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Am J Physiol Lung Cell Mol Physiol 294: L553-L562, 2008. First published December 21, 2007; doi:10.1152/ajplung.00044.2006
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IL-1β, BK, and TGF-β1 attenuate PGI2-mediated cAMP formation in human pulmonary artery smooth muscle cells by multiple mechanisms involving p38 MAP kinase and PKA

H. El-Haroun, D. L. Clarke, K. Deacon, D. Bradbury, A. Clayton, A. Sutcliffe, and Alan J. Knox

Division of Respiratory Medicine, University of Nottingham, City Hospital, Nottingham, United Kingdom

Submitted 3 February 2006 ; accepted in final form 17 December 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously shown that interleukin (IL)-1β, transforming growth factor (TGF)-β1, or bradykinin (BK) impair cAMP generation in response to prostacyclin analogs in human pulmonary artery smooth muscle (PASM), suggesting that inflammation can impair the effects of prostacyclin analogs on PASM in pulmonary hypertension. Here we explored the biochemical mechanisms involved. We found that IL-1β, BK, and TGF-β1 reduced adenylyl cyclase isoform 1, 2, and 4 mRNA, increased G{alpha}i protein levels, and reduced prostacyclin receptor (IP receptor) mRNA expression. In contrast, G{alpha}s protein levels were unchanged. Protein kinase A (PKA) (H-89, KT-2750, PKIm) and p38 mitogen-activated protein (MAP) kinase (SB-202190) inhibitors attenuated these effects, but protein kinase C (bisindolylmaleide) or phosphoinositol 3-kinase (LY-294002) inhibitors did not. Fluorescent kemptide assay and Western blotting confirmed that PKA and p38 MAP kinase were activated by IL-1β, BK, and TGF-β1. These studies suggest that IL-1β, BK, and TGF-β1 impair IP receptor-mediated cAMP accumulation by multiple effects on different components of the signaling pathway and that these effects are PKA and p38 MAP kinase dependent.

pulmonary artery smooth muscle; kinases; adenosine 3',5'-cyclic monophosphate; prostacyclin receptor

PROSTACYCLIN (PGI2) is the main arachidonic acid metabolite in the mammalian vasculature, with production being greater in pulmonary than systemic arterial segments (37). It is produced by PGI2 synthase and is a powerful vasodilator of the pulmonary vascular bed through its actions on pulmonary artery smooth muscle cells (10). Pulmonary vascular tone and remodeling is controlled by the balanced release of vasoactive mediators (44). Patients with severe pulmonary hypertension have an imbalance in the local production of vasoactive eicosanoids (3) and reduced pulmonary artery expression of PGI2 synthase (42).

The use of PGI2 and its analogs in pulmonary hypertension has been a major therapeutic advance. Long-term infusion of PGI2 or its stable analog iloprost improves survival and reduces pulmonary vascular resistance in patients with either primary (1, 3, 25) or secondary pulmonary hypertension (34). PGI2-mediated pulmonary vascular smooth muscle relaxation occurs via prostacyclin (IP) receptor activation (6, 33, 35). The IP receptor is coupled to guanosine nucleotide-binding {alpha}-stimulatory protein (G{alpha}s), which stimulates adenylyl cyclase (AC) with a resultant conversion of ATP to cAMP. cAMP then causes protein kinase A (PKA) activation and phosphorylation of key proteins leading to relaxation and reduced cell proliferation (26).

Current paradigms suggest that proinflammatory cytokines and mediators such as interleukin-1β (IL-1β), transforming growth factor-β1 (TGF-β1), and bradykinin (BK) play an important role in pathophysiology of pulmonary hypertension (11, 27, 41). Our recent studies have shown that, when human pulmonary artery smooth muscle cells were treated for long periods with IL-1β, BK, or TGF-β1, the cAMP response to subsequent application of PGI2 analogs was impaired (5). These studies were the first in any biological system to show that proinflammatory cytokines and mediators could impair PGI2 signaling pathways. Furthermore, this effect might explain the development of tolerance to the therapeutic effects of PGI2 analogs in pulmonary hypertension. In preliminary mechanistic studies, we showed that these agents were acting in part through an autocrine loop involving production of endogenous prostanoids via cyclooxygenase-2 (COX-2) and downregulation of AC isoforms 1, 2, and 4 (5).

Here, we have extended this work to perform more detailed studies of the biochemical mechanisms involved, looking at sites in the IP receptor/G protein/AC signaling cascade and the role of several protein kinases [PKA, protein kinase C (PKC), phosphoinositide 3 kinase (PI 3-kinase) and p38 mitogen-activated protein kinase (MAP kinase)] in these effects. We found that IL-1β, BK, and TGF-β1 acted at multiple sites to reduce IP receptor messenger ribose nucleic acid (mRNA) expression, increase guanosine nucleotide inhibitory protein (G{alpha}i) expression, and reduce AC isoform 1, 2, and 4 expression. These effects were dependent on PKA and p38 MAP kinase but not PKC or PI 3-kinase. These studies provide novel insights into the mechanism that cytokines and mediators use to regulate IP receptor signaling pathways.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. Human proximal pulmonary artery smooth muscle cells (HPASMC) from a 22-yr-old male were purchased from Clonetics and cultured to passage 6 in smooth muscle cell growth medium-2 Bulletkit (Clonetics BioWittaker, Wokingham, Berkshire, UK).

