2 transrepression. The pioglitazone-mediated suppression of aldosterone secretion and CYP11B2 expression were canceled by PPARY L466A/E469A mutant. Pioglitazone also suppressed potassium-mediated CYP11B2 induction, but not N6-2’-O- dibutyladenosine-3’,5’-cyclic monophosphate stimulation. Rosiglitazone and GW1929 also suppressed CYP11B2 transactivation. Mutation analysis revealed that the Ad1/CRE element in CYP11B2 5’-flanking region was responsible for the pioglitazone-mediated transrepression. Pioglitazone suppressed ionomycin and a truncated constitutively active form Ca2+/calmodulin-dependent kinase I (CaMKI)-mediated CYP11B2 transcriptional activation. A CaMK inhibitor KN-93 attenuated pioglitazone-mediated CYP11B2 transrepression. PPARY suppresses CYP11B2 expression and aldosterone secretion.
Journal of Molecular Endocrinology (2011) 46, 37-49
Introduction
Nuclear receptor peroxisome proliferator-activated receptor-y (PPARY), which is targeted by the anti- diabetic agent thiazolidinedione (TZD), is abundantly expressed in adipose tissue and acts as a key regulator for adipocyte differentiation. PPARY also is expressed in various tissues involved in metabolic functions includ- ing skeletal muscle, liver, and pancreas (Heikkinen et al. 2007).
Additionally, roles of PPARY in cardiovascular disorders including atherosclerosis/hypertension have been reported. PPAR agonists prevent macro- vascular events in type 2 diabetic patients, independent of glycemic control (Dormandy et al. 2005). PPARY agonists also prevent hypertension in high-fat-fed Zuker rats (Walker et al. 1999) and renin/angiotensi- nogen double-transgenic mice (Ryan et al. 2004). Moreover, the human PPARY dominant negative
mutant induces hypertension and metabolic dysfunc- tions (Barroso et al. 1999), and vascular smooth muscle cell (VSMC)-specific PPARy dominant negative mutant transgenic mice develop impaired vasodilation and hypertension (Sugawara et al. 2001). We have previously shown that PPARy agonists suppress angiotensin II (AII) type 1 receptor (Halabi et al. 2008) and thromboxane receptor (Sugawara et al. 2002) in VSMCs as well as macrophage activation (Jiang et al. 1998, Ricote et al. 1998) and thromboxane synthase expression in macrophages (Ikeda et al. 2000). Thus, VSMCs and macrophages are important targets of PPARY against atherosclerosis/hypertension. Additionally, some PPARY agonist is reported to decrease AII-induced plasma aldosterone levels in Sprague-Dawley rats (Diep et al. 2002).
Aldosterone is an important factor in the pro- gression of hypertension and vascular damage. Although adrenalectomy ameliorates vascular injury
DOI: 10.1677/JME-10-0088 Online version via http://www.endocrinology-journals.org
in AII/salt-treated rats, independent of systemic blood pressure, administration of aldosterone abolishes the adrenalectomy effects (Rocha et al. 2000, 2002). PPARY is shown to affect several aldosterone regulatory factors including an increase in renin expression (Todorov et al. 2007) and body fluid volume by sodium absorption from the collecting ducts in the kidney (Guan et al. 2005), and a decrease in AII secretion from adipocytes (Harte et al. 2005). Despite the fact that some PPARY agonists decrease plasma aldoster- one levels in rats (Diep et al. 2002), direct effects of PPARY on aldosterone secretion in the adrenal gland are unknown.
Steroid hormone biosynthesis is rapidly regulated by the translocation of substrate cholesterol from the outer mitochondrial membrane to the inside of mitochondria (Rainey et al. 2004). In contrast, chronic aldosterone secretion is limited by the expression of the aldosterone synthase gene, CYP11B2. In this study, we have investigated the roles of PPARY on CYP11B2 expression/aldosterone secretion using adrenocortical H295R cells.
