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Differential regulation and roles of urocortins in human adrenal H295R cells
Regulatory Peptides 162 (2010) 18–25
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r e g p e p
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Differential regulation and roles of urocortins in human adrenal H295R cells Kazunori Kageyama ⁎, Komaki Hanada, Toshihiro Suda Department of Endocrinology and Metabolism, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
a r t i c l e
i n f o
Article history: Received 23 September 2009 Received in revised form 14 January 2010 Accepted 11 February 2010 Available online 19 February 2010 Keywords: Corticotropin-releasing factor Urocortin Cortisol Aldosterone Adrenal gland
a b s t r a c t Three urocortins (Ucns) are known as members of the corticotropin-releasing factor (CRF) family of peptides and serve as natural ligands for CRF receptors. Ucn1 and Ucn3 exhibit potent effects on the adrenal system via the CRF receptors. This study aimed to explore the regulation and roles of Ucns in the adrenal system using human adrenal carcinoma H295R cells, which express Ucn1, Ucn2, Ucn3, CRF receptor type 1 (CRF1 receptor), and CRF receptor type 2a (CRF2a receptor) mRNA. Forskolin, which stimulates adenylate cyclase and then increases intracellular cAMP production, was shown to transiently decrease Ucn1 and Ucn2 mRNA levels, but increase Ucns 1–3 mRNA levels in H295R cells. Steroidogenic acute regulatory protein, Cyp11β1, and Cyp11β2 mRNA levels, and both cortisol and aldosterone secretions were elevated by Ucn1. Cell viability was reduced by both Ucn1 and Ucn3 via the CRF2 receptor in H295R cells. Ucn1 and Ucn3 increased the expression of the cAMP-response element binding protein and extracellular signal-related kinase (ERK) phosphorylations. The ERK and protein kinase A pathways were involved in Ucn3-decreased cell viability. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Three urocortins (Ucns) are known as members of the corticotropin-releasing factor (CRF) family of peptides. Ucn1 is a 40 amino acid peptide cloned from the Edinger–Westphal nucleus [1], while Ucn2 and Ucn3 have been identified in the human genome database and mouse genomic DNA, respectively [2–4]. Ucn1 and Ucn3 are expressed in various human tissues, whereas the presence of Ucn2 has been established in human endometrium or gestational tissues [5,6]. The actions of the CRF family of peptides are mediated by at least two distinct G protein-coupled receptors: CRF receptor type 1 (CRF1 receptor) [7–9] and CRF receptor type 2 (CRF2 receptor) [10–12]. Stimulation of these receptors activates adenylate cyclase to produce cAMP, which then induces physiological effects. Ucn1 binds to both the CRF1 and CRF2 receptors, while Ucn2 and Ucn3 are highly selective for the CRF2 receptor, with little or no affinity for the CRF1 receptor. A consensus cAMP-response element (CRE) site has been shown to mediate the regulation of Ucn1 expression by cAMP in both mouse and human Ucn1 promoters [13]. Raised intracellular cAMP levels induced by forskolin are known to increase Ucn1 mRNA level in human umbilical vein endothelial cells [14]. Angiotensin-II, ACTH, and urotensin II are major candidates for natural ligands in the activation of the cAMP pathway in the adrenal system [15–17]. CRF and Ucns mediate stress responses, cardiovascular function, and immune functions via the CRF receptors [18,19]. The presence of the CRF family of peptides has been reported in the human adrenal
⁎ Corresponding author. Tel.: + 81 172 39 5062; fax: + 81 172 39 5063. E-mail address: [email protected] (K. Kageyama). 0167-0115/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2010.02.006
gland [20]. Specifically, Ucn1, Ucn3, CRF1 receptor, and CRF2 receptor are expressed in the human adrenal gland [21,22], suggesting that the CRF family peptides may play important roles in regulating adrenal functions. Indeed, CRF and Ucn1 stimulate dehydroepiandrosterone sulfate production via the CRF1 receptor in human fetal adrenal cells [23], and Ucn3 inhibits cell proliferation or tumor growth via the CRF2 receptor [24,25]. Although Ucns have potent effects on the adrenal system, their possible roles and regulation have not yet been clearly determined. In the present study, we studied cAMP-dependent changes in the gene expression of Ucns in human adrenal H295R cells with the aim of investigating the effects of Ucns on steroidogenesis and cell proliferation. 2. Materials and methods 2.1. Materials Forskolin, PKA inhibitor 14–22 amide (PKAi), and PD98059 were purchased from Calbiochem (San Diego, CA, USA), and antalarmin from Sigma-Aldrich Corp. (St. Lois, MO). Human Ucn1, Ucn2, Ucn3, and angiotensin-II were purchased from the Peptide Institute (Osaka, Japan). Antisauvagine-30 was synthesized by Asahi Techno Glass (Chiba, Japan). 2.2. Cell cultures H295R cells were obtained from the American Type Culture Collection (Manassas, VA, USA), and incubated in DMEM/F12 containing 2.5% NuSerum (BD Biosciences, Bedford, MA, USA) 1% ITS Premix
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(BD Biosciences) 100 mg/mL streptomycin, and 100 U/mL penicillin at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells were plated at 3 × 103 cells/cm2 seven days before each experiment, and the medium was changed every 48 h. On day 7, cells were treated as indicated below. BE(2)C cells were incubated in DMEM/F12 medium, supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% nonessential amino acid, 100 mg/mL streptomycin, and 100 U/mL penicillin at 37 °C, in a humidified atmosphere of 5% CO2 and 95% air. At the end of each experiment, total cellular RNA or medium was collected and stored at −80 °C until the assay was performed. All treatments were performed in triplicate and repeated three times. 2.3. RNA extraction Cells were incubated with medium alone (control) or with medium containing forskolin, Ucns, and angiotensin-II for the times indicated in the graph. To examine the dose-dependent effects of forskolin or Ucns, cells were incubated for the indicated times with medium alone (control) or with medium containing increasing concentrations of forskolin (0.1–10 µM) or Ucns (1–100 nM). At the end of each experiment, cellular total RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. cDNA was synthesized from the total RNA (0.5 µg) using random hexamers as primers with the SuperScript FirstStrand Synthesis System for reverse transcriptase–polymerase chain reaction (RT–PCR) (Invitrogen Corp., Carlsbad, CA, USA), according to the manufacturer's instructions. 2.4. Reverse transcriptase–polymerase chain reaction (RT–PCR) Polymerase chain reaction (PCR) was carried out in a programmable thermal controller (Bio-Rad, Hercules, CA, USA) with the following oligonucleotide primers: the sequences of the primers employed in this study are summarized in Table 1. The primers were designed according to the publications [6,26]. PCR conditions for Ucn1, CRF1 and CRF2a receptor primers were 1 × (96 °C, 2 min), 35 × (96 °C, 15 sec; 64 °C, 30 sec; 72 °C, 1 min) and 1 × (72 °C, 5 min). CRF and CRF2b receptor primer conditions were 1 × (96 °C, 2 min), 38 × (96 °C, 15 sec; 64 °C, 30 sec; 72 °C, 1 min) and 1 × (72 °C, 5 min); Ucn2 primer conditions were 36 × (94 °C, 40 sec; 55 °C, 40 sec; 72 °C, 40 sec) and 1 × (72 °C, 10 min); Ucn3 primer conditions were 37 × (94 °C, 1 min; 63 °C, 1 min; 72 °C, 1 min) and 1 × (72 °C, 10 min); and GAPDH primer conditions were 1 × (96 °C, 2 min), 22 × (96 °C, 15 sec; 60 °C, 30 sec; 72 °C, 1 min) and 1 × (72 °C, 5 min). Products were separated by electrophoresis on a 1.5% agarose gel containing ethidium bromide. The expected sizes of
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the PCR products for CRF and Ucn1 were 678 bp and 468 bp, respectively, and those for Ucn2 and Ucn3 were 195 bp and 310 bp, respectively. The expected size of the PCR product for the CRF1 receptor was 475 bp, and those for the CRF2a and CRF2b receptors were 233 bp and 248 bp, respectively. The expected size of the PCR product for the GAPDH receptor was 969 bp.
2.5. Real-time reverse transcriptase–polymerase chain reaction (RT–PCR) Cellular total RNA extraction and cDNA synthesis were performed similarly as described earlier. The resulting cDNAs were then subjected to real-time PCR as follows. The expression levels of human Ucns mRNA were evaluated using quantitative real-time PCR based on specific sets of primers and probes (Assays-on-Demand Gene Expression Products, Applied Biosystems, Foster City, CA, USA). GAPDH was used as a housekeeping gene to standardize values, because the GAPDH mRNA levels were not changed in any treatments of these studies. Each reaction consisted of 1×TaqMan universal PCR Master Mix (Applied Biosystems), 1×Assays-on-Demand Gene Expression Products (Hs00175020_m1 for human Ucn1, Hs00264218_s1 for human Ucn2, Hs00846499_s1 for human Ucn3, Hs00264912_m1 for steroidogenic acute regulatory protein (StAR), Hs01596404_m1 for 11β-hydroxylase (Cyp11β1), Hs01597732_m1 for 18-hydroxylase (Cyp11β2), and Hs99999905_m1 for human GAPDH) and 2 µL of cDNA in a total volume of 50 µL using the following parameters on the ABI PRISM 7000 Sequence Detection System (Applied Biosystems): 95 °C for 10 min, 40 cycles at 95 °C for 15 sec and 60 °C for 1 min. The above assays involved specific sets of primers and a TaqMan probe spanning the exon/exon junction and should not, therefore, have been influenced by DNA contamination. Data were collected and recorded by ABI PRISM 7000 SDS Software (Applied Biosystems) and expressed as a function of the threshold cycle (CT). Using the diluted samples, the amplification efficacies for each gene of interest and the housekeeping gene amplimers were found to be identical.