Experimental protocols. Confluent cells were growth arrested in serum-free medium for 24 h and then incubated with serum-free medium containing IL-1β (10 ng·ml–1·24 h–1), BK (10 µmol·l–1·24 h–1), or TGF-β1 (1 ng·ml–1·36 h–1) for the times indicated. Inhibitors were added 1 h before treatment.

cAMP assay. cAMP was measured over 20 min in response to carbaprostacyclin (1 µmol/l), iloprost (1 µmol/l), and forskolin (FSK) (1 µmol/l). Cells were treated for 30 min with 3-isobutyl 1-methylxanthine (IBMX, 1 mmol/l) before stimulation. This time was chosen based on our previous studies (31). Experiments were terminated by the addition of 100 µl ice-cold 30% TCA. The resulting solution was then transferred to centrifuge tubes kept at 4°C, and the TCA was removed by amine-freon extraction. The cAMP content of the extract was determined using a protein-binding assay (31). The bound [3H]cAMP was measured using the Tri-Carb 2100TR liquid scintillation analyzer (Packard bioscience, Pangbourne, Berkshire, UK).

Western blot analysis. Western blotting for COX-1, COX-2, G{alpha}i, G{alpha}s, and p38 MAP kinase was performed as described (32). The monoclonal anti-human G{alpha}i-3 and G{alpha}s were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and monoclonal antihuman p38 MAP kinase and phosphorylated p38 MAP kinase were from Cell Signaling Technology (Beverly, MA). Each blot is representative of three to four independent experiments.

RNA isolation and RT-PCR. Total RNA was isolated using the RNeasy mini kit (Qiagen, West Sussex, UK). Total RNA (1 µg) was reverse transcribed as previously described (46). Forty cycles of PCR were performed for all nine AC isoforms (45). IP receptor studies consisted of denaturation at 95°C for 30 s, primer annealing at 58°C for 1 min, primer extension at 72°C for 1 min, and a final extension of 72°C for 10 min. Glutamine phosphate dehydrogenase was used as an internal control gene. PCR products were electrophoresed on 2% of agarose gel containing 0.5 µg/ml ethidium bromide and visualized using the GeneGenius gel documentation and analysis system (Syngene, Braintree, Essex, UK) (46). Each gel is representative of three to four independent experiments.

Nonradioactive assay for cAMP-dependent protein kinase activity. Confluent cells were growth arrested for 24 h. After treatment, the cells were washed with PBS and incubated with gentle shaking for 5 min with protein extraction buffer (0.9% NaCl, 25 mmol/l Tris-hydochloric acid, pH 7.6, 0.1% Triton X-100, 1 mmol/l phenylmethylsulfonyl fluoride, and 0.01% leupeptin). The cell extract was centrifuged (14,000 g, 4°C, 10 min). PKA assay was performed as described (14, 23). Briefly 1–5 µl of cell extract were incubated with Pep Tag peptide, f-kemptied (Promega) in PKA reaction buffer with PKA activator and peptide protector. PKA reactions were incubated for 30 min at 30°C. Reactions were stopped at 100°C for 10 min, cooled to 4°C, and then separated on a 0.8% agarose gel. Relative fluorescence was gauged on an ultraviolet transilluminator.

Materials. Recombinant human (rh) IL-1β and rh TGF-β1 were purchased from R & D Systems Europe (Abingdon, Oxon, UK), and BK, FSK, IBMX, bisindolylmaleide (BIS), TCA, amine/freon extraction, LY-294002, and H-89 were from Sigma. Carbaprostacyclin was purchased from Cayman Chemical (Alexis, Bingham, Notts, UK). Iloprost was a gift from Schering (Burgess Hill, West Sussex, UK). PKA-dependent cAMP and cAMP were from Sigma, [8-3H]cAMP (sp act 962 GBq·mmol–1·l–1) was from Amersham Life Science (Little Chalfont, Bucks, UK), the Super Signal CL-horseradish peroxidase substrate system from Pierce (Rockford, IL,), and the Bio-Rad protein assay reagent was from Bio-Rad Laboratories limited (Hemel Hempstead, Herts, UK). The p38 MAP kinase inhibitor SB-202190 and PKA inhibitors KT5720 and PKIm (PKI, 14–22 amide myristolated) were purchased from Calbiochem (Merck Biosciences, Notts, UK). Drugs were dissolved in PBS or dimethyl sulfoxide (DMSO) and appropriate concentrations of vehicle were used as controls. The final concentration of DMSO used was never greater than 0.1%.

Statistical analysis. Data were expressed as means and SE. Comparisons were by one-way ANOVA with the Tukey post hoc test where P values of <0.05 were regarded as statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Prolonged incubation with IL-1β, BK, and TGF-β1 reduces carbaprostacyclin, iloprost, and FSK-induced cAMP accumulation. Basal cAMP levels were similar in control and pretreated cells. Pretreatment with IL-1β (10 ng/ml) or BK (10 µmol/l) for 24 h (Fig. 1A) or TGF-β1 (1 ng/ml) for 36 h significantly attenuated cAMP production in response to carbaprostacyclin (1 µmol/l), iloprost (1 µmol/l), and FSK (1 µmol/l) compared with control cells (Fig. 1A). We have previously shown that the effect of TGF-β1 acts slower than IL-1β or BK, which is the result of slower induction of COX-2 and a resultant production of endogenous PGI2 that we have previously shown is involved in the response (5). We used these time points for the different agents in subsequent experiments. The results with FSK, a direct activator of AC, suggested that the effects of IL-1β, BK, and TGF-β1 were at least in part a direct effect on AC itself. To study this further, we measured their effects on AC mRNA expression.


Figure 1
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  		Fig. 1. A: interleukin (IL)-1β (10 ng·ml–1·24 h–1), bradykinin (BK) (10 µmol·l–1·24 h–1), or transforming growth factor (TGF)-β1 (1 ng·ml–1·36 h–1) inhibit cAMP accumulation in response to carbaprostacyclin (1 µmol/l), iloprost (1 µmol/l), and forskolin (1 µmol/l). Means and SE are shown; n = 8 determinations from 3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with cells without IL-1β, BK, and TGF-β1. B: IL-1β, BK, and TGF-β1 reduce adenylyl cyclase (AC) 1, 2, and 4 mRNA. Relative densities were calculated by dividing the AC by the glutamine phosphate dehydrogenase (GAPDH) bands (n = 3).

