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Endocrinology Vol. 147, No. 9 4454-4462
Copyright © 2006 by The Endocrine Society

Urocortin Induces Interleukin-6 Gene Expression via Cyclooxygenase-2 Activity in Aortic Smooth Muscle Cells

Kazunori Kageyama, Komaki Hanada, Takeshi Nigawara, Takako Moriyama, Ken Terui, Satoru Sakihara and Toshihiro Suda

Department of Endocrinology, Metabolism, and Infectious Diseases, Hirosaki University School of Medicine, Aomori 036-8562, Japan

Address all correspondence and requests for reprints to: Kazunori Kageyama, M.D., The Third Department of Internal Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan. E-mail: kkageyama{at}hkg.odn.ne.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Corticotropin-releasing factor (CRF) plays a central role in controlling stress-related activity of the hypothalamic-pituitary-adrenal axis. Four CRF-related peptides have been found in mammals: CRF and urocortins (Ucns) 1–3. Ucns bound to CRF2ß receptors have a physiological role in the cardiovascular system. We previously found that both Ucn1 and -2 induced accumulation of intracellular cAMP via CRF2ß receptor binding and significantly increased IL-6 secretion by A7r5 aortic smooth muscle cells. In the present study, we investigated Ucn effects on IL-6 gene expression and IL-6 synthesis in A7r5 aortic smooth muscle cells. Ucn1 and -2 stimulated IL-6 gene transcription and IL-6 secretion via CRF2 receptors. Indomethacin, a cyclooxygenase (COX) inhibitor, suppressed IL-6 gene transcription and IL-6 secretion by Ucn1 or -2. NS-398, a COX-2 inhibitor, suppressed IL-6 induction to the same extent as indomethacin. These results suggest that the COX-2 pathway is involved downstream in regulation of Ucn-increased IL-6 gene expression and IL-6 secretion. In addition, COX-2 expression levels were increased at 6 h with the combination of Ucn1 and IL-1, compared with single peptide activation. Ucn1 showed a potent stimulatory effect on IL-6 output, whereas IL-1 alone had no significant effects. However, when Ucn1 was simultaneously used with IL-1, it markedly potentiated the increments in IL-6 output and promoter activity produced by Ucn1. Taken together, these findings indicate that the COX-2 pathway plays a major role in increasing IL-6 levels stimulated by Ucn and IL-1 in A7r5 cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CORTICOTROPIN-RELEASING FACTOR (CRF), a 41-amino-acid polypeptide isolated originally from the ovine hypothalamus, plays a central role in controlling the hypothalamic-pituitary-adrenal axis during stressful periods (1). Four CRF-related peptides have been found in mammals: CRF, urocortin (Ucn) 1 (2), Ucn2 (3, 4), and Ucn3 (stresscopin) (3, 5). Ucn1, a 40-amino-acid peptide cloned from the Edinger-Westphal nucleus, and Ucn2 and -3, identified in the human genome database and mouse genomic DNA, have potent effects on appetite and the cardiovascular system (2, 3, 4, 5, 6, 7).

Actions of the CRF family of peptides are mediated by G protein-coupled receptors derived from two genes: CRF receptor type 1 (8, 9, 10) and CRF receptor type 2 (CRF2 receptor) (11, 12, 13). The two CRF receptors share 69% of their amino acid identify (14), but they have different tissue distributions and pharmacological properties with respect to ligands (15). CRF2 receptor has at least three alternative splice variants: CRF2{alpha} receptor, CRF2ß receptor, and CRF2{gamma} receptor (16). In the rat, CRF2{alpha} receptor mRNA is found primarily in the brain, including sites in the hypothalamus, lateral septum, raphe nuclei of the midbrain, and olfactory bulb (17). In contrast, CRF2ß receptor mRNA is expressed predominantly in peripheral sites such as the heart, blood vessels, gastrointestinal tract, and cardiac and skeletal muscle (17, 18). CRF receptors are coupled positively to adenylate cyclase, a combination that leads to induction of the intracellular secondary messenger, cAMP. The resulting increase in cAMP levels mediates the Ucn-induced vasodilatatory response and cardiac inotropic effects (19, 20), in addition to down-regulating CRF2 receptor gene expression (21).