2.6. Cortisol and aldosterone assays Cells were incubated for 6 h with medium alone (control) or with medium containing increasing concentrations of Ucn1 (1–100 nM). To examine the effects of a CRF receptor antagonist on Ucn1- and Ucn3-induced cortisol and aldosterone levels in medium-cultured H295R cells, the cells were pre-incubated with medium containing 100 nM antalarmin, antisauvagine-30 or vehicle for 30 min, and then incubated for 6 h with medium containing 1–100 nM Ucn1 and Ucn3, 10 µM forskolin or vehicle.
Table 1 Oligonucleotide primers used in the RT–PCR. Target
Oligonucleotide sequence
Size, base pairs
Reference
CRF
CRF-F: 5′-AAGGAAGACAACCTCCAGAGAAAGC-3′ CRF-R: 5′-TCCATGAGTTTCCTGTTGCTGTGAG-3′ Ucn1-F: 5′-GTTCCCCAAGGCGTCTTCA-3′ Ucn1-R: 5′-CTTGCCCACCGAGTCGAAT-3′ Ucn2-F: 5′-GTGTCGGCCACTGCTGAGCCTGAGAGA-3′ Ucn2-R: 5′-ATCTGATATGACCTGCATGACAGTGGCT-3′ Ucn3-F: 5′-TGCTGCTCCTGCTGCTGCTC-3′ Ucn3-R: 5′-GTGTCCTGGCGTGGCTTTCCC-3′ CRF1 receptor-F: 5′-CAAACAATGGCTACCGGGAG-3′ CRF1 receptor-R: 5′-ACACCCCAGCCAATGCAGA-3′ CRF2a receptor-F: 5′-GACGCGGCACTGCTCCACAG-3′ CRF2a receptor-R: 5′-GCATTCCGGGTCGTGTTGT-3′ CRF2b receptor-F: 5′-CCCTCACCAACCTCTCAGGTCC-3′ CRF2b receptor-R: 5′-CAGGTCATACTTCCTCTGCTTGTC-3′ GAPDH-F: 5′-GGTCGGAGTCAACGGATTTG-3′ GAPDH-R: 5′-ATGAGGTCCACCACCCTGTT-3′
678
[26]
468
[26]
195
[6]
310
[6]
475
[26]
233
[26]
248
[26]
969
[26]
Ucn1 Ucn2 Ucn3 CRF1 receptor CRF2a receptor CRF2b receptor GAPDH
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The culture medium was collected from the dishes and stored at −80 °C for the measurement of cortisol and aldosterone levels using a cortisol immunoassay (R&D Systems, Minneapolis, MN, USA) and an aldosterone ELISA (DRG, Mountainside, NJ, USA), respectively. All samples from each experiment were determined in the same assay. 2.7. Western blot analysis Cells were pre-incubated with medium containing 100 nM antalarmin, antisauvagine-30 or vehicle for 30 min, and incubated for a further 5 min after 100 nM Ucn1 or 100 nM Ucn3 was added to the medium. After treatment with Ucn1 or Ucn3, cells were washed twice with phosphate buffered saline (PBS) and lysed with Laemmli sample buffer. Cell debris was pelleted by centrifugation, and the supernatant was recovered. Samples were boiled and subjected to electrophoresis on a gradient (4–20%) polyacrylamide gel. Proteins were transferred to a PVDF membrane (Daiichi Kagaku, Tokyo, Japan). After blocking with Detector Block® blocking buffer (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA), the membrane was incubated for 1 h with a rabbit anti-CRE binding protein (CREB), anti-phosphorylated (p)-CREB, anti-extracellular signal-related kinases (ERK), or anti-p-ERK antibodies (Cell Signaling Technology, Beverly, MA, USA), washed with PBS containing 0.05% Tween 20, and incubated with horseradish peroxidase (HRP)-labeled anti-rabbit IgG (Daiichi Kagaku, Japan). Detection was performed using a chemiluminescent substrate Super-signal WestPico (Pierce Chemical Co., Rockford, IL USA), and the membrane was exposed to BioMax film (Eastman Kodak Co., Rochester, NY, USA) followed by
quantitative analysis using NIH image software 1.61. The results are expressed as corrected arbitrary units. 2.8. Cell proliferation assay H295R cells were seeded into 96-well plates at a density of 1 × 104 cells/well, and incubated in 100 µL of culture medium containing 100 nM antalarmin, 100 nM antisauvagine-30, 100 nM PKAi, 10 µM PD98059 or vehicle, and each Ucn or vehicle at 37 °C for 24 h. Cell viability was measured using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). The assay is based on cleavage of the tetrazolium salt WST-8 to formazan product. Absorbance was measured with a test wavelength of 450 nm and a reference wavelength of 630 nm. 2.9. Statistical analysis All values are expressed as the mean ± standard error of the mean (SEM). Statistical analyses of data were performed using one-way analysis of variance (ANOVA), followed by Bonferroni/Dunn post-hoc tests. The level of statistical significance was set at P b 0.05. 3. Results 3.1. Expression of Ucns and CRF receptor mRNA in H295R cells H295R cells were found to express Ucn1, Ucn2, and Ucn3, but not CRF mRNA (Fig. 1). CRF1 and CRF2a receptor mRNAs were also expressed (Fig. 1).
Fig. 1. Expression of Ucn and CRF receptor mRNA in H295R cells. M: marker; +: reverse transcriptase–PCR shown in duplicate; −: negative control for reverse transcriptase–PCR.
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3.2. Effects of forskolin or angiotensin-II on Ucns1–3 mRNA levels in H295R cells H295R cells were incubated with forskolin to determine whether Ucns1–3 mRNA levels were dependent on cAMP. As shown in Fig. 2A and B, real-time PCR analysis revealed that incubation with forskolin transiently decreased, then increased Ucn1 and Ucn2 mRNA levels (ANOVA; P b 0.005). Ucn1 mRNA level fell transiently to 67% of the
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control value within 2 h of the addition of 10 µM forskolin, followed by an increase to 224% of the control level within 6 h (Fig. 2A). Ucn2 mRNA level also fell transiently to 51% of the control value within 2 h of the addition of 10 µM forskolin, followed by a gradual increase to 173% of the control level within 24 h, in a dose-dependent manner (Fig. 2A and B). Forskolin increased Ucn3 mRNA level within 2 h, in a dose-dependent manner (ANOVA; P b 0.005) (Fig. 2A and B). Ucn3 mRNA level was elevated transiently to 171% of the control value within 2 h of the addition of 10 µM forskolin, and then recovered within 24 h (Fig. 2A). Angiotensin-II (100 nM) also increased Ucn1, Ucn2, and Ucn3 mRNA levels within 6 h (Fig. 2C). 3.3. Effects of Ucn1 and Ucn3 on mRNA levels of genes encoding enzymes involved in steroid hormone biosynthesis in H295R cells To determine whether Ucns induce mRNA levels of enzymes involved in steroid hormone biosynthesis, such as StAR, a crucial protein involved in steroidogenesis, Cyp11β1, a gene encoding 11βhydroxylase regulated by ACTH, and Cyp11β2, a gene encoding aldosterone synthase, H295R cells were incubated with Ucn1 or Ucn3. StAR, Cyp11β1, and Cyp11β2 mRNA levels were elevated transiently from 2 h to 6 h after the addition of 100 nM Ucn1, but not Ucn3 (Fig. 3A, C, and D). Ucn1 increased StAR mRNA level in a dose-dependent manner (ANOVA; P b 0.005, Fig. 3B). 3.4. Effects of Ucn1 and Ucn3 on cortisol level in medium from cultured H295R cells To determine whether Ucns induce cortisol secretion, H295R cells were incubated with Ucn1 or Ucn3. Incubation with Ucn1 increased cortisol level in a dose-dependent manner (ANOVA; P b 0.05, Fig. 4A). However, there was no significant increase following incubation with Ucn3. Pre-treatment with antalarmin, a selective CRF1 receptor antagonist, but not antisauvagin-30, a selective CRF2 receptor antagonist, blocked Ucn1-induced cortisol secretion (Fig. 4B). 3.5. Effects of Ucn1 and Ucn3 on aldosterone level in medium from cultured H295R cells We next determined whether Ucns induce aldosterone secretion in H295R cells. Incubation with Ucn1 increased aldosterone level in a dose-dependent manner (ANOVA; P b 0.05, Fig. 4C). However, there was no significant increase after incubation with Ucn3. Pretreatment with antalarmin, a selective CRF1 receptor antagonist, but not antisauvagin-30, a selective CRF2 receptor antagonist, blocked Ucn1-induced aldosterone secretion (Fig. 4D). 3.6. Effects of Ucn1 and Ucn3 on CREB or ERK phosphorylation
Fig. 2. Effects of forskolin or angiotensin-II on Ucns1–3 mRNA levels in H295R cells. Cells were treated in triplicate and the average of three independent experiments is shown. *P b 0.05 (compared with control). (A) Time-dependent changes in forskolininduced mRNA levels of each Ucn. Cells were incubated with medium alone (control) or with medium containing 10 µM forskolin for the times indicated in the graph. (B) Dosedependent effects of forskolin on each Ucn mRNA level. Cells were incubated for the indicated times with medium alone (control) or with medium containing increasing concentrations of forskolin (0.1–10 µM). (C) Time-dependent changes in angiotensinII-induced mRNA levels of each Ucn. Cells were incubated with medium alone (control) or with medium containing 100 nM angiotensin-II for the times indicated in the graph.