 
IL-1β, BK, and TGF-β1 reduce AC isoform 1, 2, and 4 mRNA expression. We found that HPASMC expressed AC isoforms 1, 2, 3, 4, 6, 7, and 9. Positive control experiments showed that human brain expressed all nine isoforms (data not shown). IL-1β or BK incubation for 24 h and TGF-β1 for 36 h significantly reduced AC isoform 1, 2, and 4 mRNA expression (Fig. 1, B and C) but had no effect on other isoforms (data not shown).

IL-1β, BK, and TGF-β1 reduce G{alpha}i-3 protein but not G{alpha}s protein expression. G{alpha}i-3 protein levels increased in HPASMC after exposure to 10 ng/ml IL-1β for 24 h, 10 µmol/l BK for 24 h, and 1 ng/ml TGF-β1 for 36 h (Fig. 2A) [There was no change at earlier time points (data not shown).] In contrast, 10 ng/ml IL-1β, 10 µmol/l BK, or 1 ng/ml TGF-β1 had no effect on G{alpha}s protein levels at any time point (Fig. 2B).


Figure 2
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  		Fig. 2. IL-1β (10 ng·ml–1·24 h–1), BK (10 µmol·l–1·24 h–1), or TGF-β1 (1 ng·ml–1·36 h–1) increase G{alpha}i-3 protein (A), have no effect on G{alpha}s protein (representative Western blots of 3 experiments; B), and reduce prostacyclin receptor (IP receptor) mRNA expression (representative PCR experiment of 3 experiments; C). Protein loading controls (not shown) showed equal protein loading.

 
IL-1β, BK, and TGF-β1 reduce IP receptor mRNA expression. IL-1β or BK for 24 h or TGF-β1 for 36 h all significantly attenuated IP receptor mRNA expression (Fig. 2C).

Activation of PKA is involved in IL-1β-, BK-, and TGF-β1-induced downregulation of PGI2 analog and FSK-induced cAMP formation. The reductions in carbaprostacyclin (1 µmol/l)-, iloprost (1 µmol/l)-, and FSK (1 µmol/l)-induced cAMP accumulation caused by IL-1β (10 ng·ml–1·24 h–1), BK (10 µmol·l–1·24 h–1), or TGF-β1 (1 ng·ml–1·36 h–1) were all significantly attenuated by H-89, KT-5720, and the PKA inhibitor peptide PKIm, suggesting that PKA is involved (Fig. 3, A–D). H-89, KT-5720, and PKIm did not alter cAMP accumulation in controls in the absence of cytokines. Furthermore, IL-1β (10 ng/ml), BK (10 µmol/l), and TGF-β1 (1 ng/ml) significantly increased PKA activity (Fig. 4A). Collectively, these data provide compelling evidence that PKA is involved in the pathway.


Figure 3
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  		Fig. 3. The protein kinase A (PKA) inhibitor H-89 (0.1 µmol/l) attenuates IL-1β-, BK-, and TGF-β1-induced downregulation of cAMP formation in response to carbaprostacyclin (1 µmol/l) (A), iloprost (1 µmol/l) (B), and forskolin (FSK, 1 µmol/l) (C). The PKA inhibitors KT-2750 (0.1 µmol/l) and PKIm (0.1 µmol/l) attenuate BK-induced downregulation of cAMP formation in response to iloprost (D). Mean and SE are shown; n = 8 determinations from 3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with cells without pretreatment.

 

Figure 4
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  		Fig. 4. IL-1β (10 ng/ml), BK (10 µmol/l), or TGF-β1 (1 ng/ml) administered for 1 h increase PKA activity (kemptide assay, representative experiment of 3 experiments; A), and IL-1β, BK, and TGF-β1 phosphorylate p38 MAP kinase (representatives Western blots of 3 experiments; B). pp38, phosphorylated p38. Total phosphorylated p38 MAP kinase showed no change and served as a protein loading control.

 
p38 MAP kinase is involved in IL-1β-, BK-, and TGF-β1-induced attenuation of PGI2 analog and FSK-induced cAMP formation. The reductions in carbaprostacyclin (1 µmol/l)-, iloprost (1 µmol/l)-, and FSK (1 µmol/l)-induced cAMP accumulation caused by IL-1β (10 ng·ml–1·24 h–1), BK (10 µmol·l–1·24 h–1), or TGF-β1 (1 ng·ml–1·36 h–1) were all significantly attenuated by SB-202190 (0.1 µmol/l), suggesting involvement of p38 MAP kinase (Fig. 5, A–C). SB-202190 did not alter cAMP accumulation in controls in the absence of cytokines. Furthermore, IL-1β (10 ng/ml), BK (10 µmol/l), or TGF-β1 (1 ng/ml) increased p38 phosphorylation, which peaked after 10 min of exposure, diminished after 60 min, and returned to normal after 120 min of exposure (Fig. 4B). We next perform experiments to confirm that the H-89 and SB-202190 were selectively inhibiting PKA. We found that H-89 inhibited BK-induced cAMP response element-binding protein (CREB) phosphorylation concentration dependently but had no effect on phosphorylation of a downstream target of p38 MAP kinase, mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2). In contrast, SB-202190 concentration dependently inhibited MAPKAPK2 without inhibiting CREB phosphorylation (Fig. 6).


Figure 5
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  		Fig. 5. The p38 mitogen-activated protein (MAP) kinase inhibitor (SB-202190, 0.1 µmol/l) attenuates IL-1β (10 ng/ml)-, BK (10 µmol/l)-, and TGF-β1 (1 ng/ml)-induced downregulation of cAMP formation in response to carbaprostacyclin (1 µmol/l; A), iloprost (1 µmol/l; B), and FSK (1 µmol/l; C). Means and SE are shown; n = 8 determinations from 3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with cells without pretreatment.