Materials and methods
Reagents
Pioglitazone, rosiglitazone, GW1929, and GW9662 were purchased from Alexis Biochemicals (Farmingdale, NY, USA). Human AII and N6-2’-O-dibutyladenosine-3’, 5’-cyclic monophosphate (dbcAMP) were purchased from Sigma. KN-93 was purchased from Calbiochem (La Jolla, CA, USA). HX630 was kindly provided by Dr Kagechika (Tokyo Medical and Dental University).
Plasmids
The subcloned chimeric constructs containing the human CYP11B2 genomic DNA and luciferase cDNA (pGL3-basic, Promega) were used for the transient transfection studies: - 1521/+2-luc (harboring the CYP11B2 5’-flanking region from -1521 to +2 relative to the transcription start site upstream of the luciferase cDNA in pGL3-basic); - 747/+2-luc; - 135/+2-luc; -65/+2-luc. ß-Galactosidase control plasmid in pCMV (pCMV-ß-gal) was purchased from Clontech. Murine PPARy1 expression vector (pCMX-PPARY1) was kindly provided by Dr Umesono (Kyoto University). Truncated human Ca2+ /calmodulin-dependent kinase I (CaMKI, residues 1-295; Sun et al. 1996) and murine Nur-related factor 1 (NURR1) cDNA were cloned by PCR from normal human dermal fibroblast or murine pituitary AtT20 cell RNA and cloned into the pcDNA3 expression vector (Invitrogen; CaMKI-295-pcDNA3 and NURR1-pcDNA3). Several vectors were mutated
using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA); activation function domain 2 (AF-2 domain) in pCMX-PPARy1 from L466 to A and E469 to A (pCMX-PPAR1 L466A/E469A); Ad5 element in -1521/+2-luc from 5’-CTCCAG- CCTTGACCTT-3’ to 5’-CTCCAGCCTTGAtaTc-3’ (-1521/+2-luc-Ad5-mut); Ad1/CRE element in -1521/+2-luc from 5’-TGACGTGA-3’ to 5’-gGta- ccGA-3’ (-1521/+2-luc-Ad1/CRE-mut; Bassett et al. 2004). Ad1/CRE element of CYP11B2 gene promoter (CAGTTCTCCCATGACGTGATATGTTTCC) was inser- ted into the upstream of pGL3-promoter vector (Promega; Ad1/CRE-SV-Luc).
Cell culture
H295R cells were grown with 1:1 mixture of DMEM and Ham’s F12 medium supplemented with 10% fetal bovine serum (FBS), insulin-transferrin-selenium-G supplements (Invitrogen), 1.25 mg/ml BSA (Sigma), 5.35 µg/ml linoleic acid (Sigma), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured in a humidified incubator at 37 ℃ with 5% CO2. SW-13 cells were provided by Health Science Research Resources Bank, and were grown in Leibovitz’s L-15 supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified incubator at 37 °℃ with air.
RNA preparation and quantitative reverse transcription-PCR
Human total RNAs from normal heart (pooled from three male Caucasians, aged 30-39; cause of death: trauma), liver (from a 51-year-old male Caucasian; cause of death: sudden death), kidney (from a 40-year- old female Caucasian; cause of death: sudden death), skeletal muscle (pooled from two male/female Cauca- sians, aged 43 and 46; cause of death: sudden death), and adrenal gland (pooled from 62 male/female Caucasians, aged 15-61; cause of death: sudden death) were obtained from Clontech. When H295R cells were grown to 80% confluence in six-multiwell plates, they were exposed to pioglitazone for 48-72 h in DMEM supplemented with 1% stripped FBS. Then the cells were treated with AII, KCl, or dbcAMP. H295R and SW-13 cell total RNA were extracted using TaKaRa FastPure RNA kit (Takara Bio, Ohtsu, Japan) according to the manufacturer’s instructions. Total RNAs were subjected to reverse transcription reaction using PrimeScript Reverse Transcriptase (Takara Bio) with random 6 mer and oligo dT primers according to the manufacturer’s instructions. Thereafter, obtained templates were used for quantitative real-time PCR (95 ℃, 3 min for 1 cycle; 95 ℃, 15 s; 60 ℃, 10 s; 72 ℃,