We examined H295R cells to determine if treatment with Ucns affected CREB or ERK phosphorylation. Incubation with 100 nM Ucn1 or Ucn3 had maximal increases in CREB and ERK phosphorylations 5 min after its addition (not shown). Ucn1 had a more potent effect on CREB phosphorylation than Ucn3. A CRF2 receptor antagonist, antisauvagine-30, failed to suppress the stimulating effect of Ucn1 on CREB phosphorylation, whereas it inhibited the effect of Ucn3 (Fig. 5A). Incubation with 100 nM Ucn1 and Ucn3 significantly increased ERK phosphorylation 5 min after its addition. Antisauvagine-30 partially suppressed the stimulating effect of Ucn1 on ERK phosphorylation, whereas it completely inhibited the effect of Ucn3 (Fig. 5B). 3.7. Effects of Ucn1 and Ucn3 on H295R cell viability Finally, we examined whether Ucns modulate cell viability in H295R cells. Treatment with 100 nM Ucn1 and Ucn3 decreased the
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Fig. 3. Effects of Ucn1 and Ucn3 on mRNA levels of genes encoding enzymes involved in steroid hormone biosynthesis in H295R cells. Cells were treated in triplicate and the average of three independent experiments is shown. *P b 0.05 (compared with control (C)). Concentrations (nM) used in the treatments are indicated in parentheses. (A) Time-dependent changes in Ucn-induced StAR mRNA levels. Cells were incubated with medium alone (control) or with medium containing 100 nM Ucn1 or Ucn3 for the times indicated in the graph. (B) Dose-dependent effects of Ucn1 on StAR mRNA levels. Cells were incubated for 2 h with medium alone (control) or with medium containing increasing concentrations of Ucn1 (1–100 nM). (C and D) Time-dependent changes in Ucn-induced Cyp11β1 and Cyp11β2 mRNA levels. Cells were incubated with medium alone (control) or with medium containing 100 nM Ucn1 or Ucn3 for the times indicated in the graph.
cell viability to 91% and 88%, respectively (Fig. 5C). Pre-treatment with antisauvagine-30 inhibited the Ucns-induced effects. To determine whether a protein kinase was involved in the Ucns-decreased cell viability, H295R cells were pre-incubated for 30 min with a selective protein kinase A (PKA) inhibitor, PKAi, or a selective mitogenactivated protein (MAP) kinase kinase-ERK inhibitor, PD98059, prior to the addition of Ucn3. As shown in Fig. 5D, 100 nM PKAi partially suppressed the Ucn3-decreased cell viability, whereas 10 µM PD inhibited it. 4. Discussion H295R cells express Ucn1, Ucn2, Ucn3, CRF1 receptor, and CRF2a receptor, suggesting that Ucns play an endogenous role in H295R cells via the CRF receptors. The intra-adrenal regulatory system of CRF family of peptides depends on the balance between the local concentration of CRF ligand and the availability of their receptors [27]. Our results are consistent with those of previous studies
showing that both CRF1 receptor and CRF2a receptor are expressed in human adrenocortical cells [28]. Endogenous Ucns in the adrenal system have been suggested to act in an autocrine or paracrine manner [29]. Ucns are likely to be regulated by a cAMP-dependent pathway, because both mouse and human Ucn1 promoters contain a consensus CRE site [13]. Angiotensin-II, ACTH, and urotensin II are potential candidates for natural ligands in the activation of the cAMP pathway in the adrenal system [15–17]. In our study, forskolin was shown to transiently decrease Ucn1 and Ucn2 mRNA levels, but increase Ucns 1–3 mRNA levels in H295R cells. The mechanism underlying the temporal decrease in Ucn1 and Ucn2 mRNA levels is unknown, although the expression levels of mRNA are determined mainly by transcriptional mRNA synthesis, and post-transcriptional processes such as mRNA stability and degradation. Angiotensin-II also increased Ucns1–3 mRNA levels, with different time-courses from forskolin. Angiotensin-II may be more physiological than forskolin. Other pathways, in addition to a cAMP-dependent pathway, may be involved in the regulation.
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Fig. 4. Effects of Ucn1 and Ucn3 on cortisol and aldosterone levels in medium-cultured H295R cells. Dose-dependent effects of Ucn1 on cortisol (A) and aldosterone (C) levels in medium-cultured H295R cells. Cells were incubated for 6 h with medium alone (control) or with medium containing increasing concentrations of Ucn1 (1–100 nM). Effects of a CRF receptor antagonist on Ucn1- and Ucn3-induced cortisol (B) and aldosterone (D) levels in medium-cultured H295R cells. Cells were pre-incubated with medium containing 100 nM antalarmin (ANT), antisauvagine-30 (AS) or vehicle for 30 min, and then incubated for 6 h with medium containing 1–100 nM Ucn1 and Ucn3, 10 µM forskolin (Fsk), or vehicle.
The role of Ucns in the adrenal system might be a physiological one. Sirianni et al. reported that treatment with CRF and Ucn1 stimulated dehydroepiandrosterone sulfate production via the CRF1 receptor in human fetal adrenal cells [23]. In the present study, we found that Ucn1 increased Cyp11β1, a gene encoding 11β-hydroxylase regulated by ACTH, and Cyp11β2, a gene encoding aldosterone synthase, as well as the mRNA level of StAR in H295R cells. Therefore, Ucn1 is able to regulate the biosynthesis of steroid hormones. In fact, Ucn1 induced cortisol and aldosterone secretion. Angiotensin-II and ACTH are known to promote secretion of aldosterone and cortisol. They directly or indirectly may increase synthesis and secretion of the steroid hormones. Selective antagonists have been used to clarify the
role of CRF-related peptides [30]. The use of a selective CRF1 receptor antagonist, but not a CRF2 receptor antagonist, had effects on Ucn1induced cortisol or aldosterone secretion. Taken together with the findings of Sirianni et al., our result suggests that Ucn1-induced steroid secretion is mediated mainly via the CRF1 receptor in an autocrine or paracrine manner. Our study demonstrates that both Ucn1 and Ucn3 reduce cell viability in H295R cells, and this viability was blocked by a CRF2 receptor selective antagonist. Indeed, Ucn3 and the CRF2 receptor are colocalized in more than 85% of cortical cells [22], suggesting that Ucn3 via the CRF2 receptor plays an important role in the adrenal cortex [31]. Therefore, Ucn3 directly decreases cell viability
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Fig. 5. Involvement of protein kinases in Ucns-decreased H295R cell viability. Cells were treated in triplicate and the average of three independent experiments is shown. *P b 0.05 (compared with control (C)). +P b 0.05 (compared with Ucn1 and Ucn3, respectively). (A and B) Effects of Ucn1 and Ucn3 on CREB (A) or ERK (B) phosphorylations. Cells were preincubated with medium containing 100 nM AS-30 (AS) or vehicle for 30 min, and incubated further for 5 min after 100 nM Ucn1 or 100 nM Ucn3 was added to the medium. (C) Effects of Ucn1 and Ucn3 on H295R cell viability. Cells were pre-incubated with medium containing 100 nM antalarmin (ANT), antisauvagine-30 (AS) or vehicle for 30 min, and then incubated further for 24 h with medium containing 1 nM or 100 nM Ucn1 and Ucn3, or vehicle. Concentrations (nM) used in the treatments are indicated in parentheses. Cell viability was measured by Cell Counting Kit-8. (D) Effects of a protein kinase inhibitor on Ucn3-decreased H295R cell viability. Cells were pre-incubated with medium containing 100 nM PKAi, 10 µM PD98059 (PD), or vehicle for 30 min, and then incubated for 24 h with medium containing 100 nM Ucn3, or vehicle. Cell viability was measured by Cell Counting Kit-8.
via the CRF2 receptor in H295R cells. While CRF2 receptor mediates a ‘stress-coping’ response such as anxiolysis in the brain [32], CRF2 receptor has been shown to have an inhibitory effect of cell proliferation or tumor growth [24,25]. This result supports the inhibitory effect via the CRF2 receptor [24,25], although the mechanism is still unclear.
When the adrenal cells secrete Ucn1 or Ucn3, the cells may fail to grow. Angiotensin-II or ACTH promotes adrenal cell proliferation [33]. When angiotensin-II or ACTH increases Ucn secretion, the induced Ucn directly or indirectly may decrease the cell proliferation. Growth factors, more potent than the inhibitory effect of Ucns, would stimulate the cell proliferation.