 

Figure 6
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  		Fig. 6. Selectivity of kinase inhibitors. The PKA inhibitor H-89 (0.1 µmol/l) attenuates BK-induced phosphorylation of cAMP response element-binding protein (CREB) but has little impact on p38 phosphorylation. The p38 inhibitor SB-202190 (0.1 µmol/l) inhibits phosphorylation of the downstream target mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) and has little impact on BK-induced (10 µmol/l) CREB phosphorylation (representatives Western blots of 3 experiments).

 
Inhibitors of PKA and p38 MAP kinase attenuate IL-1β, BK, and TGF-β1 effects on signaling components (AC mRNA expression, G{alpha}i protein, and IP receptor mRNA). Because we have shown that the PKA and p38 MAP kinase inhibitors attenuated IL-1β-, BK-, or TGF-β1-induced attenuation of the PGI2 analog and FSK-induced cAMP formation, we went on to determine if they inhibited the different components of the IP receptor/G protein/AC signaling cascade that was responsible for these effects. H-89 (1 µmol/l) markedly inhibited the downregulation of the AC1, AC2, and AC4 mRNA caused by IL-1β (10 ng·ml–1·24 h–1), BK (10 µmol·l–1·24 h–1), or TGF-β1 (1 ng·ml–1·36 h–1). SB-202190 (0.1 µmol/l) was also effective but less so than H-89. SB-202190 had a greater effect on AC1 than AC2 and AC4 downregulation (Fig. 7). H-89 (1 µmol/l) and SB-202190 (0.1 µmol/l) both inhibited the increased G{alpha}i-3 protein level expression caused by IL-1β (10 ng·ml–1·24 h–1), BK (10 µmol·l–1·24 h–1), and TGF-β1 (1 ng·ml–1·36 h–1) (Fig. 8A). H-89 abolished the effect, whereas SB-202190 was only partially effective. H-89 (1 µmol/l) (PKA) and SB-202190 (0.1 µmol/l) (p38 MAP kinase) abolished the downregulation of IP receptor mRNA in response to IL-1β (10 ng·ml–1·24 h–1), BK (10 µmol·l–1·24 h–1), and TGF-β1 (1 ng·ml–1·36 h–1) (Fig. 8B).


Figure 7
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  		Fig. 7. PKA (H-89, 0.1 µmol/l) and p38 MAP kinase (SB-202190, 0.1 µmol/l) inhibitors attenuate IL-1β (10 ng/ml)-, BK (10 µmol/l)-, and TGF-β1-induced (1 ng/ml) downregulation of AC mRNA expression (representative RT-PCR experiment of 3 experiments).

 

Figure 8
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  		Fig. 8. PKA (H-89, 0.1 µmol/l) and p38 MAP kinase (SB-202190, 0.1 µmol/l) inhibitors attenuate IL-1β (10 ng/ml)-, BK (10 µmol/l)-, and TGF-β1-induced (1 ng/ml) downregulation of Gi protein expression (representative Western blots of 3 experiments) [protein loading controls (not shown) showed equal protein loading; A] and IP receptor mRNA expression (representative RT-PCR experiment of 3 experiments, GAPDH control; B).

 
PKA and p38 MAP kinases are involved downstream of COX-2 induction and PGI2 generation. PGI2 is produced by the cytokines we studied via induction of COX-2, and this contributes to the desensitization process (5). PKA and p38 MAP kinase could be acting either upstream of PGI2 generation or downstream of PGI2. If these kinases are operating downstream of PGI2 generation then they should also be activated by exogenous PGI2 or its analogs. To test this possibility we performed experiments to determine the effect of iloprost on PKA and p38 MAP kinase activity. Total PKA activity was 52.7 ± 2.6 nmol·l–1·min–1·µg protein–1 in control cells. This was increased to 114 ± 8.9 after treatment with iloprost. Similarly, p38 phosphorylation assays to measure MAP kinase activation showed that iloprost activated p38 MAP kinase (Fig. 9C); this suggests that activation of PKA and p38 MAP kinase both occur at a step downstream of PGI2 generation and binding to IP receptors. Consistent with this observation, chronic iloprost (1 µmol/l) pretreatment for 24 h reduced cAMP accumulation to subsequent iloprost (1 µmol/l) or carbaprost (1 µmol/l) exposure. The reduction was significantly attenuated by H-89 and SB-202190 (Fig. 9, A and B).


Figure 9
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  		Fig. 9. PKA (H-89, 1 µmol/l; A) and p38 MAP kinase (SB-202190, 0.1 µmol/l; B) inhibitors attenuate iloprost-induced (1 µmol/l) downregulation of carbaprostacyclin (1 µmol/l) and iloprost (1 µmol/l)-induced cAMP formation. Means and SE are shown; n = 8 determinations from 3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with cells without pretreatment. C: iloprost phosphorylates p38 MAP kinase (representative Western blot of 3 experiments). Total phosphorylated p38 MAP kinase showed no change and served as a protein loading control.

 
Neither PKC nor PI 3-kinase is involved in IL-1β-, BK-, and TGF-β1-induced downregulation of PGI2 analogs and FSK-induced cAMP formation. To determine if PKC had a role, we studied the effect of the broad-spectrum PKC inhibitor BIS (0.1 µmol/l). BIS had no effect on the reduction in cAMP responses caused by IL-1β, BK, or TGF-β1 (data not shown). Similarly, the PI 3-kinase inhibitor LY-294002 (0.1 µmol/l) had no effect on the reduction in cAMP responses caused by IL-1β, BK, or TGF-β1 (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major findings in these studies are that IL-1β, BK, and TGF-β1 impaired PGI2 analog-mediated cAMP formation by acting at several sites. IL-1β, BK, and TGF-β1 downregulated AC isoforms 1, 2, and 4, increased G{alpha}i protein levels, and reduced IP receptor mRNA expression. All of these effects were dependent on PKA and p38 MAP kinase but not PKC or PI 3-kinase. These studies provide important new insight into the biochemical mechanisms used by cytokines and mediators to impair IP receptor signaling pathways.