The three Ucns may serve as natural ligands for CRF2 receptors; these peptides have considerably higher affinities for CRF2 receptor than does CRF itself. In addition, the distribution of Ucn1- and -2-like immunoreactive fibers in the rodent brain correlates with distribution of the CRF2 receptor rather than CRF receptor type 1 (3, 5). Both Ucn1 and -2 and CRF2ß receptor are expressed in the rat heart (4, 18). Furthermore, Ucn1 and -2 are more powerful inotropes than CRF, with a greater potential to increase coronary blood flow and reduce systemic blood pressure (4, 19, 20). These results suggest that endogenous Ucns in combination with CRF2ß receptor have a physiological role in regulation of the cardiovascular system.

The administration of CRF has positive inotropic effects on the heart in vivo and in vitro (22, 23). A recent study illustrated the potent effect of Ucn1 on the cardiovascular system in vivo (24). Ucn1 has more potent vasodilatory and cardiac inotropic and chronotropic effects than CRF (20, 25). Ucn1 produces vasodilation via the adenylate cyclase and protein kinase A (PKA) pathway (19). Furthermore, we demonstrated that the vasodilatory effects of both Ucn2 and -3 are more potent than CRF effects, although less potent than Ucn1 effects (26). Ucns contribute to vasodilation via the p38 MAPK as well as PKA pathways (26).

Cytokine networks are involved in the pathogenesis and progression of numerous vascular diseases such as atherosclerosis, immune arteritis, and Kaposi’s sarcoma. IL-6 is a pleiotropic cytokine with a variety of biological activities. Plasma IL-6 levels rise in response to both immune activation and nonimmune stress (27, 28), with this increase prompting lymphocytic proliferation and differentiation (29) and inducing production of acute-phase proteins in the liver (30). In addition, IL-6 stimulates the hypothalamic-pituitary-adrenal axis (31), leading to an increase in glucocorticoid levels, which in turn causes inhibition of IL-6 levels (32, 33). Therefore, IL-6 is an important mediator of the interaction between the neuroendocrine and immune systems. IL-6 is also expressed in normal arteries as well as atherosclerotic lesions, and its secretion by arterial smooth muscle cells is increased by pathophysiologically relevant factors such as lipopolysaccharide, IL-1, TNF{alpha}, and endothelin. IL-6, a proinflammatory cytokine, has been implicated in development of vascular processes such as atherogenesis.

We previously found that both Ucn1 and -2 induced accumulation of intracellular cAMP via CRF2ß receptor binding and caused a significant increase in IL-6 output levels, and we further demonstrated that both protein kinase C and p38 MAPK signaling cascades were involved downstream of the Ucn-cAMP pathway in A7r5 aortic smooth muscle (AoSM) cells. In the present study, we examined whether Ucns increase IL-6 gene expression in AoSM cells by activating CRF2ß receptors. We further explored the hypothesis that Ucns increase IL-6 gene expression and output via cyclooxygenase (COX)-dependent pathways and investigated the additive effects of IL-1 in this regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Rat or human Ucn1 was purchased from Peptide Institute (Osaka, Japan). Ucn2 and antisauvagine-30 (AS-30) were synthesized by Asahi Techno Glass (Chiba, Japan). Indomethacin and NS-398 were purchased from Calbiochem (San Diego, CA). Lipopolysaccharide was purchased from Sigma (St. Louis, MO) and IL-1ß from Endogen (Woburn, MA).

Cell culture
The rat AoSM cell line, A7r5, was obtained from American Type Culture Collection (Manassas, VA). The A7r5 cells were incubated in DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM [;scap];l[;r];-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin at 37 C in a humidified atmosphere of 5% CO2-95% air. Cells were plated at 104 cells/cm2 7 d before each experiment, with the medium changed every 48 h. On the sixth day, cells were washed and starved overnight with DMEM medium supplemented with 0.2% BSA. On the seventh day, cells were incubated in medium with added vehicle, Ucn1 or -2 with or without medium containing IL-1ß, or one of various inhibitors or an antagonist. The human AoSM cells were obtained from Cambrex Bio Science (Walkersville, MD). The human AoSM cells were incubated in smooth muscle cell basal medium (Cambrex Bio Science) supplemented with 5% FBS, 0.5 µg/ml epithelial growth factor, 5 mg/ml insulin, 1 µg/ml fibroblast growth factor-B, and GA-1000 at 37 C in a humidified atmosphere of 5% CO2-95% air. Cells were plated at 104 cells/cm2 7 d before each experiment, with the medium changed every 48 h. On the sixth day, cells were washed and starved overnight with smooth muscle cell basal medium supplemented with 1% FBS. On the seventh day, cells were incubated in medium with added vehicle, or Ucn1, with or without medium containing IL-1ß. At the end of incubation, total cellular RNA or protein was collected and stored at –80 C until assay was performed. All treatments were performed in triplicate and repeated three times.

