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Endocrinology Vol. 144, No. 6 2234-2241
Copyright © 2003 by The Endocrine Society

Urocortin-Related Peptides Increase Interleukin-6 Output via Cyclic Adenosine 5'-Monophosphate-Dependent Pathways in A7r5 Aortic Smooth Muscle Cells

Kazunori Kageyama and Toshihiro Suda

The Third Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki, 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 receptor type 2ß, expressed in the rodent cardiovascular system, is a member of the G protein-coupled receptor family. This receptor is coupled positively to adenylate cyclase and is bound preferentially by the urocortin (Ucn)-related peptides (Uncs): Ucn, Ucn II, and Ucn III. In the present study, we investigated the effects of Ucns on IL-6 levels in A7r5 aortic smooth muscle cells. In this cell line, both Ucn and Ucn II induced accumulation of intracellular cAMP via corticotropin-releasing factor receptor type 2ß and also caused a significant increase in IL-6 output levels. The adenylate cyclase inhibitor, MDL-12330A, inhibited this Ucn- or Ucn II-induced increase in IL-6 levels. Although H89 (10 µM), a protein kinase A inhibitor, had no effect on the increase in IL-6 concentration, bisindolylmaleimide I (10 nM), a protein kinase C inhibitor, was found to significantly inhibit IL-6 output levels. Blockade of Ucn- or Ucn II-induced increases in IL-6 levels by SB203580 (100 nM), a p38 MAPK inhibitor, suggested that the p38 MAPK pathway was involved in this regulation. The cAMP-mediated increase in IL-6 levels was suppressed synergistically by both bisindolylmaleimide I and SB203580. These findings demonstrate that both protein kinase C and p38 MAPK signaling cascades are involved downstream of the Ucns-cAMP pathway in A7r5 aortic smooth muscle cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTERLEUKIN-6 IS A pleiotropic cytokine with a variety of biological activities. Plasma IL-6 levels are elevated in response to both immune activation and nonimmune stress (1, 2), with this increase prompting the proliferation and differentiation of lymphocytes (3) and inducing production of acute-phase proteins in the liver (4). In addition, IL-6 stimulates the hypothalamic-pituitary-adrenal (HPA) axis (5), leading to an increase in glucocorticoid levels, which in turn cause an inhibition of IL-6 levels (6, 7). Therefore, IL-6 is an important mediator of the interaction between the neuroendocrine and immune systems.

Corticotropin-releasing factor (CRF), a 41-amino-acid polypeptide isolated originally from the ovine hypothalamus, plays a central role in controlling the HPA axis during stress (8). Four CRF-related peptides have been found in mammals and are termed CRF, urocortin (Ucn; Ref. 9), Ucn II (10, 11), and Ucn III (e.g. stresscopin; Refs. 11 and 12). Ucn, a 40-amino-acid peptide cloned from the Edinger-Westphal nucleus, and Ucn II and III, identified in the human genome database and in mouse genomic DNA, have potent effects on appetite and the cardiovascular system (9, 10, 11, 12, 13, 14).

In addition to activation of the HPA axis, CRF modulates immune responses in the periphery by both direct and indirect mechanisms (6). Both CRF and Ucn are positioned to influence immune functions, as CRF mRNA has been detected in rodent thymus and spleen (15), and Ucn mRNA and peptide have been found in human lymphocytes (16) and rat thymus and spleen (17). In vitro studies have shown that CRF stimulates B and T lymphocyte proliferation (18, 19), whereas an in vivo study in rats found that peripheral CRF family peptides initiated an increase in plasma IL-6 levels in response to stressors (20). Although induction of IL-6 by Ucn has been demonstrated in A7r5 cell culture by Gaudriault, G. E., and W. W. Vale (unpublished observations), the intracellular signaling mechanism of this induction has not been clarified.