20 s for 40 cycles) with iQ Supermix (for CYP11B2) or iQ SYBR Green Supermix (for others; Bio-Rad) by DNA Engine thermal cycler attached to Chromo4 detector (Bio-Rad). The following primer and TaqMan probe sequences were used: PPARY (forward, 5’-GACCTGAA- ACTTCAAGAGTACC-3’, reverse, 5’-TGAGGCTTAT- TGTAGAGCTGAG-3’), CYP11B2 (forward, 5’-GGCAG- AGGCAGAGATGCTG-3’, reverse, 5’-CTTGAGTTAGT- GTCTCCACCAGGA-3’, probe, 5’-CTGCACCACGTG- CTGAAGCACT-3’), ß-actin (forward, 5’-CCAACCGCG- AGAAGATGACC-3’, reverse, 5’-CCAGAGGCGTACAGG- GATAG-3’), calmodulin (CaM) 1 (forward, 5’-AACAGAA- GCTGAATTGCAGG-3’, reverse, 5’-AATTCGGGGAAGT- CAATGG-3’), CaM2 (forward,5’-GATGAAATGATCAGG- GAAGC-3’, reverse, 5’-CAAGGTCTTCACTTTGCTGT- C-3’), CaM3 (forward, 5’-GATGGCCAGAAAGATGAA- GG-3’, reverse, 5’-TGATGTAGCCATTCCCATCC-3’), Ca2+/CaMK I (sense, 5’-AAGGCAGCATGGAGAATG- AG-3’, reverse, 5’-CTACAATGTTGGGGTGCTTG-3’), and CaMKIV (forward, 5’-TGCTGCAGATGCCGTTAA- AC-3’, reverse, 5’-AGATCACGATGGACAATCCC-3’).
Immunohistochemistry
Immunohistochemistry of normal human adrenal cortex from autopsy files of Tohoku University Hospital was performed by the streptavidin-biotin amplification method using Histofine kit (Nichirei, Tokyo, Japan). Antigen retrieval was performed by heating the slides in an autoclave at 120 °℃ for 5 min in citric acid buffer (2 mM citric acid and 9 mM trisodium citrate dehydrate (pH 6-0)). Rabbit polyclonal antibody raised against PPARY was as previously described (Sato et al. 2004). The primary antibody was diluted at 1:3000, and the antigen-antibody complex was visualized with 3,3’-diaminobenzidine (DAB) solution (1 mM DAB, 50 mM Tris-HCl buffer (pH 7.6), and 0-006% H2O2). As a negative control, normal mouse or rabbit IgG was used instead of the primary antibody. Some specimens were stained with hematoxylin. Peptide pre-absorption of the antibody was performed as described previously (Sato et al. 2004).
Protein preparation and western immunoblot analysis
Nuclear protein was prepared from H295R cells using Nuclear Extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. The denatured samples were separated on an SDS-poly- acrylamide gel and transferred onto polyvinylidene fluoride (PVDF) membrane (Bio-Rad; Uruno et al. 2004, 2005, 2008). The membranes were then blocked with 5% nonfat dry milk and probed with primary antibody for PPARY (Sato et al. 2004; diluted at 1:1000), and HRP-conjugated antibody (GE Healthcare,
Waukesha, WI, USA). The bands were visualized with ECL-plus reagent (GE Healthcare). Pre-absorption test was performed for PPARy using the blocking peptide.
Measurement of aldosterone concentration
When H295R cells were grown to confluence in 24-multiwell plates, they were exposed to pioglitazone for 48-72 h in DMEM supplemented with 1% stripped FBS. AII was then added to the media at a concen- tration of 100 nmol/l, and the cells were incubated for 2 h. Next, the media were freshly changed, and the cells were further incubated in the presence of pioglitazone and AII for 2 h. Aldosterone concen- trations of the media were thereafter measured by Aldosterone EIA kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions.