IL-6 assay
Serum-starved A7r5 or 1% FBS-incubated human AoSM cells were incubated at 37 C for 48 h with the indicated concentrations of each peptide. The medium was then aspirated, and IL-6 levels in the supernatants were measured using commercial IL-6 ELISA kits (Biosource International, Camarillo, CA). All samples from each experiment were determined in the same assay.

Constructs and transfection
A 544-bp restriction fragment containing the IL-6 promoter (–523 to +21 relative to the proximal transfection start point) was obtained from rat genomic DNA. This DNA fragment was used to produce the IL-6 promoter-driven luciferase reporter construct, pIL-6-Luc, by a two-step cloning method. First, the DNA fragment was cloned into pGEM-T Easy vector (Promega Corp., Madison, WI) and then digested with KpnI and HindIII, and subcloned into KpnI and HindIII cloning sites of the pA3-Luc plasmid.

For luciferase activity assay, cells were placed in 12-well (22 mm diameter) culture dishes at 60% confluency. The next day, cells were transfected following the manufacturer’s instructions using the FuGENE 6 transfection reagent kit (Roche Diagnostics, Indianapolis, IN). FuGENE 3 µl per 1 µg DNA was used. For each well, the total amount of DNA was 0.5 µg. The culture medium was then replaced with DMEM supplemented with 10 or 5% FBS. One day before each experiment, cells were washed and starved overnight with DMEM supplemented with 0.2% BSA or 1% FBS.

IL-6 luciferase activity
Luciferase assay was performed according to the manufacturer’s protocol. At the end of each experiment, cells were washed two times with PBS without Ca2+ and Mg2+, harvested with PicaGene lysis buffer (Toyo Inki, Tokyo, Japan), and centrifuged at 12,000 rpm for 2 min. For the luciferase assay, 20 µl of each supernatant were used. The reactions were started by the injection of 100 µl of luciferin solution, PicaGene buffer. Light output was measured for 20 sec at room temperature using a luminometer (Berthold Lumat LB9501, Postfach, Germany). Activity of ß-galactosidase was used as an internal control.

Western blot analysis
After treatment with Ucn1 or -2, cells were washed twice with PBS and lysed with Laemmli sample buffer. Cell debris was pelleted by centrifugation, and supernatant was recovered. Samples were boiled and subjected to electrophoresis on a gradient (4–20%) polyacrylamide gel. Proteins were transferred to a polyvinyl difluoride membrane (Daiichi Kagaku, Tokyo, Japan). After blocking by detector block (Kirkegaard & Perry Laboratories, Gaithersburg, MD), the membrane was incubated for 1 h with a rabbit anti-COX-2 antibody or anti-ß2 microglobulin (B2M) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), washed with PBS containing 0.05% Tween 20, and incubated with horseradish peroxidase-labeled antirabbit IgG (Daiichi Kagaku). Detection was performed using a chemiluminescent substrate Supersignal WestPico (Pierce Chemical Co., Rockford, IL), and the membrane was exposed to BioMax film (Eastman Kodak Co., Rochester, NY).

RNA extraction
Cellular total RNA was extracted using the RNeasy minikit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Then cDNAs were synthesized from total RNA (0.5 µg) using random hexamer as a primer with the SuperScript first-strand synthesis system for RT-PCR kit (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s instructions.

Real-time RT-PCR
Resulting cDNAs were subjected to real-time PCR as follows. The expression level of rat COX-2 mRNA was 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). B2M was used as a housekeeping gene to normalize values. Each reaction consisted of 1x TaqMan universal PCR master mix (Applied Biosystems), 1x Assays-on-Demand gene expression products (Rn00568225 m1 for rat COX-2; Rn00560865 m1 for rat B2M), and 2 µl cDNA in a total volume of 50 µl using the following parameters with an 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 TaqMan probe spanning exon/exon junctions and therefore should not 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 threshold cycle (CT). Using diluted samples, the amplification efficacies for each gene of interest and the housekeeping gene amplimers were found to be identical.