The actions of the CRF-family peptides are mediated by G protein-coupled receptors derived from two genes, namely the CRF receptor (CRF R) type 1 (CRF R1; Refs. 21, 22, 23) and CRF R type 2 (CRF R2; Refs.24, 25, 26). These two CRF Rs share a 69% amino acid identify (21, 27) but have different tissue distributions and pharmacological properties with respect to ligands (28). CRF R2 has at least three alternative splice variants, CRF R2{alpha} (24), CRF R2ß (25, 26, 27), and CRF R2{gamma} (29). In the rat, CRF R2{alpha} mRNA is found primarily in the brain, which includes sites in the hypothalamus, lateral septum, raphe nuclei of the midbrain, and the olfactory bulb (30). In contrast, CRF R2ß mRNA is expressed predominantly in peripheral sites such as the heart, blood vessels, gastrointestinal tract, and cardiac and skeletal muscles (30, 31). CRF Rs 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 actions (32, 33) in addition to down-regulating CRF R2 gene expression (31).

Three Ucn-related peptides (Ucns) may serve as natural ligands for CRF R2, as these peptides have considerably higher affinities for CRF R2 than CRF. In addition, the distribution of Ucn- and Ucn III-like immunoreactive fibers in the rodent brain correlates with CRF R2, rather than CRF R1 (10, 12). Both Ucn/Ucn II and CRF R2ß are expressed in the rat heart (11, 31). Furthermore, Ucn and Ucn II are more powerful inotropes than CRF, with a greater potential to increase coronary blood flow and reduce overall blood pressure (12, 32, 33). These results suggest that endogenous Ucns, in combination with CRF R2ß, have a physiological role in the cardiovascular system.

In the present study, we examined whether Ucns increased IL-6 output levels in aortic smooth muscle cells by activating CRF R2ß. We confirmed the hypothesis that Ucns increase IL-6 levels via cAMP-dependent pathways and investigated the involvement of protein kinase A (PKA), protein kinase C (PKC), and MAPKs in this regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Rat CRF and Ucn were purchased from Peptide Institute (Osaka, Japan). Mouse Ucn II and antisauvagine-30 were synthesized by Asahi Techno Glass (Chiba, Japan). Bisindolylmaleimide I (BIM), MDL-12330A, H89, SB203580, and PD98059 were purchased from Calbiochem (San Diego, CA). Dibutyryl (db)-cAMP was purchased from Sigma (St. Louis, MO).

Cell culture
The aortic smooth muscle cell line, A7r5, was obtained from American Type Culture Collection (Manassas, VA). The A7r5 cells were incubated in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin at 37 C in a humidified atmosphere of 5% CO2 and 95% air. Cells were plated initially at 104 cells/cm2 7 d before each experiment, with the medium being changed every 48 h. On the sixth day, the cells were washed and starved overnight with DMEM supplemented with 0.2% BSA. On the seventh day, the cells were incubated in medium with added vehicle, cAMP analogs, or Ucns, with or without medium containing the various inhibitors. All treatments were performed in triplicate and repeated three times.

cAMP assay
Serum-starved A7r5 cells were preincubated for 20 min with 0.1 mM of 3-isobutyl-1-methylxanthine in assay medium, and then treated at 37 C for 20 min with the indicated concentrations of each peptide. The medium was aspirated and cells extracted with 1 ml of 95% ethanol containing 0.1 N HCl. cAMP content was measured in the supernatants using commercial cAMP enzyme immunoassay kits (Amersham Pharmacia Biotech, Little Chalfont, UK).

IL-6 assay
Serum-starved A7r5 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 measured using commercial IL-6 ELISA kits (BioSource International, Camarillo, CA). All the samples from each experiment were determined in the same assay.

Statistical analysis
All values are expressed as the mean ± SEM. Statistical analyses of the data were performed using one-way ANOVA, or two-way ANOVA with repeated measures with dose and treatment as the 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 CRF-family peptides on cAMP accumulation in A7r5 aortic smooth muscle cells
A7r5 aortic smooth muscle cells were incubated with CRF, Ucn, or Ucn II (Fig. 1Go). In controls, the absolute value of cAMP accumulation was 48 fmol per assay well. Incubation with 0.1 nM–1 µM of Ucn or Ucn II increased cAMP accumulation in a dose-dependent manner (ANOVA; P < 0.0001). Incubation with Ucn or Ucn II for 20 min resulted in a significant increase in cAMP accumulation compared with CRF (EC50 values for Ucn, Ucn II, and CRF were 0.3, 1.3, and 40 nM, respectively). Figure 1Go also demonstrates the effects on Ucn II (10 nM)-induced cAMP accumulation levels following antisauvagine-30 administration over the concentration range of 0.1 nM–1 µM. Antisauvagine-30 produced a significant dose-dependent decrease in Ucn II-induced cAMP accumulation (ANOVA; P < 0.0001).