Transient transfection and luciferase assay
When H295R cells were grown to 80% confluence in 24-multiwell plates, they were transiently transfected with 200 ng luciferase-reporter plasmids and 100 ng pCMV-B-gal using Lipofectamine LTX and Plus reagent (Invitrogen) for 48 h according to the manufacturer’s instructions. In some experiments, expression vectors including pCMX-PPARy1 (100 ng, if not otherwise specified), pCMX-PPARY1 L466A/E469A (100 ng), or pcDNA3-CaMKI-295 (200 ng) also were transfected. The media were changed to DMEM supplemented with 1% charcoal/resin-treated (stripped) FBS, and the cells were incubated with or without PPARY agonists for the indicated times. Then the cells were treated with AII (100 or 200 nmol/l), KCl (10-4 mmol/1, 16 mEq/1 of K+), dbcAMP (1 mmol/1), or ionomycin (1 µmol/l). After appropriated treat- ments, they were washed with PBS, and the cell extracts were prepared using Glo Lysis buffer (Promega). Luciferase activity was measured using Bright-Glo reagents (Promega), and ß-galactosidase activity was simultaneously measured. Data were normalized by B-galactosidase activity.
Measurement of intracellular calcium
H295R cells (4×104) were seeded into 96-well plates, and the cells were incubated for 24 h. They were transiently transfected with 20 ng pCMX-PPARY1 using Lipofectamine LTX and Plus reagent (Invitrogen) for 48 h, then were exposed to 3 umol/l pioglitazone in DMEM supplemented with 1% strriped FBS for 24 h. Thereafter, the cells were loaded with Fluo4-AM (Dojindo, Kumamoto, Japan; 5 µg/ml) in the presence of 1.25 mmol/1 probenecid (Dojindo) and 0-04% Pluronic F-12 (Dojindo) for 1 h.
They were then washed with PBS, and the recording medium containing 1.25 mmol/l probenecid, and AII (100 nmol/l) or KCl (40 mmol/l) was added to the media. The change of intracellular calcium was determined by fluorescent intensity (excitation at 485 nm, emission at 535 nm).
Statistical analysis
All data are presented as mean ±S.E.M. Statistical analyses were performed with ANOVA followed by Fisher’s least significant difference post hoc test.
Results
PPARY expression in normal adrenal gland and H295R cells
We first examined PPARY expression levels in the normal human adrenal gland and H295R cells. PPARY mRNA expression in the adrenal gland was lower than that in the kidney and heart, while higher than that in the liver, skeletal muscle, and H295R cells, and was rarely expressed in SW-13 adrenocortical carcinoma cells (Fig. 1A). Next, we determined the localization of PPARY in the normal human adrenal cortex by immunohistochemistry. As shown in Fig. 1B (left panel), PPARY was predominantly expressed in zona glomerulosa, while little in zona fasciculata and reticularis. The nuclear staining of human adrenal cortex with the PPARY antibody was diminished with pre-absorption by its antigen peptide (Fig. 1B, right panel). Western blot analysis using anti-PPARY antibody also revealed the expression of PPARY protein in H295R cells nuclear protein, the band of which was abrogated by pre-absorption with the blocking peptide (Fig. 1C).
Effects of pioglitazone on aldosterone secretion/ CYP11B2 expression
We next examined the effect of a TZD pioglitazone on aldosterone secretion/ CYP11B2 expression. AII treat- ment increased aldosterone secretion from H295R cells into the media, and pioglitazone co-treatment suppressed the AII-induced aldosterone secretion (Fig. 1D). To evaluate the effect of CYP11B2 gene- mediated regulation of aldosterone secretion, but not steroidogenic acute regulatory protein-mediated acute phase secretion, the media were changed once 2 h after AII stimulation. AII treatment also induced CYP11B2 mRNA as well as gene transcriptional activity in H295R cells, and pioglitazone co-treatment reduced the AII-mediated inductions (Fig. 1E and F).
Effects of PPARY on aldosterone secretion/CYP11B2 expression
We next examined the effect of PPARY overexpression on CYP11B2 transcriptional activity using previously described promoter/reporter constructs (Bassett et al. 2004). As shown in Fig. 2A, PPARY overexpression attenuated the AII-mediated CYP11B2 transactivation both in the absence or presence of pioglitazone in a dose-dependent manner. We next examined the effects of PPARY agonists/antagonist on CYP11B2 transcrip- tional activity. As shown in Fig. 2B, the AII-mediated CYP11B transactivation was also suppressed by rosiglitazone and GW1929 to a similar extent as pioglitazone. Moreover, the pioglitazone-mediated CYP11B transrepression was abrogated by a PPARY antagonist, GW9662 (Fig. 2C).