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Ucn1 and Ucn3 increased CREB and ERK phosphorylations. Ucn1induced CREB phosphorylation failed to be blocked by a CRF2 receptor selective antagonist, suggesting that the other pathway except CRF1 receptor mainly mediated in the CREB phosphorylation. On the other hand, Ucn3-induced CREB and ERK phosphorylations are blocked by a CRF2 receptor selective antagonist, suggesting that the CRF2 receptor mainly mediated in the phosphorylation, with both PKA and ERK pathways being involved in the Ucn3-decreased cell viability. A recent review showed that ERK 1/2 acts as a central integrative growth regulatory element in adrenocortical cells [34]. However, it remains to be established whether Ucn3 has therapeutic potential in patients with adrenal tumors. Additionally, it is possible that Ucns cause vasodilation via the CRF2b receptor [35]; perhaps Ucn1 and Ucn3 act locally as vasodilators in the adrenal gland. In conclusion, adrenal H295R cells were shown to express Ucn1, Ucn2, Ucn3, CRF1 receptor, and CRF2a receptor mRNA in the present study, and forskolin was shown to transiently decrease Ucn1 and Ucn2 mRNA levels, but increase Ucns 1–3 mRNA levels. StAR, Cyp11β1, and Cyp11β2 mRNA levels, and both cortisol and aldosterone secretion were elevated by Ucn1. Cell viability was reduced by both Ucn1 and Ucn3 via the CRF2 receptor in H295R cells. Ucn1 and Ucn3 have differential regulation and roles via the two types of receptors in human adrenal H295R cells. Acknowledgments We thank Dr. Iwasaki (Kochi Medical School) for providing the BE (2)C cells. We also thank the Department of Pharmacology, Hirosaki University School of Medicine, Japan for generously providing us with ABI PRISM 7000. This work was supported in part by Health and Labour Science Research Grants (Research on Measures for Intractable Diseases) from the Ministry of Health, Labour, and Welfare of Japan. References [1] Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, Chan R, Turnbull AV, Lovejoy D, Rivier C, Sawchenko PE, Vale W. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 1995;378:287–92. [2] Hsu SY, Hsueh AJ. Human stresscopin and stresscopin-related peptide are selective ligands for the type 2 corticotropin-releasing hormone receptor. Nat Med 2001;7: 605–11. [3] Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, Vaughan J, Reyes TM, Gulyas J, Fischer W, Bilezikjian L, Rivier J, Sawchenko PE, Vale WW. Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA 2001;98: 7570–5. [4] Reyes TM, Lewis K, Perrin MH, Kunitake KS, Vaughan J, Arias CA, Hogenesch JB, Gulyas J, Rivier J, Vale WW, Sawchenko PE. Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc Natl Acad Sci USA 2001;98:2843–8. [5] Florio P, Torres PB, Torricelli M, Toti P, Vale W, Petraglia F. Human endometrium expresses urocortin II and III messenger RNA and peptides. Fertil Steril 2006;86: 1766–70. [6] Imperatore A, Florio P, Torres PB, Torricelli M, Galleri L, Toti P, Occhini R, Picciolini E, Vale W, Petraglia F. Urocortin 2 and urocortin 3 are expressed by the human placenta, deciduas, and fetal membranes. Am J Obstet Gynecol 2006;195:288–95. [7] Chang CP, Pearse RI, O'Connell S, Rosenfeld MG. Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain. Neuron 1993;11:1187–95. [8] Chen R, Lewis KA, Perrin MH, Vale WW. Expression cloning of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci USA 1993;90:8967–71. [9] Vita N, Laurent P, Lefort S, Chalon P, Lelias JM, Kaghad M, Le Fur G, Caput D, Ferrara P. Primary structure and functional expression of mouse pituitary and human brain corticotrophin releasing factor receptors. FEBS Lett 1993;335:1–5.
25
[10] Lovenberg TW, Liaw CW, Grigoriadis DE, Clevenger W, Chalmers DT, De Souza EB, Oltersdolf T. Cloning and characterization of a functionally distinct corticotropinreleasing factor receptor subtype from rat brain. Proc Natl Acad Sci USA 1995;92: 836–40. [11] Perrin M, Donaldson C, Chen R, Blount A, Berggren T, Bilezikjian L, Sawchenko P, Vale W. Identification of a second CRF receptor gene and characterization of a cDNA expressed in heart. Proc Natl Acad Sci USA 1995;92:2969–73. [12] Stenzel P, Kesterson R, Yeung W, Cone RD, Rittenberg MB, Stenzel-Poore MP. Identification of a novel murine receptor for corticotropin-releasing hormone expressed in the heart. Mol Endocrinol 1995;9:637–45. [13] Zhao L, Donaldson CJ, Smith GW, Vale WW. The structures of the mouse and human urocortin genes. Genomics 1998;50:23–33. [14] Kageyama K, Hanada K, Suda T. Differential regulation of urocortins1–3 mRNA in human umbilical vein endothelial cells. Regul Pept 2009;155:131–8. [15] Foster RH. Reciprocal influences between the signalling pathways regulating proliferation and steroidogenesis in adrenal glomerulosa cells. J Mol Endocrinol 2004;32:893–902. [16] Enyeart JJ. Biochemical and Ionic signaling mechanisms for ACTH-stimulated cortisol production. Vitam Horm 2005;70:265–79. [17] Giuliani L, Lenzini L, Antonello M, Aldighieri E, Belloni AS, Fassina A, GomezSanchez C, Rossi GP. Expression and functional role of urotensin-II and its receptor in the adrenal cortex and medulla: novel insights for the pathophysiology of primary aldosteronism. J Clin Endocrinol Metab 2009;94:684–90. [18] Suda T, Kageyama K, Sakihara S, Nigawara T. Physiological roles of urocortins, human homologues of fish urotensin 1, and their receptors. Peptides 2004;25: 1689–701. [19] Kageyama K, Bradbury MJ, Zhao L, Blount AL, Vale WW. Urocortin messenger ribonucleic acid: tissue distribution in the rat and regulation in thymus by lipopolysaccharide and glucocorticoids. Endocrinology 1999;140:5651–8. [20] Suda T, Tomori N, Tozawa F, Demura H, Shizume K, Mouri T, Miura Y, Sasano N. Immunoreactive corticotropin and corticotropin-releasing factor in human hypothalamus, adrenal, lung cancer, and pheochromocytoma. J Clin Endocrinol Metab 1984;58:919–24. [21] Willenberg HS, Bornstein SR, Hiroi N, Päth G, Goretzki PE, Scherbaum WA, Chrousos GP. Effects of a novel corticotropin-releasing-hormone receptor type I antagonist on human adrenal function. Mol Psychiatry 2000;5:137–41. [22] Fukuda T, Takahashi K, Suzuki T, Saruta M, Watanabe M, Nakata T, Sasano H. Urocortin 1, urocortin 3/stresscopin, and corticotropin-releasing factor receptors in human adrenal and its disorders. J Clin Endocrinol Metab 2005;90:4671–8. [23] Sirianni R, Mayhew BA, Carr BR, Parker Jr CR, Rainey WE. Corticotropin-releasing hormone (CRH) and urocortin act through type 1 CRH receptors to stimulate dehydroepiandrosterone sulfate production in human fetal adrenal cells. J Clin Endocrinol Metab 2005;90:5393–400. [24] Hao Z, Huang Y, Cleman J, Jovin IS, Vale WW, Bale TL, Giordano FJ. Urocortin2 inhibits tumor growth via effects on vascularization and cell proliferation. Proc Natl Acad Sci USA 2008;105:3939–44. [25] Wang J, Xu Y, Xu Y, Zhu H, Zhang R, Zhang G, Li S. Urocortin's inhibition of tumor growth and angiogenesis in hepatocellular carcinoma via corticotrophin-releasing factor receptor 2. Cancer Invest 2008;26:359–68. [26] Kimura Y, Takahashi K, Totsune K, Muramatsu Y, Kaneko C, Darnel AD, Suzuki T, Ebina M, Nukiwa T, Sasano H. Expression of urocortin and corticotropin-releasing factor receptor subtypes in the human heart. J Clin Endocrinol Metab 2002;87: 340–6. [27] Tsatsanis C, Dermitzaki E, Venihaki M, Chatzaki E, Minas V, Gravanis A, Margioris AN. The corticotropin-releasing factor (CRF) family of peptides as local modulators of adrenal function. Cell Mol Life Sci 2007;64:1638–55. [28] Willenberg HS, Haase M, Papewalis C, Schott M, Scherbaum WA, Bornstein SR. Corticotropin-releasing hormone receptor expression on normal and tumorous human adrenocortical cells. Neuroendocrinology 2005;82:274–81. [29] Audya T, Jain R, Hollander CS. Receptor mediated immunomodulation by corticotropin-releasing factor. Cell Immunol 1991;134:77–84. [30] Zoumakis E, Rice KC, Gold PW, Chrousos GP. Potential uses of corticotropinreleasing hormone antagonists. Ann N Y Acad Sci 2006;1083:239–51. [31] Takahashi K, Totsune K, Saruta M, Fukuda T, Suzuki T, Hirose T, Imai Y, Sasano H, Murakami O. Expression of urocortin 3/stresscopin in human adrenal glands and adrenal tumors. Peptides 2006;27:178–82. [32] Hauger RL, Risbrough V, Brauns O, Dautzenberg FM. Corticotropin releasing factor (CRF) receptor signaling in the central nervous system: new molecular targets. CNS Neurol Disord Drug Targets 2006;5:453–79. [33] Gill GN, Ill CR, Simonian MH. Angiotensin stimulation of bovine adrenocortical cell growth. Proc Natl Acad Sci USA 1977;74:5569–73. [34] Hoeflich A, Bielohuby M. Mechanisms of adrenal gland growth: signal integration by extracellular signal regulated kinases1/2. J Mol Endocrinol 2009;42:191–203. [35] Kageyama K, Furukawa K-I, Miki I, Terui K, Motomura S, Suda T. Vasodilative effects of urocortin II via protein kinase A and a mitogen-activated protein kinase in rat thoracic aorta. J Cardiovasc Pharmacol 2003;42:561–5.