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  		Figure 1. Effects of CRF-family peptides on cAMP accumulation in A7r5 aortic smooth muscle cells. Cells were preincubated for 20 min with medium containing 0.1 mM of 3-isobutyl-1-methylxanthine, followed by the addition of each peptide. Cells were incubated for 20 min with medium alone (control), or with medium containing CRF (open triangle), Ucn (open circle), Ucn II (open square), antisauvagine-30 (closed triangle), and 10 nM of Ucn II with increasing concentrations of antisauvagine-30 (closed square). Intracellular cAMP was measured by EIA. Results shown are representative of three independent experiments. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post hoc test.

 
Effects of CRF-family peptides on IL-6 levels in A7r5 aortic smooth muscle cells
To examine whether Ucns induced a functional response in the smooth muscle cells, we examined the effects of CRF family peptides on IL-6 levels (Fig. 2Go). Incubation with 0.1 nM–1 µM of Ucn or Ucn II increased IL-6 levels in a dose-dependent manner (ANOVA; P < 0.0001). The effect of Ucn II on IL-6 level was more potent than CRF but less potent than Ucn (EC50 values for Ucn, Ucn II, and CRF were 1.2, 7.9, and 316 nM, respectively). Antisauvagine-30 suppressed the Ucn (100 nM)- or Ucn II (100 nM)-increased IL-6 levels significantly in a dose-dependent manner (ANOVA; P < 0.0001).



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  		Figure 2. Effects of CRF family peptides on interleukin-6 levels in A7r5 aortic smooth muscle cells. Cells were incubated for 48 h with medium alone (control) or with medium containing CRF (open triangle), Ucn (open circle), Ucn II (open square), antisauvagine-30 (closed triangle), 100 nM of Ucn with increasing concentrations of antisauvagine-30 (closed circle), and 100 nM of Ucn II with increasing concentrations of antisauvagine-30 (closed square). IL-6 levels in the medium were measured by ELISA. Results shown are representative of three independent experiments. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post hoc test.

 
Effects of MDL-12330A on Ucn- or Ucn II-induced IL-6 levels in A7r5 aortic smooth muscle cells
To determine whether adenylate cyclase was involved in the Ucns-induced IL-6 pathway, A7r5 cells were incubated with 0.02–200 µM of MDL-12330A, an adenylate cyclase inhibitor, and either Ucn or Ucn II. As shown in Fig. 3Go, MDL-12330A blocked the effects of Ucn (100 nM) and Ucn II (100 nM) on IL-6 levels in a dose-dependent manner (ANOVA; P < 0.0001).



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  		Figure 3. Effects of MDL-12330A on Ucn- or Ucn II-induced IL-6 levels in A7r5 aortic smooth muscle cells. Cells were incubated for 48 h in either medium-added vehicle, Ucn (100 nM), or Ucn II (100 nM), with or without medium containing increasing concentrations of MDL-12330A (0.02–200 µM). Cells treated with MDL-12330A alone are indicated as MDL. Results shown are representative of three independent experiments. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post hoc test. *, P < 0.0001 (compared with Ucn). #, P < 0.05 (compared with Ucn II). ##, P < 0.0001 (compared with Ucn II).

 
Effects of H89 or BIM following Ucn or Ucn II treatment on IL-6 levels in A7r5 aortic smooth muscle cells
To determine which protein kinase is involved in the regulation of IL-6 output levels, A7r5 cells were incubated with various protein kinase inhibitors and either Ucn (100 nM) or Ucn II (100 nM). Only the maximal dose (30 µM), but not 0.1–10 µM, of H89, a PKA inhibitor, blocked the ability of either Ucn or Ucn II to increase IL-6 levels (Fig. 4AGo). On the other hand, BIM, a PKC inhibitor, dose-dependently (0.01–1 µM) inhibited Ucn- or Ucn II-induced IL-6 levels (ANOVA; P < 0.0001; Fig. 4BGo).