We next investigated the effect of PPARY AF-2 domain mutation on CYP11B2 transcriptional activity/ aldosterone secretion. In the presence of wild-type PPARY, pioglitazone suppressed both the AII-mediated CYP11B2 transactivation (Fig. 2D) and aldosterone secretion (Fig. 2E), while the pioglitazone-mediated suppression was canceled in the presence of PPARY L466A/E469A mutant. Next, we examined the role of retinoid X receptor (RXR) on CYP11B2 tran- scriptional activity. As shown in Fig. 2F, an RXR agonist HX630 also suppressed the AII-induced CYP11B2 transcriptional activity and additively decreased the pioglitazone-mediated transrepression. These data suggest that pioglitazone suppressed CYP11B2 transcriptional activity and aldosterone secretion through PPARY.
Pharmacological properties of pioglitazone on CYP11B2 transcriptional activity
We next determined the pharmacological properties of pioglitazone on the AII-induced CYP11B2 tran- scriptional activity. Time-course analysis revealed that pioglitazone suppressed the AII-mediated CYP11B2 transactivation from 0-5 h, with maximal suppression at 24 h (Fig. 3A). Pioglitazone suppressed the AII- mediated CYP11B2 transactivation in a concentration- dependent manner (Fig. 3B).
We next examined whether pioglitazone suppresses potassium- and cAMP-mediated CYP11B2 expression. In addition to AII, potassium and the cAMP analogue dbcAMP increased both CYP11B2 transcriptional activity (Fig. 3C) and mRNA expression (Fig. 3D). Pioglitazone suppressed not only the AII- but also the potassium-mediated upregulation of CYP11B2 tran- scriptional activity (Fig. 3C) and mRNA expression (Fig. 3D), while it did not suppress the dbcAMP- mediated stimulation.
A
120
PPARy mRNA level
100
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60
40
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Heart
Adrenal
Liver
Skeletal
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75
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35
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Aldosterone secretion
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Role of Ad1/CRE element on pioglitazone-mediated CYP11B2 transrepression
To explore the mechanism(s) for the pioglitazone- mediated CYP11B2 transrepression, we next examined the transcriptional activity of the CYP11B2 5’-flanking
region using its deletion mutants. Both the AII-induced (Fig. 4A) and potassium-induced (Fig. 4B) CYP11B2 transactivation and the pioglitazone-mediated sup- pression were observed in -1521/+2-luc, -747/ +2-luc, and -135/+2-luc. However, the AII- and potassium-mediated CYP11B2 transactivation and the
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pioglitazone-mediated transrepression were not observed in -65/+2-luc, which lacks both the Ad5 and Ad1/CRE elements. We thereafter examined the transcriptional activity of the Ad5 and Ad1/CRE elements in CYP11B2 5’-flanking region using their mutants. As shown in Fig. 4C, mutation of the Ad5 element (-1521/+2-luc-Ad5-mut) partially abrogated both the AII- and potassium-induced CYP11B2 transcriptional activity comparing to the wild-type -1521/+2-luc, but did not cancel the pioglitazone- mediated CYP11B2 transrepression (AII, 40% sup- pression in wild-type versus 34% suppression in Ad5-mut; potassium, 42% suppression in wild-type versus 46% suppression in Ad5-mut). On the other hand, mutation of the Ad1/CRE element (-1521/ +2-luc-Ad1/CRE-mut) strongly reduced both the AII- and potassium-induced CYP11B2 transcriptional activity, and abrogated the pioglitazone-mediated CYP11B2 transrepression (AII, 40% suppression in wild-type versus 13% suppression in Ad1/CRE-mut; potassium, 42% suppression in wild-type versus 19%
suppression in Ad1/CRE-mut). AII also increased the transcriptional activity of Ad1/CRE-SV-Luc, and piogli- tazone suppressed the AII-mediated induction (Fig. 4D). AII or pioglitazone did not alter the transcriptional activity of pGL3-promoter (Fig. 4D). These data suggest that the Ad1/CRE element is probably responsible for the pioglitazone-mediated CYP11B2 transrepression. We next examined the effect of pioglitazone on the AII-induced expression of nerve growth factor-induced clone B (NGFIB) and NURR1, both of which are nuclear orphan receptors binding to the Ad5 element and positively regulate CYP11B2 transcriptional activity. Although AII increased both NGFIB and NURR1 mRNA expression, pioglitazone did not affect their expression (Supplementary Figure 1, see section on supplementary data given at the end of this article), indicating their lesser involvement in the transrepression. Consistent with the notion, pio- glitazone had little effect on the NURR1-mediated CYP11B2 transactivation (Fig. 4E).