Relative quantitative gene expression
Relative quantitative gene expression was calculated with the 2–{Delta}{Delta}CT method (34). In brief, for each sample assayed, the CT for reactions amplifying a gene of interest (rat COX-2) and a housekeeping gene (rat B2M) was determined. The gene of interest CT for each sample was corrected by subtracting the CT for the housekeeping gene ({Delta}CT). Untreated controls were chosen as reference samples, and the {Delta}CT for all experimental samples was subtracted by the average {Delta}CT for the control samples ({Delta}{Delta}CT). Finally, experimental mRNA abundance relative to control mRNA abundance was calculated with use of the formula 2–{Delta}{Delta}CT.

Statistical analysis
All values are expressed as the mean ± SEM. Statistical analyses of data were performed using one-way or two-way ANOVA with repeated measures with dose and treatment as dependent variables, followed by Scheffé’s F post hoc test. The level of statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of Ucn1 or -2 on IL-6 luciferase activity in A7r5 cells
Time- and dose-dependent changes in IL-6 luciferase activity were examined to determine Ucn1 or -2 effects. As shown in Fig. 1Go, A and B, Ucn1 and -2 potently stimulated IL-6 gene expression in a time- and dose-dependent manner. The time-course study showed that the maximal effect was observed at 24–96 h, with an approximately 2.5- and 2.2-fold increase, compared with the basal level, after stimulation with Ucn1 or -2, respectively (Fig. 1AGo). The dose-response study showed that the significant stimulatory effects of Ucn1 and -2 were observed at 0.1 nM and were maximal above 100 nM (ANOVA; P < 0.0001) (Fig. 1BGo). AS-30, a potent and selective CRF2 receptor antagonist suppressed Ucn1-increased IL-6 gene expression significantly in a dose-dependent manner (ANOVA; P < 0.0001). These results indicate that Ucn1 and -2 stimulate IL-6 gene expression at the transcriptional level via CRF2 receptor.


Figure 1
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  		FIG. 1. Effects of Ucn1 or -2 on IL-6 luciferase activity in A7r5 cells. Cells were treated in triplicate, with the mean of three independent experiments (an average in triplicate was considered n = 1; three experiments n = 3) shown in figures. Statistical analyses were performed using one-way ANOVA followed by Scheffé’s F post hoc test. A, Time-dependent changes in Ucn1- or -2-induced IL-6 luciferase activity. Cells were incubated with medium containing 100 nM Ucn1 or -2 for the durations shown. B, Dose-dependent changes in Ucn1- or -2-induced IL-6 luciferase activity. Cells were incubated for 24 h with medium alone (control) or medium containing Ucn1 (open circle), Ucn2 (closed circle), AS-30 (closed square), or 100 nM Ucn1 with increasing concentrations of AS-30 (open square).

 
Effects of COX inhibitors on Ucn1- or -2-induced IL-6 output or luciferase activity
To determine whether a COX pathway is involved in regulation of IL-6 output levels by Ucn, A7r5 cells were incubated with different types of COX inhibitors and either Ucn1 or 2 (100 nM). Both indomethacin (1 µM), a nonselective COX inhibitor, and NS-398 (10 µM), a selective COX-2 inhibitor, blocked the ability of either Ucn1 or -2 to increase IL-6 levels (Fig. 2AGo). Furthermore, Ucn1 and -2 effects on IL-6 promoter activity were determined. As shown in the IL-6 output study, the stimulatory effects of both hormones were completely abolished under treatment with indomethacin or NS-398 (Fig. 2BGo). There was no significant difference between the two inhibitors. These results suggest that the effects of Ucn1 and -2 on IL-6 output and gene expression are mediated through a COX, especially a COX-2, pathway.