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  		Figure 4. Effects of H89 or BIM on Ucn- or Ucn II-induced IL-6 levels in A7r5 aortic smooth muscle cells. Results shown are representative of three independent experiments. Statistical analyses were carried out using one-way ANOVA, followed by Scheffé’s F post hoc test. *, P < 0.0001 (compared with Ucn). #, P < 0.05 (compared with Ucn II). ##, P < 0.0001 (compared with Ucn II). A, Cells were incubated for 48 h with either medium-added vehicle, Ucn (100 nM), or Ucn II (100 nM), with or without medium containing increasing concentrations of H89. Cells treated with H89 alone are indicated as H89. B, Cells were incubated for 48 h with either medium-added vehicle, Ucn (100 nM), or Ucn II (100 nM), with or without medium containing increasing concentrations of BIM. Cells treated with BIM alone are indicated as BIM.

 
Effects of SB203580 or PD98059 following Ucn or Ucn II treatment on IL-6 levels in A7r5 aortic smooth muscle cells
We investigated, by incubating A7r5 cells with either SB203580 or PD98059 and either Ucn or Ucn II, whether MAPK was involved in the Ucn-induced IL-6 pathway. As shown in Fig. 5Go, A and B, 0.1–10 µM of SB203580, but not PD98059, significantly attenuated the effects of Ucn (100 nM) or Ucn II (100 nM) on IL-6 levels.



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  		Figure 5. Effects of SB203580 or PD98059 on Ucn- or Ucn II-induced IL-6 levels in A7r5 aortic smooth muscle cells. Results shown are representative of three independent experiments. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post hoc test. *, P < 0.005 (compared with Ucn or Ucn II). **, P < 0.001 (compared with Ucn or Ucn II). A, Cells were incubated for 48 h with medium-added vehicle or Ucn (100 nM), with or without medium containing increasing concentrations of SB203580 or PD98059. Cells treated with SB203580 alone or PD98059 alone are indicated as SB or PD, respectively. B, Cells were incubated for 48 h with medium-added vehicle or Ucn II (100 nM), with or without medium containing increasing concentrations of SB203580 or PD98059.

 
Synergistic effects of BIM and SB203580 on Ucn- or Ucn II-induced IL-6 levels in A7r5 aortic smooth muscle cells
To examine whether BIM and SB203580 inhibited Ucn (100 nM)- or Ucn II (100 nM)-induced IL-6 output levels synergistically, both compounds were incubated with either Ucn or Ucn II. Figure 6Go shows that both BIM (1 µM) and SB203580 (10 µM) caused a significant suppression of IL-6 induction, whereas concomitant administration of BIM (1 µM) and SB203580 (10 µM) completely blocked IL-6 output levels (two-way ANOVA; P < 0.0001, followed by Scheffé’s F post hoc test).



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  		Figure 6. Synergistic effects of BIM and SB203580 on Ucn- or Ucn II-induced IL-6 levels in A7r5 aortic smooth muscle cells. Cells were incubated for 48 h with medium-added vehicle, Ucn, or Ucn II (100 nM), with or without medium containing increasing concentrations of BIM and/or SB203580. Results shown are representative of three independent experiments. Statistical analyses were performed using two-way ANOVA, followed by Scheffé’s F post hoc test. *, P < 0.0001 (compared with Ucn). +, P < 0.05 (compared with Ucn II). ++, P < 0.0001 (compared with Ucn II). #, P < 0.0001 (compared with BIM + Ucn or SB + Ucn). ##, P < 0.0001 (compared with BIM + Ucn II or SB + Ucn II).

 
Effects of various protein kinase inhibitors on cAMP-induced IL-6 levels in A7r5 aortic smooth muscle cells
Because both Ucn and Ucn II induced intracellular cAMP accumulation in this cell line, we investigated whether cAMP was directly involved in the regulation of IL-6 output levels. As shown in Fig. 7AGo, a cAMP analog, db-cAMP, also caused a dose-dependent increase in IL-6 levels (ANOVA; P < 0.0001). A further experiment involved incubating the cells for 48 h in medium containing db-cAMP (100 µM) and increasing concentrations of BIM and/or SB203580, or H89 (Fig. 7BGo). Both BIM and SB203580 inhibited cAMP-induced IL-6 concentration, similar to the effect of Ucn or Ucn II. Concomitant administration of BIM (10 µM) and SB203580 (10 µM) completely blocked cAMP-increased IL-6 levels. Only the maximal dose (30 µM), but not 0.1–10 µM, of H89 blocked the ability of cAMP to increase IL-6 levels.



