Sustained exposure to prostaglandin D₂ augments the contraction induced by acetylcholine via a DP₁ receptor-mediated activation of p38 in bronchial smooth muscle of naive mice

Abstract

Prostaglandin D₂ (PGD₂), one of the key lipid mediators of allergic airway inflammation, is increased in the airways of asthmatics. However, the role of PGD₂ in the pathogenesis of asthma is not fully understood. In the present study, effects of PGD₂ on smooth muscle contractility of the airways were determined to elucidate its role in the development of airway hyperresponsiveness (AHR). In a murine model of allergic asthma, antigen challenge to the sensitized animals caused a sustained increase in PGD₂ levels in bronchoalveolar lavage (BAL) fluids, indicating that smooth muscle cells of the airways are continually exposed to PGD₂ after the antigen exposure. In bronchial smooth muscles (BSMs) isolated from naive mice, a prolonged incubation with PGD₂ (10⁻⁵ M, for 24 h) induced an augmentation of contraction induced by acetylcholine (ACh): the ACh concentration-response curve was significantly shifted upward by the 24-h incubation with PGD₂. Application of PGD₂ caused phosphorylation of ERK1/2 and p38 in cultured BSM cells: both of the PGD₂-induced events were abolished by laropiprant (a DP₁ receptor antagonist) but not by fevipiprant (a DP₂ receptor antagonist). In addition, the BSM hyperresponsiveness to ACh induced by the 24-h incubation with PGD₂ was significantly inhibited by co-incubation with SB203580 (a p38 inhibitor), whereas U0126 (a ERK1/2 inhibitor) had no effect on it. These findings suggest that prolonged exposure to PGD₂ causes the BSM hyperresponsiveness via the DP₁ receptor-mediated activation of p38. A sustained increase in PGD₂ in the airways might be a cause of the AHR in allergic asthmatics.

Introduction

Heightened responsiveness to non-specific stimuli of the airways, called airway hyperresponsiveness (AHR), is a characteristic feature in patients with allergic asthma. It has been suggested that the augmented contraction of airway smooth muscle is one of the causes of the AHR (1,2,3,4,5). Clinical observation that the inhalation of short-acting β₂-stimulant is largely effective for airway limitation in asthma attack also suggests an increased airway smooth muscle contraction in the airway obstruction. It is thus important for development of asthma therapy to understand the disease-related changes in the contractile signaling of airway smooth muscle cells.

In allergic asthma, an involvement of prostaglandin D₂ (PGD₂), one of the cyclooxygenase (COX) metabolites that released from mast cells in allergic reaction, has been suggested in its pathogenesis. In animal models of allergic asthma, the PGD₂ levels in bronchoalveolar lavage (BAL) fluids were increased (6,7,8). In people with asthma, an increase in PGD₂ in BAL fluids was observed after allergen challenge to the airways (9, 10). An involvement of PGD₂ in the allergic inflammation, including the airway inflammation in asthma, has also been suggested. In a DP₁ receptor (one of the receptors for PGD₂) gene knockout mice, both cytokines increase in BAL fluids and airway eosinophilia induced by allergen challenge were weakened as compared to wild-type animals (11). In Th2 lymphocytes, PGD₂ also has an ability to cause cytokine release via an activation of DP₂ receptor (also called as CRTH2 receptor) (12). However, the functional role of PGD₂ on airway smooth muscle remains largely unclear.

We show here that PGD₂ levels in the airways were increased immediately after antigen challenge and were sustained during the development of antigen-induced AHR. To imitate the antigen-induced sustained increase in PGD₂ in vitro, the BSM tissues isolated from naive mice were incubated with PGD₂ for 24 h. Using the PGD₂-treated BSM tissues, change in the BSM contractility was evaluated by measuring isometric contractions induced by acetylcholine (ACh) and high K⁺ depolarization. The receptor subtypes and signaling molecules involved in the PGD₂-induced BSM hyperresponsiveness were also identified pharmacologically.

Methods

Animals

Male BALB/c mice were purchased from Tokyo Laboratory Animals Science Co., Ltd. (Tokyo, Japan) and housed in a pathogen-free facility. All animal experiments were approved by the Animal Care Committee of Hoshi University (Tokyo, Japan).