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Role of pioglitazone on calcium signaling
Since the Ad1/CRE element is essential for the AII- and potassium-mediated CYP11B2 transactivation via the Ca2+-CaM-CaMKI signaling pathway (Clyne et al. 1997, Pezzi et al. 1997, Condon et al. 2002), we therefore investigated the role of pioglitazone on this pathway. As shown in Fig. 5A and B, both AII and potassium increased intracellular calcium ion concentration in H295R cells. Although pioglitazone did not affect the AII-mediated intracellular Ca2+ increase, it enhanced the potassium-mediated one. We next examined the effect of pioglitazone on the Ca2+-mediated CYP11B2 transcriptional activity. As shown in Fig. 5C, ionomycin increased CYP11B2 transcriptional activity, while pioglitazone diminished the increase. We next exam- ined the effects of pioglitazone on CaMs mRNA expre- ssion. As shown in Fig. 5D, the mRNA level of CaM3 was significantly higher than that of CaM1 and CaM2. Pioglitazone weakly increased the mRNA expression
of both CaM1 and CaM2, while it did not affect that of CaM3. These data indicate that pioglitazone partially enhances, but not suppresses, the Ca2+-CaM signals.
Role of CaMK on pioglitazone-mediated CYP11B2 transrepression
Since CaMKI is known to stimulate CYP11B2 transcrip- tional activity through the Ad1/CRE element down- stream of the Ca2+-CaM signals (Condon et al. 2002), we next examined the role of CaMKI on the pioglitazone-mediated CYP11B2 transrepression. In addition to CaMKI, CaMKIV is also reported to increase CYP11B2 transcriptional activity weakly (Condon et al. 2002). Therefore, we evaluated the expression level of CaMKI and IV. As shown in Fig. 6A, the mRNA expression of CaMKI in H295R cells was much higher than that of CaMKIV. Pioglitazone did not affect their mRNA expression level. Overexpression of a truncated
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WT: 5’ -CTCCAGCCTTGACCTT-3’
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Vehicle
Pio
constitutively active form of CaMKI (CaMKI-295) increased CYP11B2 transcriptional activity (Fig. 6B), and pioglitazone completely abrogated the increase. As shown in Fig. 6C, the AII- or potassium-mediated CYP11B2 transactivation was not observed in the presence of a CaMK inhibitor KN-93. Additionally, the pioglitazone-mediated transrepression was also can- celed in the presence of KN-93. These data indicate that pioglitazone suppresses CYP11B2 transactivation probably via the suppression of signal transduction from CaMKI to CYP11B2 promoter.
Discussion
The present data demonstrate that PPARY decreases CYP11B2 expression/aldosterone secretion through the suppression of CaMKI function that stimulates the Ad1/CRE element in CYP11B2 5’-flanking region in the human adrenal H295R cells.