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r e g p e p
Short communication
Differential regulation and roles of urocortins in human adrenal H295R cells Kazunori Kageyama ⁎, Komaki Hanada, Toshihiro Suda Department of Endocrinology and Metabolism, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
a r t i c l e
i n f o
Article history: Received 23 September 2009 Received in revised form 14 January 2010 Accepted 11 February 2010 Available online 19 February 2010 Keywords: Corticotropin-releasing factor Urocortin Cortisol Aldosterone Adrenal gland
a b s t r a c t Three urocortins (Ucns) are known as members of the corticotropin-releasing factor (CRF) family of peptides and serve as natural ligands for CRF receptors. Ucn1 and Ucn3 exhibit potent effects on the adrenal system via the CRF receptors. This study aimed to explore the regulation and roles of Ucns in the adrenal system using human adrenal carcinoma H295R cells, which express Ucn1, Ucn2, Ucn3, CRF receptor type 1 (CRF1 receptor), and CRF receptor type 2a (CRF2a receptor) mRNA. Forskolin, which stimulates adenylate cyclase and then increases intracellular cAMP production, was shown to transiently decrease Ucn1 and Ucn2 mRNA levels, but increase Ucns 1–3 mRNA levels in H295R cells. Steroidogenic acute regulatory protein, Cyp11β1, and Cyp11β2 mRNA levels, and both cortisol and aldosterone secretions were elevated by Ucn1. Cell viability was reduced by both Ucn1 and Ucn3 via the CRF2 receptor in H295R cells. Ucn1 and Ucn3 increased the expression of the cAMP-response element binding protein and extracellular signal-related kinase (ERK) phosphorylations. The ERK and protein kinase A pathways were involved in Ucn3-decreased cell viability. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Three urocortins (Ucns) are known as members of the corticotropin-releasing factor (CRF) family of peptides. Ucn1 is a 40 amino acid peptide cloned from the Edinger–Westphal nucleus [1], while Ucn2 and Ucn3 have been identified in the human genome database and mouse genomic DNA, respectively [2–4]. Ucn1 and Ucn3 are expressed in various human tissues, whereas the presence of Ucn2 has been established in human endometrium or gestational tissues [5,6]. The actions of the CRF family of peptides are mediated by at least two distinct G protein-coupled receptors: CRF receptor type 1 (CRF1 receptor) [7–9] and CRF receptor type 2 (CRF2 receptor) [10–12]. Stimulation of these receptors activates adenylate cyclase to produce cAMP, which then induces physiological effects. Ucn1 binds to both the CRF1 and CRF2 receptors, while Ucn2 and Ucn3 are highly selective for the CRF2 receptor, with little or no affinity for the CRF1 receptor. A consensus cAMP-response element (CRE) site has been shown to mediate the regulation of Ucn1 expression by cAMP in both mouse and human Ucn1 promoters [13]. Raised intracellular cAMP levels induced by forskolin are known to increase Ucn1 mRNA level in human umbilical vein endothelial cells [14]. Angiotensin-II, ACTH, and urotensin II are major candidates for natural ligands in the activation of the cAMP pathway in the adrenal system [15–17]. CRF and Ucns mediate stress responses, cardiovascular function, and immune functions via the CRF receptors [18,19]. The presence of the CRF family of peptides has been reported in the human adrenal
⁎ Corresponding author. Tel.: + 81 172 39 5062; fax: + 81 172 39 5063. E-mail address: [email protected] (K. Kageyama). 0167-0115/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2010.02.006
gland [20]. Specifically, Ucn1, Ucn3, CRF1 receptor, and CRF2 receptor are expressed in the human adrenal gland [21,22], suggesting that the CRF family peptides may play important roles in regulating adrenal functions. Indeed, CRF and Ucn1 stimulate dehydroepiandrosterone sulfate production via the CRF1 receptor in human fetal adrenal cells [23], and Ucn3 inhibits cell proliferation or tumor growth via the CRF2 receptor [24,25]. Although Ucns have potent effects on the adrenal system, their possible roles and regulation have not yet been clearly determined. In the present study, we studied cAMP-dependent changes in the gene expression of Ucns in human adrenal H295R cells with the aim of investigating the effects of Ucns on steroidogenesis and cell proliferation. 2. Materials and methods 2.1. Materials Forskolin, PKA inhibitor 14–22 amide (PKAi), and PD98059 were purchased from Calbiochem (San Diego, CA, USA), and antalarmin from Sigma-Aldrich Corp. (St. Lois, MO). Human Ucn1, Ucn2, Ucn3, and angiotensin-II were purchased from the Peptide Institute (Osaka, Japan). Antisauvagine-30 was synthesized by Asahi Techno Glass (Chiba, Japan). 2.2. Cell cultures H295R cells were obtained from the American Type Culture Collection (Manassas, VA, USA), and incubated in DMEM/F12 containing 2.5% NuSerum (BD Biosciences, Bedford, MA, USA) 1% ITS Premix
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(BD Biosciences) 100 mg/mL streptomycin, and 100 U/mL penicillin at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells were plated at 3 × 103 cells/cm2 seven days before each experiment, and the medium was changed every 48 h. On day 7, cells were treated as indicated below. BE(2)C cells were incubated in DMEM/F12 medium, supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% nonessential amino acid, 100 mg/mL streptomycin, and 100 U/mL penicillin at 37 °C, in a humidified atmosphere of 5% CO2 and 95% air. At the end of each experiment, total cellular RNA or medium was collected and stored at −80 °C until the assay was performed. All treatments were performed in triplicate and repeated three times. 2.3. RNA extraction Cells were incubated with medium alone (control) or with medium containing forskolin, Ucns, and angiotensin-II for the times indicated in the graph. To examine the dose-dependent effects of forskolin or Ucns, cells were incubated for the indicated times with medium alone (control) or with medium containing increasing concentrations of forskolin (0.1–10 µM) or Ucns (1–100 nM). At the end of each experiment, cellular total RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. cDNA was synthesized from the total RNA (0.5 µg) using random hexamers as primers with the SuperScript FirstStrand Synthesis System for reverse transcriptase–polymerase chain reaction (RT–PCR) (Invitrogen Corp., Carlsbad, CA, USA), according to the manufacturer's instructions. 2.4. Reverse transcriptase–polymerase chain reaction (RT–PCR) Polymerase chain reaction (PCR) was carried out in a programmable thermal controller (Bio-Rad, Hercules, CA, USA) with the following oligonucleotide primers: the sequences of the primers employed in this study are summarized in Table 1. The primers were designed according to the publications [6,26]. PCR conditions for Ucn1, CRF1 and CRF2a receptor primers were 1 × (96 °C, 2 min), 35 × (96 °C, 15 sec; 64 °C, 30 sec; 72 °C, 1 min) and 1 × (72 °C, 5 min). CRF and CRF2b receptor primer conditions were 1 × (96 °C, 2 min), 38 × (96 °C, 15 sec; 64 °C, 30 sec; 72 °C, 1 min) and 1 × (72 °C, 5 min); Ucn2 primer conditions were 36 × (94 °C, 40 sec; 55 °C, 40 sec; 72 °C, 40 sec) and 1 × (72 °C, 10 min); Ucn3 primer conditions were 37 × (94 °C, 1 min; 63 °C, 1 min; 72 °C, 1 min) and 1 × (72 °C, 10 min); and GAPDH primer conditions were 1 × (96 °C, 2 min), 22 × (96 °C, 15 sec; 60 °C, 30 sec; 72 °C, 1 min) and 1 × (72 °C, 5 min). Products were separated by electrophoresis on a 1.5% agarose gel containing ethidium bromide. The expected sizes of
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the PCR products for CRF and Ucn1 were 678 bp and 468 bp, respectively, and those for Ucn2 and Ucn3 were 195 bp and 310 bp, respectively. The expected size of the PCR product for the CRF1 receptor was 475 bp, and those for the CRF2a and CRF2b receptors were 233 bp and 248 bp, respectively. The expected size of the PCR product for the GAPDH receptor was 969 bp.
2.5. Real-time reverse transcriptase–polymerase chain reaction (RT–PCR) Cellular total RNA extraction and cDNA synthesis were performed similarly as described earlier. The resulting cDNAs were then subjected to real-time PCR as follows. The expression levels of human Ucns mRNA were evaluated using quantitative real-time PCR based on specific sets of primers and probes (Assays-on-Demand Gene Expression Products, Applied Biosystems, Foster City, CA, USA). GAPDH was used as a housekeeping gene to standardize values, because the GAPDH mRNA levels were not changed in any treatments of these studies. Each reaction consisted of 1×TaqMan universal PCR Master Mix (Applied Biosystems), 1×Assays-on-Demand Gene Expression Products (Hs00175020_m1 for human Ucn1, Hs00264218_s1 for human Ucn2, Hs00846499_s1 for human Ucn3, Hs00264912_m1 for steroidogenic acute regulatory protein (StAR), Hs01596404_m1 for 11β-hydroxylase (Cyp11β1), Hs01597732_m1 for 18-hydroxylase (Cyp11β2), and Hs99999905_m1 for human GAPDH) and 2 µL of cDNA in a total volume of 50 µL using the following parameters on the ABI PRISM 7000 Sequence Detection System (Applied Biosystems): 95 °C for 10 min, 40 cycles at 95 °C for 15 sec and 60 °C for 1 min. The above assays involved specific sets of primers and a TaqMan probe spanning the exon/exon junction and should not, therefore, have been influenced by DNA contamination. Data were collected and recorded by ABI PRISM 7000 SDS Software (Applied Biosystems) and expressed as a function of the threshold cycle (CT). Using the diluted samples, the amplification efficacies for each gene of interest and the housekeeping gene amplimers were found to be identical.