Sensitization and antigenic challenge

Preparation of a murine model of allergic bronchial asthma, which has an in vivo AHR (13), was performed as described previously (14). In brief, BALB/c mice (8 weeks of age) were actively sensitized by intraperitoneal injections of 8 µg ovalbumin (OA; Sigma-Aldrich, St. Louis, MO, USA) with 2 mg Imject Alum (Pierce Biotechnology, Inc., Rockfold, IL, USA) on day 0 and day 5. The sensitized mice were challenged with aerosolized OA-saline solution (5 mg/ml) for 30 min on days 12, 16 and 20. A control group of mice received the same immunization procedure but inhaled saline aerosol instead of OA challenge. The aerosol was generated with a Mini Elite nebulizer (Philips Respironics, Amsterdam, Netherlands) and introduced to a Plexiglas chamber box (130 × 200 mm, 100 mm height) in which the mice were placed. Mice were sacrificed 1, 3, 6, 12 and 24 h after the last challenge by exsanguination from abdominal aorta under urethane (1.6 g/kg, i.p.; Sigma, St. Louis, MO, USA) anesthesia.

RNA extraction from bronchial smooth muscle (BSM) tissues

Total RNA of the mouse BSM tissue was isolated from an aliquot of the tissue using NucleoSpin™ miRNA (TaKaRa Bio, Inc., Shiga, Japan) according to the manufacturer’s instruction. cDNAs were prepared from the total RNA by using PrimeScript^TM RT reagent Kit (TaKaRa) according to the manufacturer’s instruction.

Real-time RT-PCR

The mRNA levels were examined by real-time RT-PCR. cDNA was subjected to real-time PCR analyses using Fast SYBR Green Master Mix (Applied biosystems, Foster, CA, USA) according to the manufacturer’s instruction. The PCR primer sets used are shown in Table 1, which was designed from published database, BLAST. The thermal cycle profile used was 1) denaturing for 3 s at 95 °C, 2) annealing and extending the primers for 30 s at 60 °C, and the reaction was run for 43 cycles.

Table 1. Primer sequences for RT-PCR used in the present study.

Gene name	RefSeq Accession		Sequence	Amplicon size
mouse Pla2g4c	NM_001168504	Sense	5’-GGACCGTTGCGTTTTTGTGA-3’
		Antisense	5’-GCAAAACCAGCATCCACCAG-3’	150 bp
mouse Ptgs2	NM_0011198	Sense	5’-CCGTGGGGAATGTATGAGCA-3’
		Antisense	5’-GGGTGGGCTTCAGCAGTAAT-3’	128 bp
mouse Hpgds	NM_019455	Sense	5’-TTCCCATGGGCAGAGAAAGA-3’
		Antisense	5’-GCCCAGGTTACATAATTGCCT-3’	143 bp
mouse Gapdh	NM_008084	Sense	5’-CCTCGTCCCGTAGACAAAATG-3’
		Antisense	5’-TCTCCACTTTGCCACTGCAA-3’	100 bp

Analyses of bronchoalveolar lavage fluids

After the exsanguinations, the chest of each animal was opened and a 20-gauge blunt needle was tied into the proximal trachea. Bronchoalveolar lavage fluids (BALFs) was obtained by intratracheal instillation of 1 ml/animal of phosphate-buffered saline (PBS; pH7.5, room temperature) into the lung while it was kept located within the thoracic cavity. The lavage was reinfused into the lung twice before final collection. The resultant supernatants of the lavage fluids were subjected to protein analyses. The level of PGD₂ was measured by Prostaglandin D₂-MOX, EIA Kit (Cayman Chemical Company, Ann Arbor, MI, USA), according to the manufacturer’s instructions.