Figure 2
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  		FIG. 2. Effects of COX inhibitors on Ucn1- or -2-induced IL-6 output or luciferase activity in A7r5 cells. Cells were treated in triplicate, with the mean of three independent experiments (an average in triplicate was considered n = 1; three experiments n = 3) shown in figures. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post hoc test. A, Effects on Ucn1- or -2-induced IL-6 output. Cells were preincubated with medium containing indomethacin (INDO), NS-398 (NS), or vehicle for 30 min and then incubated for 48 h with medium containing 100 nM Ucn1 or -2 or vehicle. *, P < 0.0005 (compared with only Ucn1); #, P < 0.0005 (compared with only Ucn2). B, Effects on Ucn1- or -2-induced IL-6 luciferase activity. Cells were preincubated with medium containing indomethacin (INDO), NS-398 (NS), or vehicle for 30 min and then incubated for 24 h with medium containing 100 nM Ucn1 or -2 or vehicle. *, P < 0.05 (compared with only Ucn1); #, P < 0.05 (compared with only Ucn2).

 
Effects of Ucn1 or -2 on C0X-2 protein levels
We then determined whether COX-2 protein levels were changed by treatment with Ucn1 or -2. Figure 3Go, A and B, shows the effects of Ucn1 and -2 on COX-2 protein levels, respectively. As shown in Fig. 3Go, A and B, incubation with 100 nM of Ucn1 or -2 increased COX-2 protein levels from 1 to 6 h after the addition. Incubation with Ucn1 or -2 for 6 h significantly increased COX-2 protein levels in a dose-dependent manner. A CRF2 receptor antagonist, AS-30, suppressed the Ucn effect on increasing COX-2 protein levels (Fig. 3CGo).


Figure 3
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  		FIG. 3. Effects of Ucn1 or -2 on C0X-2 protein levels in A7r5 cells. A, Time- and dose-dependent changes in Ucn1-induced COX-2 protein levels. For time, cells were incubated with medium containing 100 nM Ucn1 for durations shown. For dose, cells were incubated for 6 h with medium containing 1 nM-1 mM Ucn1. Cells treated with medium alone are indicated as C. B, Time- and dose-dependent changes in Ucn2-induced COX-2 protein levels. For time, cells were incubated with medium containing 100 nM Ucn2 for durations shown. For dose, cells were incubated for 6 h with medium containing 1 nM to 1 mM Ucn2. Cells treated with medium alone are indicated as C. C, Effects of AS-30 on Ucn1 or -2-induced COX-2 protein changes. Cells were preincubated with medium containing 100 nM AS-30 or vehicle for 30 min and then incubated for 6 h with medium containing 100 nM Ucn1 or -2 or vehicle. B2-MG, ß2 Microglobulin.

 
Effects of Ucn1 or -2 on COX-2 mRNA
To examine whether COX-2 mRNA levels were changed by treatment with Ucn1 or -2, A7r5 cells were incubated with Ucn1 or -2. As shown in Fig. 4AGo, incubation with Ucn1 or -2 significantly decreased COX-2 mRNA levels (ANOVA; P < 0.0001). COX-2 mRNA levels transiently fell to 31.9 ± 1.6 and 22.0 ± 1.5% of the control value within 2 h after treatment with 100 nM Ucn1 and -2, respectively, before levels recovered to control values after 12 h of treatment (Fig. 4AGo). A 2-h incubation with Ucn1 or -2 significantly decreased COX-2 mRNA levels in a dose-dependent manner (ANOVA; P < 0.0001) (Fig. 4BGo). AS-30 suppressed Ucn1- or -2-mediated decreases in COX-2 mRNA levels (Fig. 4CGo).


Figure 4
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  		FIG. 4. Effects of Ucn1 or -2 on COX-2 mRNA levels in A7r5 cells. Cells were treated in triplicate, with the mean of three independent experiments (an average in triplicate was considered n = 1; three experiments n = 3) shown in figures. Statistical analyses were performed using one-way ANOVA followed by Scheffé’s F post hoc test. A, Time-dependent changes in Ucn1- or -2-induced COX-2 mRNA levels. Cells were incubated with medium containing 100 nM Ucn1 or -2 for the durations shown. B, Dose-dependent changes in Ucn1- or 2-induced COX-2 mRNA levels. Cells were incubated for 2 h with medium containing increasing 0.01–100 nM Ucn1 or -2. *, P < 0.05; **, P < 0.005; ***, P < 0.0005 (compared with control). C, Effects of AS-30 on Ucn1- or -2-induced COX-2 mRNA levels. Cells were preincubated with medium containing 100 nM AS-30 or vehicle for 30 min and then incubated for 2 h with medium containing 100 nM Ucn1 or -2 or vehicle. *, P < 0.01 (compared with control); +, P < 0.01 (compared with Ucn1 or -2).