Expression of PPARY in mouse (Kliewer et al. 1994) and human (Ferruzzi et al. 2005) adrenal gland has previously been demonstrated. Although PPARY agonists are reported to suppress androgen
A
2000
1750
Basal
2000
Fluorescent change
1500
AII
1750
T
T
1250
Pio+AII
Fluorescent change
1500
1000
1250
750
1000
500
750
250
500
0
250
-250
0
T
-60
0
60
120
180
240
300
360
Basal
AII
Pio+AII
Time (s)
B
3500
3500
3000
†
Fluorescent change
Basal
2500
KCl
T
2000
Pio+KCl
Fluorescent change
3000
2500
T
1500
2000
1000
1500
500
1000
0
500
-500
-60
0
60
120
180
240
300
360
0
Basal
KCl
Pio+KCl
Time (s)
C
D
300
200
**
T
Relative luciferase activity
250
†
*
150
CaMs mRNA levels
T
200
-
T
100
150
100
T
50
50
0
0
Control
Iono
Pio+
Pio
+
+
+
–
–
–
Iono
CaM1
CaM2
CaM3
production and expression of 17a-hydroxylase (CYP17) and 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2) in H295R cells (Kempna et al. 2007), as well as inhibit adrenal cancer cell line proliferation (Betz et al. 2005, Ferruzzi et al. 2005), these effects are shown to be independent of PPARY. Thus, the function of PPARY in adrenal gland still remains uncertain. The PPARY expression level in the normal human adrenal gland is reported to be lower than that in carcinoma as determined by immunohistochemistry
(Ferruzzi et al. 2005). In this study, we also demon- strated that the expression level of PPARY in the human adrenal gland was higher than that in the liver and skeletal muscle (Fig. 1A). Additionally, immuno- histochemistry of the normal human adrenal cortex revealed that PPARY was predominantly localized in the zona glomerulosa (Fig. 1B). We therefore explored the roles of PPARY in aldosterone pro- duction and CYP11B2 expression in the human adrenal cells.
A
B
160
120
Relative luciferase activity
T
140
CaMK mRNA levels
100
T
120
†
100
T
80
60
80
60
40
40
20
20
0
0
Pio
+
+
CaMKI-295
+
+
+
+
–
–
–
CaMKI
CaMKIV
Pio
+
+
–
–
-1502/+2-luc
pGL3-Basic
C
350
Relative luciferase activity
300
T
250
200
†
†
T
150
100
-
T
50
0
AII
KCl
AII
KCl
+
+
+
+
+
+
+
+
–
–
–
–
Pio
+
+
+
+
–
–
–
–
–
–
–
–
KN-93 (-)
KN-93 (+)
Overexpression of PPARY reduced the AII-induced CYP11B2 transcriptional activity as well as enhanced the suppressive effect of pioglitazone (Fig. 2A). Addition- ally, other PPARY agonists rosiglitazone and GW1992 also suppressed the AII-induced CYP11B2 transcrip- tional activity (Fig. 2B). The effect of pioglitazone on CYP11B2 transcriptional activity was canceled by PPARY antagonist GW9662 (Fig. 2C). These data therefore indicate that the pioglitazone-mediated suppression of aldosterone secretion and CYP11B2 expression are dependent on PPARY.
CaMKI is reported to be abundantly expressed in the human adrenal zona glomerulosa, and stimulate CYP11B2 transcriptional activity via the Ad1/CRE element in H295R cells more potently than CaMKIV (Condon et al. 2002). Additionally, we demonstrated in this study that the expression level of CaMKI in H295R cells was much higher than that of CaMKIV (Fig. 6A). Therefore, CaMKI is most likely a very important regulator for the Ad1/CRE element in the CYP11B2 promoter. The Ad1/CRE element in the CYP11B2 5’-flanking region is bound by cAMP
response-element-binding protein (CREB), activating transcriptional factor (ATF) 1, and ATF2 (Bassett et al. 2000). Interestingly, CREB and ATF1 are reported to be stimulated by CaMKI (Sun et al. 1996). Additionally, CREB is known to cooperate with coactivators inclu- ding steroid receptor coactivator-1 (SRC-1) and p300/ CREB-binding protein (CBP) in the AII-induced gene expression (Sahar et al. 2007). Moreover, the pioglitazone-mediated CYP11B2 transrepression was canceled by PPARY1 L466A/E469A mutant (Fig. 2D) whose function for the ligand-dependent recruitment of coactivators including SRC-1 and CBP is impaired (Gurnell et al. 2000). Therefore, these coactivators may be involved in the PPARy-mediated suppression of CYP11B2 expression.