We found that the three agents we studied all had very similar effects with the exception that the effects of TGF-β1 were slightly delayed. Our previous work showed that the delayed effects of TGF-β1 are due to a slower onset of COX-2 induction and endogenous prostanoid formation than IL-1β or BK, which we have previously shown play a critical role in the effects of these agents in downregulating PGI2 receptor-mediated cAMP accumulation. The times of incubation chosen (24 h for IL-1β or BK, 36 h for TGF-β1) were based on our previous studies (5). The changes were robust and consistent across cell passages.

It is well recognized that G protein-coupled receptors can become desensitized or downregulated during chronic stimulation. This has been studied extensively with the β2-adrenoceptor but can also be seen with the PGI2 receptor pathway (30). This can be agonist-specific or homologous desensitization that usually occurs by receptor downregulation or effects on G protein coupling (12) or non-agonist-specific heterologous desensitization mediated by changes in AC or downstream phosphodiesterase enzymes (19). Because all of our cAMP experiments were performed in the presence of IBMX, an effect on phosphodiesterases is unlikely within our experimental design but may contribute additional effects in intact cells. To determine the mechanism of the effect in our system, we first studied the effect of IL-1β, BK, and TGF-β1 on AC isoform mRNA expression. We found that isoforms 1, 2, and 4 were all downregulated, but other AC isoforms were unchanged. This is likely to be responsible for the reduction of FSK-induced cAMP accumulation, since FSK is a direct activator of AC. Consistent with our findings, IL-1β reduced AC activity in rat airway smooth muscle cells (17), TGF-β1 reduced AC activity in cardiomyocytes (29), and BK inhibited AC activity in rat myometrial cells (22) and guinea pig ileum membrane (21). However, none of these studies looked at the contribution of specific AC isoforms. We did not measure AC isoform protein levels due to a lack of reliable antibodies although this is clearly an area that could be looked at in future studies. We considered whether changes in intracellular calcium might be responsible for the changes we saw. We feel this is unlikely, since BK would be expected to cause a much greater change in intracellular calcium than the two cytokines due to mobilization of inositol phospholipids.

We next looked to see if there were additional effects upstream of AC either on G{alpha}i or G{alpha}s. IL-1β increased G{alpha}i, consistent with studies that have focused on β2-adrenoceptor rather than IP receptor signaling in rat lung membrane (24), guinea pig muscle (9), rabbit tracheal smooth muscle (8), and rat airway smooth muscle (17) cells. In contrast, IL-1β had no effect on G{alpha}s in our studies nor in previous studies in human airway smooth muscle cells (38) unlike in CD4 cells where G{alpha}s was reduced (15), suggesting that the effects of IL-1β on G{alpha}s are cell specific. We found that BK or TGF-β1 also increased G{alpha}i but not G{alpha}s protein levels. Few studies have looked at this directly in other systems although G{alpha}i was implicated in BK-induced endothelium-derived relaxing factor release in bovine aortic endothelial cells (20). TGF-β1 decreased G{alpha}i-2 expression in chick atrial cells (43) but increased G{alpha}i and G{alpha}s in osteoblasts (36). The differences between ours and previous studies with BK and TGF-β1 likely reflect species differences or different biological cell functions.

To determine whether the cytokines and mediator of interest could have additional effects on IP receptors, we measure IP receptor mRNA. We found that IL-1β, BK, and TGF-β1 all reduced IP receptor mRNA. This effect is likely to magnify the functional effects of AC and G{alpha}i alterations on cAMP levels. No previous studies have looked at the effect of IL-1β, BK, and TGF-β1 on IP receptor levels in this or other biological systems.

Protein kinases are important intracellular regulatory pathways, several of which have been shown to be involved in heterologous or homogenous desensitization of a number of seven transmembrane G protein-coupled receptors. To determine their role in the cytokine/mediator effects on IP receptor signaling, we first investigated whether PKA, PKC, PI 3-kinase, and p38 MAP kinase inhibitors could inhibit the effects of IL-1β, BK, and TGF-β1 on PGI2 analog and FSK-mediated cAMP accumulation. PKA (H-89) and p38 MAP kinase (SB-202190) inhibitors attenuated the effects, whereas PKC (BIS) or PI 3-kinase (LY-294002) inhibitors did not. We confirmed that the effect of H-89 was likely to be via PKA inhibition by using two PKA inhibitors that are structurally distinct from H-89, KT-2750 (a small-molecular-weight inhibitor of PKA) and PKIm (a myristolated peptide of 14–22 amino acids of the PKA inhibitor PKI), using the reduction in iloprost-induced cAMP synthesis after chronic BK treatment. Both PKIm and KT-2750 treatment prior to BK addition increased cAMP synthesis in response to iloprost (Fig. 3D) implying that PKA induced by BK has a role in the reduction in cAMP synthesis in response to iloprost. We then studied kinase inhibitor effects on the individual components of the signaling cascade. H-89, the PKA inhibitor, abolished the changes in AC isoforms 1, 2 and 4, G{alpha}i and IP receptor mRNA. SB-202190, the p38 MAP kinase inhibitor had similar effects but was less effective and reduced AC1 down-regulation to a greater extent than AC2 and -4. Collectively these results suggest that PKA and p38 MAP kinases mediated the effects of IL-1β, BK and TGF-β1. To provide further evidence, we confirmed that PKA and p38 MAP kinase were activated by IL-1β, BK, and TGF-β1. The fact that the isoforms are differentially regulated by p38 MAP kinase is consistent with these AC isoforms having differences in structure and control mechanisms.