 
Effects of IL-1 on Ucn1-induced IL-6 output or luciferase activity
CRF family peptides are known to have a network with cytokines such as IL-1. Furthermore, IL-1 works cooperatively with IL-6 in inflammation. Therefore, to examine possible cooperation between cytokines and Ucn, the synergistic effects of the combination of Ucn1 and IL-1 on COX-2 protein and IL-6 gene expression and output were examined. COX-2 protein expression levels showed an additional increase at 6 h with Ucn1 and IL-1, whereas COX-2 mRNA levels showed an additional and significant decrease with Ucn1 and IL-1 (Fig. 5AGo). Ucn1 showed a potent stimulatory effect on IL-6 output, whereas IL-1 alone had no significant effects in A7r5 cells (Fig. 5BGo). When IL-1 was simultaneously used with Ucn1, it markedly potentiated the increments produced by Ucn1 alone in a dose-dependent manner (ANOVA; P < 0.0001) (Fig. 5BGo). Similarly, there was a tend toward a greater increase in IL-6 promoter activity when Ucn1 was simultaneously used with IL-1, compared with the activity produced by Ucn1 alone (Fig. 5CGo). In human AoSM cells, Ucn1 showed no significant effects on IL-6 output, whereas IL-1 alone had a potent stimulatory effect (Fig. 6AGo). When IL-1 was simultaneously used with Ucn1, the combination showed an additive effect on IL-6 output (Fig. 6AGo). Similarly, Ucn1 showed a stimulatory, but not significantly, effect on IL-6 promoter activity in human AoSM cells, whereas IL-1 alone had a potent stimulatory effect (Fig. 6BGo). The combination also showed a greater increase in IL-6 promoter activity when Ucn1 was simultaneously used with IL-1, compared with the activity produced by Ucn1 or IL-1 alone, in human AoSM cells (Fig. 6BGo).


Figure 5
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  		FIG. 5. Effects of IL-1 on Ucn1-induced IL-6 output or luciferase activity in A7r5 cells. Cells were treated in triplicate, with the mean of three independent experiments (an average in triplicate was considered n = 1; three experiments n = 3) shown in figures. Statistical analyses were performed using one-way ANOVA followed by Scheffé’s F post hoc test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. A, Synergistic effects of IL-1 and Ucn1 on COX-2 protein (left panel) and mRNA (right panel) levels. Cells were incubated for 6 h with medium containing 500 pM IL-1 and 100 nM Ucn1 or vehicle. B, Effects of IL-1 on Ucn1-induced IL-6 output. Cells were incubated for 48 h with medium alone (control) or medium containing 100 nM Ucn1 and 500 pM IL-1 or 100 nM Ucn1 with increasing concentrations of IL-1 (5–500 pM). C, Effects of IL-1 on Ucn1-induced IL-6 luciferase activity. Cells were incubated for 24 h with medium alone (control) or medium containing 100 nM Ucn1, 500 pM IL-1, or 100 nM Ucn1 with increasing concentrations of IL-1 (5–500 pM).

 

Figure 6
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  		FIG. 6. Effects of IL-1 on Ucn1-induced IL-6 output or luciferase activity in human AoSM cells. Cells were treated in triplicate, with the mean of three independent experiments (an average in triplicate was considered n = 1; three experiments n = 3) shown in figures. Statistical analyses were performed using one-way ANOVA followed by Scheffé’s F post hoc test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. A, Effects of IL-1 on Ucn1-induced IL-6 output. Cells were incubated for 24 h with medium alone (control) or medium containing 100 nM Ucn1 and/or 5 pM IL-1. B, Effects of IL-1 on Ucn1-induced IL-6 luciferase activity. Cells were incubated for 24 h with medium alone (control) or medium containing 100 nM Ucn1 and/or 5 pM IL-1.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here the effects of Ucn1 and -2 on transcription of the IL-6 gene using A7r5 rat AoSM cells transfected with the IL-6 5'-promoter-luciferase fusion gene. The luciferase assay would closely reflect real-time changes in promoter activity because reporter protein activity reflects only changes in transcription rate. Using this model system, we found that Ucn1 and -2, which have similar activities, increased IL-6 5'-promoter activity in these AoSM cells. These results are in agreement with those of previous studies, which showed that IL-6 gene expression was increased by Ucn (35). Furthermore, we found that Ucn1-increased IL-6 5'-promoter activity was completely blocked by AS-30, a potent and selective CRF2 receptor antagonist. Together, Ucn1 and -2 stimulate IL-6 gene transcription and secretion via CRF2 receptor action.