2.6. Cortisol and aldosterone assays Cells were incubated for 6 h with medium alone (control) or with medium containing increasing concentrations of Ucn1 (1–100 nM). To examine the effects of a CRF receptor antagonist on Ucn1- and Ucn3-induced cortisol and aldosterone levels in medium-cultured H295R cells, the cells were pre-incubated with medium containing 100 nM antalarmin, antisauvagine-30 or vehicle for 30 min, and then incubated for 6 h with medium containing 1–100 nM Ucn1 and Ucn3, 10 µM forskolin or vehicle.
Table 1 Oligonucleotide primers used in the RT–PCR. Target
Oligonucleotide sequence
Size, base pairs
Reference
CRF
CRF-F: 5′-AAGGAAGACAACCTCCAGAGAAAGC-3′ CRF-R: 5′-TCCATGAGTTTCCTGTTGCTGTGAG-3′ Ucn1-F: 5′-GTTCCCCAAGGCGTCTTCA-3′ Ucn1-R: 5′-CTTGCCCACCGAGTCGAAT-3′ Ucn2-F: 5′-GTGTCGGCCACTGCTGAGCCTGAGAGA-3′ Ucn2-R: 5′-ATCTGATATGACCTGCATGACAGTGGCT-3′ Ucn3-F: 5′-TGCTGCTCCTGCTGCTGCTC-3′ Ucn3-R: 5′-GTGTCCTGGCGTGGCTTTCCC-3′ CRF1 receptor-F: 5′-CAAACAATGGCTACCGGGAG-3′ CRF1 receptor-R: 5′-ACACCCCAGCCAATGCAGA-3′ CRF2a receptor-F: 5′-GACGCGGCACTGCTCCACAG-3′ CRF2a receptor-R: 5′-GCATTCCGGGTCGTGTTGT-3′ CRF2b receptor-F: 5′-CCCTCACCAACCTCTCAGGTCC-3′ CRF2b receptor-R: 5′-CAGGTCATACTTCCTCTGCTTGTC-3′ GAPDH-F: 5′-GGTCGGAGTCAACGGATTTG-3′ GAPDH-R: 5′-ATGAGGTCCACCACCCTGTT-3′
678
[26]
468
[26]
195
[6]
310
[6]
475
[26]
233
[26]
248
[26]
969
[26]
Ucn1 Ucn2 Ucn3 CRF1 receptor CRF2a receptor CRF2b receptor GAPDH
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The culture medium was collected from the dishes and stored at −80 °C for the measurement of cortisol and aldosterone levels using a cortisol immunoassay (R&D Systems, Minneapolis, MN, USA) and an aldosterone ELISA (DRG, Mountainside, NJ, USA), respectively. All samples from each experiment were determined in the same assay. 2.7. Western blot analysis Cells were pre-incubated with medium containing 100 nM antalarmin, antisauvagine-30 or vehicle for 30 min, and incubated for a further 5 min after 100 nM Ucn1 or 100 nM Ucn3 was added to the medium. After treatment with Ucn1 or Ucn3, cells were washed twice with phosphate buffered saline (PBS) and lysed with Laemmli sample buffer. Cell debris was pelleted by centrifugation, and the supernatant was recovered. Samples were boiled and subjected to electrophoresis on a gradient (4–20%) polyacrylamide gel. Proteins were transferred to a PVDF membrane (Daiichi Kagaku, Tokyo, Japan). After blocking with Detector Block® blocking buffer (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA), the membrane was incubated for 1 h with a rabbit anti-CRE binding protein (CREB), anti-phosphorylated (p)-CREB, anti-extracellular signal-related kinases (ERK), or anti-p-ERK antibodies (Cell Signaling Technology, Beverly, MA, USA), washed with PBS containing 0.05% Tween 20, and incubated with horseradish peroxidase (HRP)-labeled anti-rabbit IgG (Daiichi Kagaku, Japan). Detection was performed using a chemiluminescent substrate Super-signal WestPico (Pierce Chemical Co., Rockford, IL USA), and the membrane was exposed to BioMax film (Eastman Kodak Co., Rochester, NY, USA) followed by
quantitative analysis using NIH image software 1.61. The results are expressed as corrected arbitrary units. 2.8. Cell proliferation assay H295R cells were seeded into 96-well plates at a density of 1 × 104 cells/well, and incubated in 100 µL of culture medium containing 100 nM antalarmin, 100 nM antisauvagine-30, 100 nM PKAi, 10 µM PD98059 or vehicle, and each Ucn or vehicle at 37 °C for 24 h. Cell viability was measured using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). The assay is based on cleavage of the tetrazolium salt WST-8 to formazan product. Absorbance was measured with a test wavelength of 450 nm and a reference wavelength of 630 nm. 2.9. Statistical analysis All values are expressed as the mean ± standard error of the mean (SEM). Statistical analyses of data were performed using one-way analysis of variance (ANOVA), followed by Bonferroni/Dunn post-hoc tests. The level of statistical significance was set at P b 0.05. 3. Results 3.1. Expression of Ucns and CRF receptor mRNA in H295R cells H295R cells were found to express Ucn1, Ucn2, and Ucn3, but not CRF mRNA (Fig. 1). CRF1 and CRF2a receptor mRNAs were also expressed (Fig. 1).
Fig. 1. Expression of Ucn and CRF receptor mRNA in H295R cells. M: marker; +: reverse transcriptase–PCR shown in duplicate; −: negative control for reverse transcriptase–PCR.
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3.2. Effects of forskolin or angiotensin-II on Ucns1–3 mRNA levels in H295R cells H295R cells were incubated with forskolin to determine whether Ucns1–3 mRNA levels were dependent on cAMP. As shown in Fig. 2A and B, real-time PCR analysis revealed that incubation with forskolin transiently decreased, then increased Ucn1 and Ucn2 mRNA levels (ANOVA; P b 0.005). Ucn1 mRNA level fell transiently to 67% of the
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control value within 2 h of the addition of 10 µM forskolin, followed by an increase to 224% of the control level within 6 h (Fig. 2A). Ucn2 mRNA level also fell transiently to 51% of the control value within 2 h of the addition of 10 µM forskolin, followed by a gradual increase to 173% of the control level within 24 h, in a dose-dependent manner (Fig. 2A and B). Forskolin increased Ucn3 mRNA level within 2 h, in a dose-dependent manner (ANOVA; P b 0.005) (Fig. 2A and B). Ucn3 mRNA level was elevated transiently to 171% of the control value within 2 h of the addition of 10 µM forskolin, and then recovered within 24 h (Fig. 2A). Angiotensin-II (100 nM) also increased Ucn1, Ucn2, and Ucn3 mRNA levels within 6 h (Fig. 2C). 3.3. Effects of Ucn1 and Ucn3 on mRNA levels of genes encoding enzymes involved in steroid hormone biosynthesis in H295R cells To determine whether Ucns induce mRNA levels of enzymes involved in steroid hormone biosynthesis, such as StAR, a crucial protein involved in steroidogenesis, Cyp11β1, a gene encoding 11βhydroxylase regulated by ACTH, and Cyp11β2, a gene encoding aldosterone synthase, H295R cells were incubated with Ucn1 or Ucn3. StAR, Cyp11β1, and Cyp11β2 mRNA levels were elevated transiently from 2 h to 6 h after the addition of 100 nM Ucn1, but not Ucn3 (Fig. 3A, C, and D). Ucn1 increased StAR mRNA level in a dose-dependent manner (ANOVA; P b 0.005, Fig. 3B). 3.4. Effects of Ucn1 and Ucn3 on cortisol level in medium from cultured H295R cells To determine whether Ucns induce cortisol secretion, H295R cells were incubated with Ucn1 or Ucn3. Incubation with Ucn1 increased cortisol level in a dose-dependent manner (ANOVA; P b 0.05, Fig. 4A). However, there was no significant increase following incubation with Ucn3. Pre-treatment with antalarmin, a selective CRF1 receptor antagonist, but not antisauvagin-30, a selective CRF2 receptor antagonist, blocked Ucn1-induced cortisol secretion (Fig. 4B). 3.5. Effects of Ucn1 and Ucn3 on aldosterone level in medium from cultured H295R cells We next determined whether Ucns induce aldosterone secretion in H295R cells. Incubation with Ucn1 increased aldosterone level in a dose-dependent manner (ANOVA; P b 0.05, Fig. 4C). However, there was no significant increase after incubation with Ucn3. Pretreatment with antalarmin, a selective CRF1 receptor antagonist, but not antisauvagin-30, a selective CRF2 receptor antagonist, blocked Ucn1-induced aldosterone secretion (Fig. 4D). 3.6. Effects of Ucn1 and Ucn3 on CREB or ERK phosphorylation
Fig. 2. Effects of forskolin or angiotensin-II on Ucns1–3 mRNA levels in H295R cells. Cells were treated in triplicate and the average of three independent experiments is shown. *P b 0.05 (compared with control). (A) Time-dependent changes in forskolininduced mRNA levels of each Ucn. Cells were incubated with medium alone (control) or with medium containing 10 µM forskolin for the times indicated in the graph. (B) Dosedependent effects of forskolin on each Ucn mRNA level. Cells were incubated for the indicated times with medium alone (control) or with medium containing increasing concentrations of forskolin (0.1–10 µM). (C) Time-dependent changes in angiotensinII-induced mRNA levels of each Ucn. Cells were incubated with medium alone (control) or with medium containing 100 nM angiotensin-II for the times indicated in the graph.