PKA plays an important role in cAMP-activated signal transduction. IP receptor activation by PGI2 activates AC to convert ATP into cAMP, which activates PKA. PKA can phosphorylate β2-adrenoceptors, thereby mediating their desensitization (19) and also AC itself (13, 18). The effects we saw on AC isoforms, G{alpha}i protein, and IP receptors were clearly unlikely to be due to direct PKA phosphorylation. PKA can activate CREB and alter gene transcription (4, 40). Further studies are required to determine if PKA is acting via CREB to stimulate transcription of AC isoforms and G{alpha}i while switching off the transcription of IP receptors. Although no previous studies have looked at the role of PKA in the effects of IL-1β, BK, or TGF-β1 on IP receptor signaling, we have shown previously that their effects in our cells are due to endogenous PGE2 and PGI2. Studies in rat pulmonary artery cells have shown that the PGI2 analog cicaprost can cause desensitization of responses to subsequent cicaprost stimulation. This effect was due to reduction of AC isoforms 5 and 6 and was PKA dependent (39). PKA has also been shown to regulate AC5 levels in heart cells (13). Our studies are the first to show that PKA can regulate AC1, -2, and -4.

Our studies with the PKC inhibitor (BIS) indicated that it has no role in IL-1β-, BK-, and TGF-β1-induced downregulation of PGI2 analog and FSK-induced cAMP formation. The concentration of BIS used has previously been shown to have an effect on AC and G{alpha}i protein in rat pulmonary artery (39). Our data contrast with data suggesting a role for PKC in downregulation of β2-adrenoceptor signaling. For example, PKC reduced cAMP production in response to isoproterenol and desensitized β2-adrenoceptors and AC in HEK293 cells (7). PKC phosphorylated G{alpha}i-1 and G{alpha}i-2 but not G{alpha}i-3 in intestinal smooth muscle (28). The most likely explanations for the differences between these studies and ours are species and tissue differences and the fact that we were looking at IP receptor rather than β2-adrenoceptor signaling.

Similarly, our studies with LY-294002 suggest that PI 3-kinase has no role in IL-1β-, BK-, and TGF-β1-induced downregulation of PGI2 analog and FSK-induced cAMP formation. The concentration we used has previously been shown to inhibit PI 3-kinase in HPASMC (2). There is little information regarding the role of PI 3-kinase in such mechanisms from other biological systems although IL-1β, BK, and TGF-β1 can all activate PI 3-kinase. Furthermore, inhibition of PI 3-kinase led to activation of the cyclic nucleotide-dependent signaling pathway in bovine carotid artery (16).

Our results with SB-202190 suggest that p38 MAP kinases are involved in the effects of IL-1β, BK, and TGF-β1. The reduction in AC1 was more sensitive than AC2 and AC4 to SB-202190, suggesting that p38 MAP kinase may play a great role in the regulation of AC1 than other isoforms. We found that IL-1β, BK, and TGF-β1 activated p38 MAP kinase. IL-1β and TGF-β1 have been shown to activate p38 MAP kinase in other biological systems, but BK has not. We performed additional studies to determine the site where p38 MAP kinase is involved. We found that iloprost-activated p38 MAP kinase and furthermore that iloprost-induced downregulation of responses to itself or carbaprostacyclin could be inhibited by SB-202190, suggesting that p38 MAP kinase is activated downstream of COX-2 induction and endogenous PGI2 release.

We performed experiments using chronic iloprost exposure rather than cytokines/mediator to downregulate iloprost responses to determine if the site of action of p38 MAP kinase and PKA was downstream of endogenous PGI2 generation (via COX-2), which we have previously shown is involved in their effects (5). We found that iloprost mimicked the effect of cytokines/mediator treatment on iloprost-induced cAMP generation and that this was inhibited by both p38 MAP kinase and PKA inhibitors. Consistent with this, both kinases were activated by iloprost, suggesting that they are indeed activated downstream of COX-2 induction and PGI2 generation.