Determination of bronchial smooth muscle (BSM) responsiveness

Mice were killed by exsanguination from abdominal aorta under urethane (1.6 g/kg, i.p.) anesthesia. Then the airway tissues under the larynx to lungs were immediately removed. About 3 mm length of the left main bronchus was isolated. The resultant tissue ring preparation was then suspended in a 5 ml-organ bath by two stainless-steel wires (0.2 mm diameter) passed through the lumen. For all tissues, one end was fixed to the bottom of the organ bath while the other was connected to a force-displacement transducer (TB-612T, Nihon Kohden) for the measurement of isometric force. A resting tension of 5.0 mN was applied. The buffer solution contained modified Krebs-Henseleit solution with the following composition (mM); NaCl 118.0, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, NaHCO₃ 25.0, KH₂PO₄ 1.2 and glucose 10.0. The buffer solution was maintained at 37°C and oxygenated with 95% O₂-5% CO₂. After the equilibration period, the concentration-response curve to ACh (10^–7–10^–3 M in final concentration) was constructed cumulatively. In another series of experiment, isotonic K⁺ solution (10–90 mM in final concentration) was cumulatively administrated in the presence of atropine (10^–6 M) to determine the BSM responsiveness to high K⁺-depolarization.

Tissue culture of isolated bronchial smooth muscles (BSMs)

After the determination of BSM responsiveness described above, the tissue preparations were immediately immersed in SmBM™ Medium (SmBM; Cambrex Bio Science Walkersville, Inc., MD, USA) containing 50 U/ml penicillin and 50 µg streptomycin (Invitrogen Corp., Grand Island, NY, USA) and cultured at 37 °C under a 5% CO₂ atmosphere in the presence or absence of PGD₂ (Cayman Chemical Company) for 24 h. After the incubation period, BSM responsiveness was determined as described above. In another series of experiments, the 24-h PGD₂ incubation was carried out in the presence or absence of pharmacological agent. The agents used in the present study were U0126, an ERK1/2 inhibitor (15), and SB203580, a p38 inhibitor (16).

Cell culture and sample collection

Normal human BSM cells (hBSMCs; Cambrex Bio Sience Walkersville, Inc., Walkersville, MD, USA) were maintained in SmBM medium supplemented with 5% fetal bovine serum, 0.5 ng/ml human epidermal growth factor (hEGF), 5 µg/ml insulin, 2 ng/ml human fibroblast growth factor-basic (hFGF-b), 50 µg/ml gentamicin and 50 ng/ml amphotericin B. Cells were maintained at 37 °C in a humidified atmosphere (5% CO₂), fed every 48–72 h, and passaged when cells reached 90–95% confluence. Then the hBSMCs (passages 4–7) were seeded in 6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) at density of 3,500 cells/cm² and, when 80–85% confluence was observed, cells were cultured without serum for 24 h before the addition of PGD₂. At the indicated time after the treatment with PGD₂, hBSMCs were immediately collected and disrupted with 1× SDS sample buffer (150 µl/well), and used for Western blot analyses.

Western blot analyses

Protein samples were subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins were then electrophoretically transferred to PVDF membrane. After blocking with EzBlock Chemi (ATTO, Japan), the PVDF membrane was incubated with the primary antibody. The primary antibodies used in the present study were monoclonal mouse anti-phospho-ERK1/2 (1:1,000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-ERK1/2 (1:1,000 dilution; Santa Cruz Biotechnology, Inc.) and monoclonal rabbit anti-phospho-p38 (1:10,000 dilution; Cell Signaling Technology Japan) antibodies. Then the membrane was incubated with HRP-linked anti-rabbit IgG (1:5,000 dilution; Cell Signaling Technology) or anti-mouse IgG (1:5,000 dilution; Santa Cruz Biotechnology, Inc.).

Statistical analyses

All the data are expressed as the mean ± S.E.M. Statistical significance of difference was determined by one-way analysis of variance (ANOVA) with post hoc Bonferroni/Dunn or two-way ANOVA (PrismTM 5 for Mac OS X; GraphPad Software, Inc., La Jolla, CA, USA). A value of P<0.05 was considered to be significant.

Results

Change in prostaglandin D₂ (PGD₂) production in the airways after antigen exposure

In the present study, we used our well-established murine model of allergic bronchial asthma (13, 14, 17,18,19). In this animal model of asthma, mRNAs for Pla2g4c (phospholipase A₂ group 4c), Ptgs2 (cyclooxygenase 2: COX2) and Hpgds (hematopoietic prostaglandin D synthase) were significantly increased in the BSM tissues: the peak increments were observed at 6, 1 and 12 h after the last antigen challenge, respectively (Fig. 1). The findings indicate that the AA metabolism is shifted toward PGD₂ production in the airways. So next, PGD₂ levels in bronchoalveolar lavage fluids (BALFs) were measured using the ELISA system. As shown in Fig. 2, the time-course analyses of PGD₂ levels in BALFs revealed that PGD₂ levels in the airways were increased immediately after antigen challenge and were sustained during the development of antigen-induced AHR. Interestingly, the time-course change of PGD₂ level (Fig. 2) was almost similar to that of Hpgds expression (Fig. 1C).