The orphan nuclear receptors NGFIB, NURR1, and steroidogenic factor-1 (SF-1) bind to the Ad5 element in the CYP11B2 5’-flanking region. NGFIB and NURR1 are known to be upregulated by AII or KCl and positively regulate CYP11B2 expression, while SF-1 negatively regulates CYP11B2 expression (Bassett et al. 2002). Since the AII/KCI-mediated upregulation of NURR1 is mediated via CaMK, the Ad5 element may possibly be influenced by CaMK (Bassett et al. 2004). However, the AII-induced NGFIB and NURR1 expressions were not suppressed by pioglitazone (Supplementary Figure 1), and the pioglitazone- mediated transrepression was not canceled by mutation of the Ad5 element in CYP11B2 5’-flanking region (Fig. 4C). Therefore, the Ad5 element may not contribute to the pioglitazone-mediated CYP11B2 transrepression.
PPARY1 L466A/E469A double mutant (L468A/ E471A in humans) is known to act similarly to PPARY1 L466A single mutant (Park et al. 2003), but that in knockin mice develops hypertension (Freedman et al. 2005). Additionally, the PPARY1 P467L natural mutant in the AF-2 domain (P465L in mice) induces insulin resistance and hypertension in humans (Barroso et al. 1999). Since mice expressing human PPARY1 P467L mutant in VSMCs demonstrate vascular constriction and hypertension (Halabi et al. 2008), direct vascular effect may contribute to the progression of hypertension in this mutant. In the presence of PPARY1 L466A/E469A mutant, impairment of ligand- mediated suppression of aldosterone secretion was observed (Fig. 2E). Therefore, dysregulation of aldosterone production may be involved in the etiology of the PPARY mutant-induced hypertension.
HX630 augmented the pioglitazone-mediated CYP11B2 transrepression (Fig. 2F). PPARY hetero- dimerizes with RXR (Gearing et al. 1993), and binds to and activates PPAR-response elements (PPRE) in the promoter of target genes (IJpenberg et al. 1997). However, the Ad1/CRE element, the responsive element of the pioglitazone-mediated CYP11B2
transrepression, does not contain any consensus PPRE referred to as direct repeat 1 element. Therefore, PPARY may suppress CYP11B2 transcriptional activity as a heterodimer with RXR without direct biding to the Ad1/CRE element.
In this study, we used pioglitazone at concen- trations of 3 or 10 umol/l. The experimental concentrations of TZDs are above 10 umol/l, but TZDs exert some effects, which are PPARy-indepen- dent in NIC-h295 cells at high concentrations (10 µmol/l; Betz et al. 2005). Plasma concentration of pioglitazone reaches approximately to 4 umol/1 by 30 mg oral administration in healthy male humans (Takeda Pharmaceuticals, Osaka, Japan). We tested the effects of pioglitazone at 10 umol/l on aldoster- one secretion and CYP11B2 gene regulation that may include both PPARy-dependent and -indepen- dent effects (Figs 1 and 2A). Therefore, we used pioglitazone at 3 umol/l, which is similar to the human plasma concentration after oral adminis- tration, to exclude PPARY-independent effects of pioglitazone in most experiments. However, in Fig. 2F, we treated the cells with pioglitazone at 10 umol/1 for 24 h to compare with HX630 at the same pharmacological conditions.
Pioglitazone potentially increased dbcAMP-induced CYP11B2 transcriptional activity and mRNA expression (Fig. 3C and D). Pioglitazone may increase the dbcAMP-mediated activation of CREB/ATF1, and further studies are needed to be examined.
In conclusion, our observation that PPARY sup- presses CYP11B2 expression/aldosterone secretion through CaMK may account for the suppressive effects of PPARY on vascular events associated with athero- sclerosis and hypertension.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/ 10.1677/JME-10-0088.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to AU, no. 17790545; to AS, no. 16590898; and to SI, no. 17390245); grants-in-aid from the Ministry of Health, Labor, and Welfare of Japan (to AS); and research grants from Smoking Research Foundation (to AS).
Acknowledgements
We thank Dr T Suzuki (Tohoku University) for providing H295R cells and Dr H Kagechika (Tokyo Medical and Dental University) for providing HX630.
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Received in final form 31 October 2010 Accepted 24 November 2010 Made available online as an Accepted Preprint 24 November 2010