We examined H295R cells to determine if treatment with Ucns affected CREB or ERK phosphorylation. Incubation with 100 nM Ucn1 or Ucn3 had maximal increases in CREB and ERK phosphorylations 5 min after its addition (not shown). Ucn1 had a more potent effect on CREB phosphorylation than Ucn3. A CRF2 receptor antagonist, antisauvagine-30, failed to suppress the stimulating effect of Ucn1 on CREB phosphorylation, whereas it inhibited the effect of Ucn3 (Fig. 5A). Incubation with 100 nM Ucn1 and Ucn3 significantly increased ERK phosphorylation 5 min after its addition. Antisauvagine-30 partially suppressed the stimulating effect of Ucn1 on ERK phosphorylation, whereas it completely inhibited the effect of Ucn3 (Fig. 5B). 3.7. Effects of Ucn1 and Ucn3 on H295R cell viability Finally, we examined whether Ucns modulate cell viability in H295R cells. Treatment with 100 nM Ucn1 and Ucn3 decreased the
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Fig. 3. Effects of Ucn1 and Ucn3 on mRNA levels of genes encoding enzymes involved in steroid hormone biosynthesis in H295R cells. Cells were treated in triplicate and the average of three independent experiments is shown. *P b 0.05 (compared with control (C)). Concentrations (nM) used in the treatments are indicated in parentheses. (A) Time-dependent changes in Ucn-induced StAR mRNA levels. Cells were incubated with medium alone (control) or with medium containing 100 nM Ucn1 or Ucn3 for the times indicated in the graph. (B) Dose-dependent effects of Ucn1 on StAR mRNA levels. Cells were incubated for 2 h with medium alone (control) or with medium containing increasing concentrations of Ucn1 (1–100 nM). (C and D) Time-dependent changes in Ucn-induced Cyp11β1 and Cyp11β2 mRNA levels. Cells were incubated with medium alone (control) or with medium containing 100 nM Ucn1 or Ucn3 for the times indicated in the graph.
cell viability to 91% and 88%, respectively (Fig. 5C). Pre-treatment with antisauvagine-30 inhibited the Ucns-induced effects. To determine whether a protein kinase was involved in the Ucns-decreased cell viability, H295R cells were pre-incubated for 30 min with a selective protein kinase A (PKA) inhibitor, PKAi, or a selective mitogenactivated protein (MAP) kinase kinase-ERK inhibitor, PD98059, prior to the addition of Ucn3. As shown in Fig. 5D, 100 nM PKAi partially suppressed the Ucn3-decreased cell viability, whereas 10 µM PD inhibited it. 4. Discussion H295R cells express Ucn1, Ucn2, Ucn3, CRF1 receptor, and CRF2a receptor, suggesting that Ucns play an endogenous role in H295R cells via the CRF receptors. The intra-adrenal regulatory system of CRF family of peptides depends on the balance between the local concentration of CRF ligand and the availability of their receptors [27]. Our results are consistent with those of previous studies
showing that both CRF1 receptor and CRF2a receptor are expressed in human adrenocortical cells [28]. Endogenous Ucns in the adrenal system have been suggested to act in an autocrine or paracrine manner [29]. Ucns are likely to be regulated by a cAMP-dependent pathway, because both mouse and human Ucn1 promoters contain a consensus CRE site [13]. Angiotensin-II, ACTH, and urotensin II are potential candidates for natural ligands in the activation of the cAMP pathway in the adrenal system [15–17]. In our study, forskolin was shown to transiently decrease Ucn1 and Ucn2 mRNA levels, but increase Ucns 1–3 mRNA levels in H295R cells. The mechanism underlying the temporal decrease in Ucn1 and Ucn2 mRNA levels is unknown, although the expression levels of mRNA are determined mainly by transcriptional mRNA synthesis, and post-transcriptional processes such as mRNA stability and degradation. Angiotensin-II also increased Ucns1–3 mRNA levels, with different time-courses from forskolin. Angiotensin-II may be more physiological than forskolin. Other pathways, in addition to a cAMP-dependent pathway, may be involved in the regulation.
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Fig. 4. Effects of Ucn1 and Ucn3 on cortisol and aldosterone levels in medium-cultured H295R cells. Dose-dependent effects of Ucn1 on cortisol (A) and aldosterone (C) levels in medium-cultured H295R cells. Cells were incubated for 6 h with medium alone (control) or with medium containing increasing concentrations of Ucn1 (1–100 nM). Effects of a CRF receptor antagonist on Ucn1- and Ucn3-induced cortisol (B) and aldosterone (D) levels in medium-cultured H295R cells. Cells were pre-incubated with medium containing 100 nM antalarmin (ANT), antisauvagine-30 (AS) or vehicle for 30 min, and then incubated for 6 h with medium containing 1–100 nM Ucn1 and Ucn3, 10 µM forskolin (Fsk), or vehicle.
The role of Ucns in the adrenal system might be a physiological one. Sirianni et al. reported that treatment with CRF and Ucn1 stimulated dehydroepiandrosterone sulfate production via the CRF1 receptor in human fetal adrenal cells [23]. In the present study, we found that Ucn1 increased Cyp11β1, a gene encoding 11β-hydroxylase regulated by ACTH, and Cyp11β2, a gene encoding aldosterone synthase, as well as the mRNA level of StAR in H295R cells. Therefore, Ucn1 is able to regulate the biosynthesis of steroid hormones. In fact, Ucn1 induced cortisol and aldosterone secretion. Angiotensin-II and ACTH are known to promote secretion of aldosterone and cortisol. They directly or indirectly may increase synthesis and secretion of the steroid hormones. Selective antagonists have been used to clarify the
role of CRF-related peptides [30]. The use of a selective CRF1 receptor antagonist, but not a CRF2 receptor antagonist, had effects on Ucn1induced cortisol or aldosterone secretion. Taken together with the findings of Sirianni et al., our result suggests that Ucn1-induced steroid secretion is mediated mainly via the CRF1 receptor in an autocrine or paracrine manner. Our study demonstrates that both Ucn1 and Ucn3 reduce cell viability in H295R cells, and this viability was blocked by a CRF2 receptor selective antagonist. Indeed, Ucn3 and the CRF2 receptor are colocalized in more than 85% of cortical cells [22], suggesting that Ucn3 via the CRF2 receptor plays an important role in the adrenal cortex [31]. Therefore, Ucn3 directly decreases cell viability
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Fig. 5. Involvement of protein kinases in Ucns-decreased H295R cell viability. Cells were treated in triplicate and the average of three independent experiments is shown. *P b 0.05 (compared with control (C)). +P b 0.05 (compared with Ucn1 and Ucn3, respectively). (A and B) Effects of Ucn1 and Ucn3 on CREB (A) or ERK (B) phosphorylations. Cells were preincubated with medium containing 100 nM AS-30 (AS) or vehicle for 30 min, and incubated further for 5 min after 100 nM Ucn1 or 100 nM Ucn3 was added to the medium. (C) Effects of Ucn1 and Ucn3 on H295R cell viability. Cells were pre-incubated with medium containing 100 nM antalarmin (ANT), antisauvagine-30 (AS) or vehicle for 30 min, and then incubated further for 24 h with medium containing 1 nM or 100 nM Ucn1 and Ucn3, or vehicle. Concentrations (nM) used in the treatments are indicated in parentheses. Cell viability was measured by Cell Counting Kit-8. (D) Effects of a protein kinase inhibitor on Ucn3-decreased H295R cell viability. Cells were pre-incubated with medium containing 100 nM PKAi, 10 µM PD98059 (PD), or vehicle for 30 min, and then incubated for 24 h with medium containing 100 nM Ucn3, or vehicle. Cell viability was measured by Cell Counting Kit-8.
via the CRF2 receptor in H295R cells. While CRF2 receptor mediates a ‘stress-coping’ response such as anxiolysis in the brain [32], CRF2 receptor has been shown to have an inhibitory effect of cell proliferation or tumor growth [24,25]. This result supports the inhibitory effect via the CRF2 receptor [24,25], although the mechanism is still unclear.
When the adrenal cells secrete Ucn1 or Ucn3, the cells may fail to grow. Angiotensin-II or ACTH promotes adrenal cell proliferation [33]. When angiotensin-II or ACTH increases Ucn secretion, the induced Ucn directly or indirectly may decrease the cell proliferation. Growth factors, more potent than the inhibitory effect of Ucns, would stimulate the cell proliferation.