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  		Figure 7. Effects of various protein kinase inhibitors on cAMP-induced IL-6 levels in A7r5 aortic smooth muscle cells. Results shown are representative of three independent experiments. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post hoc test. A, Dose-dependent effects of db-cAMP on IL-6 levels. Cells were incubated for 48 h with medium alone (control), or with medium containing 1, 10, or 100 µM, or 1 mM db-cAMP. *, P < 0.0001 (compared with control). **, P < 0.0001 (compared with 100 µM db-cAMP). B, Cells were incubated for 48 h with medium-added vehicle or db-cAMP (100 µM), with or without medium containing increasing concentrations of BIM and/or SB203580 or H89. Cells treated with BIM alone, SB203580 alone, or H89 alone are indicated as BIM, SB, or H89, respectively. *, P < 0.01 (compared with cAMP). **, P < 0.001 (compared with cAMP). ***, P < 0.0001 (compared with cAMP). +, P < 0.05 (compared with cAMP + BIM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrated that Ucn and Ucn II increased IL-6 output levels in aortic smooth muscle cells by stimulating CRF R2. The finding that antisauvagine-30, a selective CRF R2 antagonist, caused blockade of the receptor thereby preventing consequent intracellular signal transduction, and increased IL-6 output levels provided further evidence of this mechanism. We also found that stimulation of adenylate cyclase contributed to the biological effect, as MDL-12330A, an adenylate cyclase inhibitor, suppressed IL-6 induction. This latter observation indicated that Ucns-stimulated adenylate cyclase increased intracellular cAMP levels in A7r5 cells, which in turn resulted in enhanced IL-6 levels.

Both Ucn and Ucn II caused a greater accumulation of intracellular cAMP in A7r5 cells than CRF. Similarly, the effect of Ucn II on IL-6 concentration was more potent than CRF, but less potent than Ucn. This may suggest that the potential to increase IL-6 levels, in part, reflects the amount of cAMP produced following Ucns induction. However, the incomplete correlations might exist between the activity of IL-6 stimulation and the extent of cAMP accumulations. This result might suggest the effects of cAMP accumulations are amplified to increase IL-6 levels in the downstream of signaling pathway. Treatment with a high dose (100 µM) of db-cAMP, a cAMP analog, also directly increased IL-6 levels. Although continuous cAMP production through activation of CRF receptors would be required to increase IL-6 levels, the endogenous net quantity of cAMP produced by the Ucns was not determined in our study.

We also attempted to clarify which protein kinase was involved in the Ucns-induced increase in IL-6 levels by using specific inhibitors of the different pathways. From previous studies, it is known that BIM inhibits PKC (IC50 = 10 nM) but requires a much higher concentration to inhibit PKA (IC50 = 2 µM) (34). In the present study, IL-6 output induced by either Ucn or Ucn II was reversed by 10 nM of BIM, suggesting that PKC is involved in the regulation of IL-6 levels. A high dose of H89 (30 µM) was required to block the ability of Ucn and Ucn II to increase IL-6 levels. Such a high dose of H89 would be expected to suppress multiple protein kinases including PKA and PKC, as established IC50 values for PKA and PKC are 48 nM and 31.7 µM, respectively (35). Taken together, these findings indicate a cross-signaling pathway between PKC and cAMP that plays a major role in increasing IL-6 levels in A7r5 cells (Fig. 8Go). However, due to limitations and nonspecificity of inhibitor studies, it is possible that other kinases may also be involved in this pathway.



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  		Figure 8. An illustrated scheme of the pathways involved in Ucn/Ucn II, cAMP induction, and IL-6 output. Ucns increase intracellular cAMP levels via the stimulation of adenylate cyclase. To increase IL-6 output, both PKC and p38 MAPK signaling cascades are involved downstream of the Ucn/Ucn II-cAMP pathway in A7r5 cells.