Fig. 1.

Time-course changes in the mRNA levels for phospholipase A₂ group 4c (Pla2g4c: A), cyclooxygenase 2 (Ptgs2: B) and hematopoietic prostaglandin D synthase (Hpgds: C) in bronchial smooth muscle tissues of mice after the last antigen challenge determined by real-time reverse transcriptase-polymerase chain reaction. The relative gene expression to the sensitized control animals (SC) was calculated by the 2^-∆∆CT methods using Gapdh as an internal control. Results are presented as mean ± SEM from five animals, respectively. *P<0.05, **P<0.01 and ***P<0.001 versus SC group by one-way ANOVA with post hoc Bonferroni/Dunnett’s test.

Fig. 2.

Time-course changes in the prostaglandin D₂ (PGD₂) levels in bronchoalveolar lavage fluids (BALFs) after the last antigen challenge determined by ELISA. Results are presented as mean ± S.E.M. from five animals, respectively. **P<0.01 and ***P<0.001 versus sensitized control (SC) by one-way ANOVA with post hoc Bonferroni/Dunnett’s test.

Effects of a 24-h incubation with prostaglandin D₂ (PGD₂) on bronchial smooth muscle (BSM) contractility

The findings that PGD₂ levels in BALFs sustainedly increased (Fig. 2) indicate that smooth muscle cells of the airways are continually exposed to PGD₂. To imitate the antigen-induced sustained increase in PGD₂in vitro, the BSM tissues isolated from naive mice were incubated with PGD₂ for 24 h as described above. As shown in Fig. 3A, in the BSM tissues isolated from naive animals, the 24-h incubation with PGD₂ caused a hyperresponsiveness to acetylcholine (ACh): the ACh concentration-response curve was significantly shifted upward by the 24-h PGD₂ incubation (P<0.01 by two-way ANOVA; Fig. 3A). On the other hand, no significant change in the contractile responsiveness to high K⁺ depolarization (in the presence of 10⁻⁶ M atropine) was observed in the PGD₂-incubated BSM tissues (Fig. 3B).

Fig. 3.

Effects of in vitro incubation with prostaglandin D₂ (PGD₂) on bronchial smooth muscle (BSM) responsiveness to acethylcholine (ACh) in naive mice. The BSM tissues isolated from naive animals were incubated with PGD₂ (10⁻⁵ M: closed circles) or its vehicle (0.1% DMSO: open circles) for 24 h. The BSM responsiveness to cumulatively applied acetylcholine (ACh: A) or K⁺ (in the presence of 10⁻⁶ M atropine: B) were measured as described in METHODS. The BSM responsiveness to ACh but not K⁺ was significantly increased when the BSMs were incubated with PGD₂. Results are presented as mean ± S.E.M. from 8 animals, respectively. **P<0.01 versus vehicle by two-way ANOVA.

Phosphorylation of ERK1/2 and p38 by prostaglandin D₂ (PGD₂) in bronchial smooth muscle (BSM) cells

To identify the signaling molecules activated by PGD₂ in the BSM cells, cultured BSM cells were stimulated with PGD₂. As shown in Fig. 4, immunoblot analyses revealed that PGD₂ caused a distinct phosphorylation of ERK1/2 and p38, i.e., activation of these molecules, in the BSM cells. Both the PGD₂-induced phosphorylation of ERK1/2 and p38 were abolished by laropiprant (a DP₁ receptor antagonist), whereas fevipiprant (a DP₂ receptor antagonist) had no effect on their phosphorylation (Fig. 4). On the other hand, in the BSM cells, PGD₂ did not cause activation of other molecules, such as JNK and STAT3 (data not shown), that were reportedly activated in various types of the cells (7, 20,21,22,23).

Fig. 4.