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Ucn1 and Ucn3 increased CREB and ERK phosphorylations. Ucn1induced CREB phosphorylation failed to be blocked by a CRF2 receptor selective antagonist, suggesting that the other pathway except CRF1 receptor mainly mediated in the CREB phosphorylation. On the other hand, Ucn3-induced CREB and ERK phosphorylations are blocked by a CRF2 receptor selective antagonist, suggesting that the CRF2 receptor mainly mediated in the phosphorylation, with both PKA and ERK pathways being involved in the Ucn3-decreased cell viability. A recent review showed that ERK 1/2 acts as a central integrative growth regulatory element in adrenocortical cells [34]. However, it remains to be established whether Ucn3 has therapeutic potential in patients with adrenal tumors. Additionally, it is possible that Ucns cause vasodilation via the CRF2b receptor [35]; perhaps Ucn1 and Ucn3 act locally as vasodilators in the adrenal gland. In conclusion, adrenal H295R cells were shown to express Ucn1, Ucn2, Ucn3, CRF1 receptor, and CRF2a receptor mRNA in the present study, and forskolin was shown to transiently decrease Ucn1 and Ucn2 mRNA levels, but increase Ucns 1–3 mRNA levels. StAR, Cyp11β1, and Cyp11β2 mRNA levels, and both cortisol and aldosterone secretion were elevated by Ucn1. Cell viability was reduced by both Ucn1 and Ucn3 via the CRF2 receptor in H295R cells. Ucn1 and Ucn3 have differential regulation and roles via the two types of receptors in human adrenal H295R cells. Acknowledgments We thank Dr. Iwasaki (Kochi Medical School) for providing the BE (2)C cells. We also thank the Department of Pharmacology, Hirosaki University School of Medicine, Japan for generously providing us with ABI PRISM 7000. This work was supported in part by Health and Labour Science Research Grants (Research on Measures for Intractable Diseases) from the Ministry of Health, Labour, and Welfare of Japan. References [1] Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, Chan R, Turnbull AV, Lovejoy D, Rivier C, Sawchenko PE, Vale W. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 1995;378:287–92. [2] Hsu SY, Hsueh AJ. Human stresscopin and stresscopin-related peptide are selective ligands for the type 2 corticotropin-releasing hormone receptor. Nat Med 2001;7: 605–11. [3] Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, Vaughan J, Reyes TM, Gulyas J, Fischer W, Bilezikjian L, Rivier J, Sawchenko PE, Vale WW. Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA 2001;98: 7570–5. [4] Reyes TM, Lewis K, Perrin MH, Kunitake KS, Vaughan J, Arias CA, Hogenesch JB, Gulyas J, Rivier J, Vale WW, Sawchenko PE. Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc Natl Acad Sci USA 2001;98:2843–8. [5] Florio P, Torres PB, Torricelli M, Toti P, Vale W, Petraglia F. Human endometrium expresses urocortin II and III messenger RNA and peptides. Fertil Steril 2006;86: 1766–70. [6] Imperatore A, Florio P, Torres PB, Torricelli M, Galleri L, Toti P, Occhini R, Picciolini E, Vale W, Petraglia F. Urocortin 2 and urocortin 3 are expressed by the human placenta, deciduas, and fetal membranes. Am J Obstet Gynecol 2006;195:288–95. [7] Chang CP, Pearse RI, O'Connell S, Rosenfeld MG. Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain. Neuron 1993;11:1187–95. [8] Chen R, Lewis KA, Perrin MH, Vale WW. Expression cloning of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci USA 1993;90:8967–71. [9] Vita N, Laurent P, Lefort S, Chalon P, Lelias JM, Kaghad M, Le Fur G, Caput D, Ferrara P. Primary structure and functional expression of mouse pituitary and human brain corticotrophin releasing factor receptors. FEBS Lett 1993;335:1–5.
25
[10] Lovenberg TW, Liaw CW, Grigoriadis DE, Clevenger W, Chalmers DT, De Souza EB, Oltersdolf T. Cloning and characterization of a functionally distinct corticotropinreleasing factor receptor subtype from rat brain. Proc Natl Acad Sci USA 1995;92: 836–40. [11] Perrin M, Donaldson C, Chen R, Blount A, Berggren T, Bilezikjian L, Sawchenko P, Vale W. Identification of a second CRF receptor gene and characterization of a cDNA expressed in heart. Proc Natl Acad Sci USA 1995;92:2969–73. [12] Stenzel P, Kesterson R, Yeung W, Cone RD, Rittenberg MB, Stenzel-Poore MP. Identification of a novel murine receptor for corticotropin-releasing hormone expressed in the heart. Mol Endocrinol 1995;9:637–45. [13] Zhao L, Donaldson CJ, Smith GW, Vale WW. The structures of the mouse and human urocortin genes. Genomics 1998;50:23–33. [14] Kageyama K, Hanada K, Suda T. Differential regulation of urocortins1–3 mRNA in human umbilical vein endothelial cells. Regul Pept 2009;155:131–8. [15] Foster RH. Reciprocal influences between the signalling pathways regulating proliferation and steroidogenesis in adrenal glomerulosa cells. J Mol Endocrinol 2004;32:893–902. [16] Enyeart JJ. Biochemical and Ionic signaling mechanisms for ACTH-stimulated cortisol production. Vitam Horm 2005;70:265–79. [17] Giuliani L, Lenzini L, Antonello M, Aldighieri E, Belloni AS, Fassina A, GomezSanchez C, Rossi GP. Expression and functional role of urotensin-II and its receptor in the adrenal cortex and medulla: novel insights for the pathophysiology of primary aldosteronism. J Clin Endocrinol Metab 2009;94:684–90. [18] Suda T, Kageyama K, Sakihara S, Nigawara T. Physiological roles of urocortins, human homologues of fish urotensin 1, and their receptors. Peptides 2004;25: 1689–701. [19] Kageyama K, Bradbury MJ, Zhao L, Blount AL, Vale WW. Urocortin messenger ribonucleic acid: tissue distribution in the rat and regulation in thymus by lipopolysaccharide and glucocorticoids. Endocrinology 1999;140:5651–8. [20] Suda T, Tomori N, Tozawa F, Demura H, Shizume K, Mouri T, Miura Y, Sasano N. Immunoreactive corticotropin and corticotropin-releasing factor in human hypothalamus, adrenal, lung cancer, and pheochromocytoma. J Clin Endocrinol Metab 1984;58:919–24. [21] Willenberg HS, Bornstein SR, Hiroi N, Päth G, Goretzki PE, Scherbaum WA, Chrousos GP. Effects of a novel corticotropin-releasing-hormone receptor type I antagonist on human adrenal function. Mol Psychiatry 2000;5:137–41. [22] Fukuda T, Takahashi K, Suzuki T, Saruta M, Watanabe M, Nakata T, Sasano H. Urocortin 1, urocortin 3/stresscopin, and corticotropin-releasing factor receptors in human adrenal and its disorders. J Clin Endocrinol Metab 2005;90:4671–8. [23] Sirianni R, Mayhew BA, Carr BR, Parker Jr CR, Rainey WE. Corticotropin-releasing hormone (CRH) and urocortin act through type 1 CRH receptors to stimulate dehydroepiandrosterone sulfate production in human fetal adrenal cells. J Clin Endocrinol Metab 2005;90:5393–400. [24] Hao Z, Huang Y, Cleman J, Jovin IS, Vale WW, Bale TL, Giordano FJ. Urocortin2 inhibits tumor growth via effects on vascularization and cell proliferation. Proc Natl Acad Sci USA 2008;105:3939–44. [25] Wang J, Xu Y, Xu Y, Zhu H, Zhang R, Zhang G, Li S. Urocortin's inhibition of tumor growth and angiogenesis in hepatocellular carcinoma via corticotrophin-releasing factor receptor 2. Cancer Invest 2008;26:359–68. [26] Kimura Y, Takahashi K, Totsune K, Muramatsu Y, Kaneko C, Darnel AD, Suzuki T, Ebina M, Nukiwa T, Sasano H. Expression of urocortin and corticotropin-releasing factor receptor subtypes in the human heart. J Clin Endocrinol Metab 2002;87: 340–6. [27] Tsatsanis C, Dermitzaki E, Venihaki M, Chatzaki E, Minas V, Gravanis A, Margioris AN. The corticotropin-releasing factor (CRF) family of peptides as local modulators of adrenal function. Cell Mol Life Sci 2007;64:1638–55. [28] Willenberg HS, Haase M, Papewalis C, Schott M, Scherbaum WA, Bornstein SR. Corticotropin-releasing hormone receptor expression on normal and tumorous human adrenocortical cells. Neuroendocrinology 2005;82:274–81. [29] Audya T, Jain R, Hollander CS. Receptor mediated immunomodulation by corticotropin-releasing factor. Cell Immunol 1991;134:77–84. [30] Zoumakis E, Rice KC, Gold PW, Chrousos GP. Potential uses of corticotropinreleasing hormone antagonists. Ann N Y Acad Sci 2006;1083:239–51. [31] Takahashi K, Totsune K, Saruta M, Fukuda T, Suzuki T, Hirose T, Imai Y, Sasano H, Murakami O. Expression of urocortin 3/stresscopin in human adrenal glands and adrenal tumors. Peptides 2006;27:178–82. [32] Hauger RL, Risbrough V, Brauns O, Dautzenberg FM. Corticotropin releasing factor (CRF) receptor signaling in the central nervous system: new molecular targets. CNS Neurol Disord Drug Targets 2006;5:453–79. [33] Gill GN, Ill CR, Simonian MH. Angiotensin stimulation of bovine adrenocortical cell growth. Proc Natl Acad Sci USA 1977;74:5569–73. [34] Hoeflich A, Bielohuby M. Mechanisms of adrenal gland growth: signal integration by extracellular signal regulated kinases1/2. J Mol Endocrinol 2009;42:191–203. [35] Kageyama K, Furukawa K-I, Miki I, Terui K, Motomura S, Suda T. Vasodilative effects of urocortin II via protein kinase A and a mitogen-activated protein kinase in rat thoracic aorta. J Cardiovasc Pharmacol 2003;42:561–5.