We also attempted to clarify which COX pathway was involved in regulation of IL-6 gene transcription and secretion by Ucn1 or -2 by using the inhibitors. From previous studies, it is known that indomethacin inhibits both COX-1 (IC50 = 740 nM) and COX-2 (IC50 = 970 nM) (36). We established that a COX pathway is involved downstream in regulation of IL-6 gene transcription and secretion by Ucn1 or -2 because indomethacin (1 µM) suppressed their effects. NS-398 is a selective inhibitor of COX-2 because it inhibits COX-2 (IC50 = 3.8 µM), whereas COX-1 activity is unaffected at concentrations up to 100 µM in vitro (36). Therefore, in particular, it is clear that COX-2 contributes to increased IL-6 gene expression and output levels because NS-398 (10 µM), a COX-2 inhibitor, suppressed IL-6 induction to the same extent as indomethacin. We then studied whether Ucn1 and -2 would increase COX-2 protein levels. Both Ucn1 and -2 increased COX-2 protein levels in a time- and dose-dependent manner. Taken together, these observations indicate that Ucn stimulated IL-6 gene expression and output levels via COX-2 activity in A7r5 cells. COX-2 expression represents an early and sensitive response of smooth muscle cells to inflammatory injury, and it is possible that COX-2-derived metabolites influence many vascular functions, such as vascular tone, thromboresistance, and vascular growth.

We previously demonstrated that both Ucn1 and -2 induced accumulation of intracellular cAMP via CRF2ß receptors and caused a significant increase in IL-6 output levels (35). Whereas a PKA inhibitor had no effect on the increase in IL-6 concentration, a protein kinase C inhibitor significantly inhibited IL-6 output levels. Blockade of Ucn-induced increases in IL-6 levels by a p38 MAPK inhibitor suggests that the p38 MAPK pathway is involved in this regulation. The cAMP-mediated increase in IL-6 levels was suppressed synergistically by both inhibitors. Ucn may stimulate protein kinase C via cAMP indirectly because the cAMP would prompt the increase in intracellular Ca2+ or diacylglycerol production (37). Therefore, it is possible that COX-2 is regulated by the cAMP-mediated protein kinase C and p38 MAPK pathway, but not PKA, through CRF2ß receptor in A7r5 cells. Furthermore, both protein kinase C and p38 MAPK pathways contribute to COX-2 expression in human tracheal smooth muscle cells (38). In AoSM cells, protein kinase C pathway is involved in the increase in COX-2 levels induced in bradykinin (39). Together, these findings from the current study may suggest that both protein kinase C and p38 MAPK signaling cascades are involved in the COX-2 regulation pathway in the smooth muscle cells.

In this study, COX-2 protein levels increased after Ucn stimulation of A7r5 cells, whereas mRNA levels decreased after treatment. Expression levels of mRNA are determined mainly by both transcriptional activity, such as synthesis of mRNA, and posttranscriptional activity, such as the rate of mRNA degradation (mRNA stability) (40, 41). Therefore, in A7r5 cells, it is possible that stimulation of translation by CRF increases COX-2 mRNA use and degradation of its mRNA. We previously found such discrimination in murine corticotrophs in the regulation of G protein-coupled receptor kinase 2 after treatment with CRF (42). CRF family peptides may prompt targeted mRNA use and the degradation of mRNA, resulting in decreases in mRNA levels.