In conclusion, our studies suggest that IL-1β-, BK-, and TGF-β1-induced attenuation of PGI2-mediated cAMP formation in HPASMC occurs via effects on specific AC isoforms (1, 2, and 4), G{alpha}i stimulation, and IP receptor downregulation and that PKA and p38 MAP kinase play critical roles in these processes. It should be acknowledged that the pulmonary artery smooth muscle cells used in this study are derived from the proximal pulmonary vasculature and may not be representative of the physiology of the complete pulmonary artery smooth muscle population, and any possible variation may be a subject for future investigation; however, these studies have important implications for the management of pulmonary hypertension.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. J. Knox, Division of Respiratory Medicine, Univ. of Nottingham, Clinical Science Bldg., City Hospital, Nottingham, NG5 1PB, United Kingdom (e-mail: alan.knox{at}nottingham.ac.uk.)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Barst RJ, Rubin LJ, Long WA, McGoon MD, Rich S, Badesch DB, Groves BM, Tapson VF, Bourge RC, Brundage BH. A comparison of continuous intravenous epoprostenol (prostacyclin) with conventional therapy for primary pulmonary hypertension. The Primary Pulmonary Hypertension Study Group. N Engl J Med 334: 296–302, 1996.[Abstract/Free Full Text]
  2. Bradbury DA, Corbett L, Knox AJ. PI 3-kinase and MAP kinase regulate bradykinin induced prostaglandin E(2) release in human pulmonary artery by modulating COX-2 activity. FEBS Lett 560: 30–34, 2004.[CrossRef][Web of Science][Medline]
  3. Christman BW, McPherson CD, Newman JH, King GA, Bernard GR, Groves BM, Loyd JE. An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension. N Engl J Med 327: 70–75, 1992.[Abstract]
  4. Dash PK, Karl KA, Colicos MA, Prywes R, Kandel ER. cAMP response element-binding protein is activated by Ca2+/calmodulin- as well as cAMP-dependent protein kinase. Proc Natl Acad Sci USA 88: 5061–5065, 1991.[Abstract/Free Full Text]
  5. El Haroun H, Bradbury D, Clayton A, Knox AJ. Interleukin-1beta, transforming growth factor-beta1, and bradykinin attenuate cyclic AMP production by human pulmonary artery smooth muscle cells in response to prostacyclin analogues and prostaglandin E2 by cyclooxygenase-2 induction and downregulation of adenylyl cyclase isoforms 1, 2, and 4. Circ Res 94: 353–361, 2004.[Abstract/Free Full Text]
  6. Fullerton DA, Hahn AR, Banerjee A, Harken AH. Pulmonary vascular smooth muscle relaxation by cGMP- versus cAMP-mediated mechanisms. J Surg Res 57: 259–263, 1994.[CrossRef][Web of Science][Medline]
  7. Guimond J, Mamarbachi AM, Allen BG, Rindt H, Hebert TE. Role of specific protein kinase C isoforms in modulation of beta1- and beta2-adrenergic receptors. Cell Signal 17: 49–58, 2005.[CrossRef][Web of Science][Medline]
  8. Hakonarson H, Herrick DJ, Serrano PG, Grunstein MM. Autocrine role of interleukin 1beta in altered responsiveness of atopic asthmatic sensitized airway smooth muscle. J Clin Invest 99: 117–124, 1997.[Web of Science][Medline]
  9. Hirata F, Lee JY, Sakamoto T, Nomura A, Uchida Y, Hirata A, Hasegawa S. IL-1 beta regulates the expression of the Gi2 alpha gene via lipid mediators in guinea pig tracheal muscle. Biochem Biophys Res Commun 203: 1889–1896, 1994.[CrossRef][Web of Science][Medline]
  10. Hoeper MM, Schwarze M, Ehlerding S, Adler-Schuermeyer A, Spiekerkoetter E, Niedermeyer J, Hamm M, Fabel H. Long-term treatment of primary pulmonary hypertension with aerosolized iloprost, a prostacyclin analogue. N Engl J Med 342: 1866–1870, 2000.[Abstract/Free Full Text]
  11. Humbert M, Monti G, Brenot F, Sitbon O, Portier A, Grangeot-Keros L, Duroux P, Galanaud P, Simonneau G, Emilie D. Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension. Am J Respir Crit Care Med 151: 1628–1631, 1995.[Abstract]
  12. Inglese J, Freedman NJ, Koch WJ, Lefkowitz RJ. Structure and mechanism of the G protein-coupled receptor kinases. J Biol Chem 268: 23735–23738, 1993.[Free Full Text]
  13. Iwami G, Kawabe J, Ebina T, Cannon PJ, Homcy CJ, Ishikawa Y. Regulation of adenylyl cyclase by protein kinase A. J Biol Chem 270: 12481–12484, 1995.[Abstract/Free Full Text]
  14. Karege F, Schwald M, Lambercy C, Murama JJ, Cisse M, Malafosse A. A non-radioactive assay for the cAMP-dependent protein kinase activity in rat brain homogenates and age-related changes in hippocampus and cortex. Brain Res 903: 86–93, 2001.[CrossRef][Web of Science][Medline]
  15. Kim MH, Agrawal DK. Effect of interleukin-1beta and tumor necrosis factor-alpha on the expression of G-proteins in CD4+ T-cells of atopic asthmatic subjects. J Asthma 39: 441–448, 2002.[CrossRef][Web of Science][Medline]
  16. Komalavilas P, Mehta S, Wingard CJ, Dransfield DT, Bhalla J, Woodrum JE, Molinaro JR, Brophy CM. PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways. J Appl Physiol 91: 1819–1827, 2001.[Abstract/Free Full Text]
  17. Koto H, Mak JC, Haddad EB, Xu WB, Salmon M, Barnes PJ, Chung KF. Mechanisms of impaired beta-adrenoceptor-induced airway relaxation by interleukin-1beta in vivo in the rat. J Clin Invest 98: 1780–1787, 1996.[Web of Science][Medline]
  18. Kunkel MW, Friedman J, Shenolikar S, Clark RB. Cell-free heterologous desensitization of adenylyl cyclase in S49 lymphoma cell membranes mediated by cAMP-dependent protein kinase. FASEB J 3: 2067–2074, 1989.[Abstract]
  19. Lefkowitz RJ, Hausdorff WP, Caron MG. Role of phosphorylation in desensitization of the beta-adrenoceptor. Trends Pharmacol Sci 11: 190–194, 1990.[CrossRef][Medline]
  20. Liao JK, Homcy CJ. The G proteins of the G alpha i and G alpha q family couple the bradykinin receptor to the release of endothelium-derived relaxing factor. J Clin Invest 92: 2168–2172, 1993.[Web of Science][Medline]
  21. Liebmann C, Mammery K, Graness A. Bradykinin inhibits adenylate cyclase activity in guinea pig ileum membranes via a separate high-affinity bradykinin B2 receptor. Eur J Pharmacol 288: 35–43, 1994.[CrossRef][Web of Science][Medline]