 
There is evidence that MAPKs have a role in the regulation of G protein-coupled receptors (36, 37). A recent study reported that MAPK-dependent signaling pathways are activated by stimulation of CRF R2 (38), leading us to investigate whether MAPKs are involved in IL-6 regulation. At least four distinctly regulated groups of MAPKs have been identified, namely, ERK-1/2, Jun amino-terminal kinases 1/2/3), p38 proteins, and ERK5 (39). Although an earlier study (40) demonstrated that SB203580, a highly specific inhibitor of p38 MAPK (IC50 = 600 nM), did not inhibit the other MAPK homologs, in our study this inhibitor was found to significantly attenuate the effects of Ucn and Ucn II on IL-6 levels. However, PD98059, a MAPK kinase 1/2 inhibitor, had no effect on this induction in our experiments. SB203580 also suppressed cAMP-increased IL-6 levels significantly, suggesting a cross-signaling between MAPK and cAMP (41). Taken together, these results support the hypothesis that p38 MAPK, but not the MAPK kinase 1/2-ERK1/2, mediates in part the effects of the Ucns-cAMP pathway on IL-6 induction (Fig. 8Go). The involvements of Jun amino-terminal kinases 1/2/3 and ERK5 were not determined in this study, because of the lack of their specific inhibitors. To verify that the PKC and MAPK cascades are independent pathways for cAMP stimulation, the effect of combined BIM and SB203580 treatment was compared with that of each inhibitor. The inhibitors suppressed IL-6 output levels synergistically, a result that suggested the existence of two distinct downstream pathways that regulate IL-6 output in A7r5 cells following cAMP stimulation (Fig. 8Go).

It has been demonstrated previously that CRF modulates the immune or inflammatory response by stimulating leukocytes to produce cytokines such as IL-1, -2, and -6 (42, 43). Ucn has more potent effects than CRF, with an in vivo study in rats (20) showing the peptide-increased serum IL-6 levels. Similarly, human peripheral blood mononuclear cells are known to produce IL-1ß and IL-6 following stimulation with Ucn (44). The increase that we observed in IL-6 levels following Ucns treatment of A7r5 cells suggests that smooth muscle cells may be a source of IL-6 secretion that occurs under stress conditions.

Although the endogenous role of Ucn in the immune system remains to be fully understood, it is possible that Ucn may act on lymphocytes in an autocrine or paracrine manner, as these cells are known to possess CRF binding sites (45). In an earlier study, we found that Ucn mRNA levels in the thymus increased in a glucocorticoid-dependent manner. This finding suggests that Ucn produced during stress may possibly modulate cellular immunity, or differentiation and/or proliferation of T lymphocytes in the thymus. Increased IL-6 output induced by Ucn would also be expected to modify both humoral and cellular immunity (46). In addition, IL-6 is able to stimulate ACTH and glucocorticoid secretion (5). This combination of actions implies that increased IL-6 levels may have direct and indirect affects on immune and other stress modulations. We have demonstrated previously that Ucn down-regulates CRF R2ß mRNA levels directly (31, 47). Furthermore, because cytokines such as IL-1 and -6 both decrease CRF R2ß mRNA expression (31, 48), it is possible that Ucn and IL-6 contribute cooperatively in regulating the levels of CRF R2ß mRNA.

In conclusion, this study demonstrated that Ucns-induced intracellular cAMP accumulation contributed to IL-6 output levels, and both PKC and p38 MAPK signaling cascades are involved downstream of the Ucns-cAMP pathway in A7r5 aortic smooth muscle cells.


   Acknowledgments
 
We are grateful to Dr. G. E. Gaudriault and Dr. W. W. Vale for critical suggestions and Dr. T. Moriyama, K. Hanada, and A. Chiba for technical assistance.


   Footnotes
 
This work was supported by grants to K.K. from the Ministry of Education, Science, and Culture of Japan (no. 14770582).

Abbreviations: BIS, Bisindolylmaleimide I; CRF, corticotropin-releasing factor; CRF R CRF receptor; db, dibutyryl; HPA, hypothalamic-pituitary-adrenal; PKA, protein kinase A; PKC, protein kinase C; Ucn, urocortin; Ucns, Ucn-related peptides.

Received November 11, 2002.

Accepted for publication March 12, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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