CRF family peptides have an associated network with cytokines (43). IL-1 works cooperatively with IL-6 in inflammation. IL-1 is known to be a major stimulus for both IL-6 production and COX-2 expression in other vascular smooth muscle cells (44). IL-1 potentiates CRF-induced proopiomelanocortin gene regulation (45). We therefore tried to clarify synergistic effects of Ucn and IL-1 on COX-2 protein and mRNA and IL-6 output and gene expression levels. COX-2 expression levels were higher 6 h after treatment with Ucn1 and IL-1, compared with the increase seen with single peptide activation, whereas COX-2 mRNA levels were decreased after treatment with Ucn1 and IL-1. This result may indicate that in A7r5 cells, stimulation of translation by Ucn1 and IL-1 facilitates COX-2 mRNA use and the degradation of its mRNA, resulting in decreases in its mRNA levels. Ucn1 showed a potent stimulatory effect on IL-6 output, whereas IL-1 alone had no significant effects in A7r5 cells. However, when Ucn1 was simultaneously used with IL-1, it markedly potentiated the increments in IL-6 output and promoter activity produced by Ucn1. IL-1 also down-regulates CRF2ß receptor mRNA levels (46). In present studies, it is possible that, after stimulation of CRF2ß receptor by Ucn1, the upstream of COX-2 activation pathway contributes to the additional effect of IL-1, although it is unclear whether IL-1 receptor can form a complex with CRF2 receptor. Taken together, these findings indicate that the COX-2 pathway plays a major role in A7r5 cells in increasing IL-6 levels after exposure to Ucn and IL-1. Although IL-1 alone had a minimal effect on IL-6 production, IL-1 had an effect additional to that of Ucn on regulation of both IL-6 synthesis and secretion via the COX-2 pathway.

We further tried to extend to a more physiological and human model by using human AoSM cells. In this human model, IL-1 alone, but not Ucn1, had a potent effect on IL-6 production. Even so, the additional effect of IL-1 and Ucn1 was produced in the human cells. The human cells required at least 1% FBS. It is unclear whether this discrepancy may be caused by the differences between cell types or between the cell culture circumstances including FBS. Further studies in the human, therefore, would be required to elucidate whether the responses produced by Ucns in the rat are shown in the human.

Vasodilatory effects of Ucn have been demonstrated in rat tail and basilar arteries (19). In our previous study, Ucn1, -2, and -3 were more potent vasodilators than CRF in a rat thoracic aorta model. In vascular smooth muscle cells, stimulation of CRF2ß receptors results in increased cAMP accumulation via activation of adenylate cyclase (21). It is at least possible that increased cAMP levels contribute to vasorelaxant responses, although the role of cGMP remains unclear.

In addition, Ucn1 and -2 had strong effects on IL-6 gene expression and secretion. The increase that we observed in IL-6 levels after Ucn treatment of A7r5 cells suggests that smooth muscle cells may be a source of IL-6 secretion under physiological stress conditions. The significance of high IL-6 production by Ucn in vascular cells is, however, still unclear. IL-6 is involved in atherosclerosis and other inflammatory processes in blood vessels. We demonstrated previously that Ucn directly down-regulates CRF2ß receptor mRNA levels (18). Because cytokines such as IL-1 and IL-6 both decrease CRF2ß mRNA expression (18, 46), it is possible that Ucn and IL-6 contribute cooperatively to regulate the levels of CRF2ß receptor mRNA in vascular cells. IL-6 may act as an autocrine and paracrine factor in the vessel wall.

In conclusion, this study demonstrated that Ucn1 and -2 stimulate IL-6 gene transcription and secretion via CRF2 receptor activity. The COX-2 pathway is involved downstream in regulation of IL-6 gene transcription and secretion by Ucn1 or -2 in our model, A7r5 AoSM cells. Ucns are important and unique modulators of vascular smooth muscle cells and act directly or indirectly as autocrine and paracrine factors in the vessel wall.


   Footnotes
 
We are grateful to Dr. G. Gaudriault and Dr. W. Vale for critical suggestions.

This work was supported in part by Health and Labor Science research grants (Research on Measures for Intractable Diseases) from the Ministry of Health, Labor, and Welfare of Japan and Grant 15590966 from the Ministry of Education, Science and Culture of Japan (to T.S.). K.K. is supported by a grant from the Funds for the Promotion of Aomori Medical Research and the Promotion of International Scientific Research.

There was no National Institutes of Health funding.

Disclosure: K.K., K.H., T.N., T.M., K.T., S.S., and T.S. have nothing to declare.

First Published Online June 15, 2006

Abbreviations: AoSM, Aortic smooth muscle; AS-30, antisauvagine-30; B2M, ß2 microglobulin; COX, cyclooxygenase; CRF, corticotropin-releasing factor; CRF2 receptor, CRF receptor type 2; CT, threshold cycle; FBS, fetal bovine serum; PKA, protein kinase A; Ucn, urocortin.

Received January 4, 2006.

Accepted for publication June 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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