  22. Liebmann C, Reissmann S. Bradykinin inhibits rat myometrial adenylate cyclase activity via a high-affinity receptor. Biomed Biochim Acta 49: 1231–1234, 1990.[Web of Science][Medline]
  23. MacAla LJ, Hayslett JP, Smallwood JI. TECHNICAL NOTE: measurement of cAMP-dependent protein kinase activity using a fluorescent-labeled kemptide. Kidney Int 54: 1746–1750, 1998.[CrossRef][Web of Science][Medline]
  24. Mak JC, Hisada T, Salmon M, Barnes PJ, Chung KF. Glucocorticoids reverse IL-1beta-induced impairment of beta-adrenoceptor-mediated relaxation and up-regulation of G-protein-coupled receptor kinases. Br J Pharmacol 135: 987–996, 2002.[CrossRef][Web of Science][Medline]
  25. McCulloch KM, Docherty C, MacLean MR. Endothelin receptors mediating contraction of rat and human pulmonary resistance arteries: effect of chronic hypoxia in the rat. Br J Pharmacol 123: 1621–1630, 1998.[CrossRef][Web of Science][Medline]
  26. Miggin SM, Kinsella BT. Investigation of the mechanisms of G protein: effector coupling by the human and mouse prostacyclin receptors. Identification of critical species-dependent differences. J Biol Chem 277: 27053–27064, 2002.[Abstract/Free Full Text]
  27. Morrell NW, Yang X, Upton PD, Jourdan KB, Morgan N, Sheares KK, Trembath RC. Altered growth responses of pulmonary artery smooth muscle cells from patients with primary pulmonary hypertension to transforming growth factor-beta(1) and bone morphogenetic proteins. Circulation 104: 790–795, 2001.[Abstract/Free Full Text]
  28. Murthy KS, Grider JR, Makhlouf GM. Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of G(i-1) and G(i-2). Am J Physiol Cell Physiol 279: C925–C934, 2000.[Abstract/Free Full Text]
  29. Nair BG, Yu Y, Rashed HM, Sun H, Patel TB. Transforming growth factor-beta 1 modulates adenylyl cyclase signaling elements and epidermal growth factor signaling in cardiomyocytes. J Cell Physiol 164: 232–239, 1995.[CrossRef][Web of Science][Medline]
  30. Narumiya S. Prostanoid receptors. Structure, function, distribution. Ann NY Acad Sci 744: 126–138, 1994.[Web of Science][Medline]
  31. Pang L, Holland E, Knox AJ. Impaired cAMP production in human airway smooth muscle cells by bradykinin: role of cyclooxygenase products. Am J Physiol Lung Cell Mol Physiol 275: L322–L329, 1998.[Abstract/Free Full Text]
  32. Pang L, Holland E, Knox AJ. Role of cyclo-oxygenase-2 induction in interleukin-1beta induced attenuation of cultured human airway smooth muscle cell cyclic AMP generation in response to isoprenaline. Br J Pharmacol 125: 1320–1328, 1998.[CrossRef][Web of Science][Medline]
  33. Priest RM, Hucks D, Ward JP. Potentiation of cyclic AMP-mediated vasorelaxation by phenylephrine in pulmonary arteries of the rat. Br J Pharmacol 127: 291–299, 1999.[CrossRef][Web of Science][Medline]
  34. Rosenzweig EB, Kerstein D, Barst RJ. Long-term prostacyclin for pulmonary hypertension with associated congenital heart defects. Circulation 99: 1858–1865, 1999.[Abstract/Free Full Text]
  35. Savineau JP, Gonzalez DLF, Marthan R. Effect of vascular smooth muscle relaxants on the protein kinase C-mediated contraction in the rat pulmonary artery. Eur J Pharmacol 249: 191–198, 1993.[CrossRef][Web of Science][Medline]
  36. Schneider HG, Michelangeli VP, Frampton RJ, Grogan JL, Ikeda K, Martin TJ, Findlay DM. Transforming growth factor-beta modulates receptor binding of calciotropic hormones and G protein-mediated adenylate cyclase responses in osteoblast-like cells. Endocrinology 131: 1383–1389, 1992.[Abstract/Free Full Text]
  37. Shaul PW, Kinane B, Farrar MA, Buja LM, Magness RR. Prostacyclin production and mediation of adenylate cyclase activity in the pulmonary artery. Alterations after prolonged hypoxia in the rat J Clin Invest 88: 447–455, 1991.
  38. Shore SA, Laporte J, Hall IP, Hardy E, Panettieri RA Jr. Effect of IL-1 beta on responses of cultured human airway smooth muscle cells to bronchodilator agonists. Am J Respir Cell Mol Biol 16: 702–712, 1997.[Abstract]
  39. Sobolewski A, Jourdan KB, Upton PD, Long L, Morrell NW. Mechanism of cicaprost-induced desensitization in rat pulmonary artery smooth muscle cells involves a PKA-mediated inhibition of adenylyl cyclase. Am J Physiol Lung Cell Mol Physiol 287: L352–L359, 2004.[Abstract/Free Full Text]
  40. Tabuchi A, Sakaya H, Kisukeda T, Fushiki H, Tsuda M. Involvement of an upstream stimulatory factor as well as cAMP-responsive element-binding protein in the activation of brain-derived neurotrophic factor gene promoter I. J Biol Chem 277: 35920–35931, 2002.[Abstract/Free Full Text]
  41. Taraseviciene-Stewart L, Gera L, Hirth P, Voelkel NF, Tuder RM, Stewart JM. A bradykinin antagonist and a caspase inhibitor prevent severe pulmonary hypertension in a rat model. Can J Physiol Pharmacol 80: 269–274, 2002.[CrossRef][Web of Science][Medline]
  42. Tuder RM, Voelkel NF. Pulmonary hypertension and inflammation. J Lab Clin Med 132: 16–24, 1998.[CrossRef][Web of Science][Medline]
  43. Ward SM, Gadbut AP, Tang D, Papageorge AG, Wu L, Li G, Barnett JV, Galper JB. TGFbeta regulates the expression of G alpha(i2) via an effect on the localization of ras. J Mol Cell Cardiol 34: 1217–1226, 2002.[CrossRef][Web of Science][Medline]
  44. Wort SJ, Evans TW. The role of the endothelium in modulating vascular control in sepsis and related conditions. Br Med Bull 55: 30–48, 1999.[Abstract/Free Full Text]
  45. Xu D, Isaacs C, Hall IP, Emala CW. Human airway smooth muscle expresses 7 isoforms of adenylyl cyclase: a dominant role for isoform V. Am J Physiol Lung Cell Mol Physiol 281: L832–L843, 2001.[Abstract/Free Full Text]
  46. Zhu YM, Bradbury DA, Pang L, Knox AJ. Transcriptional regulation of interleukin (IL)-8 by bradykinin in human airway smooth muscle cells involves prostanoid-dependent activation of AP-1 and nuclear factor (NF)-IL-6 and prostanoid-independent activation of NF-kappaB. J Biol Chem 278: 29366–29375, 2003.[Abstract/Free Full Text]




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