Activation of ERK1/2 (A) and p38 (B) induced by prostaglandin D₂ (PGD₂) in cultured bronchial smooth muscle (BSM) cells. After a starvation period, the BSM cells were stimulated with prostaglandin D₂ (PGD₂: 10⁻⁵ M). At the indicated time after the stimulation, the protein expressions of ERK1/2 and p38 and their phosphorylated forms (p-ERK1/2 and p-p38) were determined from immunoblots. Cells were also pretreated with laropiprant (a DP₁ receptor antagonist), fevipiprant (a DP₂ receptor antagonist), or their vehicle (0.1% DMSO) 15 min before addition of PGD₂.

Effects of inhibitors for ERK1/2 and p38 on the bronchial smooth muscle (BSM) hyperresponsiveness induced by prostaglandin D₂ (PGD₂)

To determine the roles of ERK1/2 and p38 on the BSM hyperresponsiveness induced by PGD₂, the BSM tissues isolated from naive mice were co-incubated with an ERK1/2 inhibitor U0126 or a p38 inhibitor SB203580 during the 24-h PGD₂ incubation period. As shown in Fig. 5A, co-incubation with U0126 had no significant effect on the ACh responsiveness in the 24-h PGD₂-incubated BSMs. However, the BSM hyperresponsiveness to ACh induced by the 24-h PGD₂ incubation was attenuated by the co-incubation with SB203580 (Fig. 5B): the ACh concentration-response curve was significantly shifted downward when the BSM tissues were incubated in the presence of SB203580 (P<0.01 by two-way ANOVA; Fig. 5B).

Fig. 5.

Effects of co-incubation with U0126 (an ERK1/2 inhibitor: A) and SB203580 (a p38 inhibitor: B) on the prostaglandin D₂ (PGD₂)-induced bronchial smooth muscle (BSM) hyperresponsiveness to acetylcholine (ACh) in mice. The BSM tissues isolated from naive BALB/c mice were incubated with PGD₂ (10⁻⁵ M, for 24 h) in the absence or presence of respective inhibitors. After the incubation period, the BSM responsiveness to ACh was determined as described in METHODS. Results are presented as mean ± S.E.M. from at least five animals. *P<0.05 and **P<0.01 versus control by two-way ANOVA.

Discussion

Consistently with the previous report (8), the mRNA expressions for Pla2g4c, Ptgs2 and Hpgds were significantly increased in the bronchial smooth muscle (BSM) tissues (Fig. 1), indicating that arachidonic acid metabolism is shifted towards prostaglandin D₂ (PGD₂) production in mice with allergic asthma. Indeed, a sustained increase in PGD₂ was observed in the airways after the antigen stimulation (Fig. 2). The BSM hyperresponsiveness reported in experimental asthma (14, 18) was replicated when the BSM tissues isolated from naive animals were incubated with PGD₂ for 24 h (Fig. 3). PGD₂ caused phosphorylation of ERK1/2 and p38 in the BSM cells via an activation of DP₁ receptor (Fig. 4). The BSM hyperresponsiveness induced by the 24-h PGD₂ incubation was inhibited by a p38 inhibitor SB203580, but not by an ERK1/2 inhibitor U0126 (Fig. 5). These findings suggest that a sustained increase in PGD₂ level in the airways is a cause of the AHR in asthma, and that the DP₁-mediated activation of p38 is responsible for the BSM hyperresponsiveness induced by PGD₂.

Our previous microarray analysis of differentially expressed genes (GEO accession No. GSE116504) demonstrated a significant change in the arachidonic acid (AA) metabolism pathway (KEGG map00590) in the BSM tissues of asthma (8). In particular, the AA metabolism was largely shifted towards PGD₂ production in the diseased BSMs (8). Currently, time-course studies of the gene expression revealed that Pla2g4c, Ptgs2 and Hpgds were up-regulated in the relatively early period after antigen challenge (Fig. 1). Notably, time-course change of the PGD₂ level in BALF (Fig. 2) seems to be parallel with that of up-regulation of Hpgds (Fig. 1C), indicating that the hematopoietic prostaglandin D synthase (HPGDS) is responsible for the increased PGD₂ production in the diseased airways. The idea is also supported by the observation that the lipocalin-type PGDS (LPGDS), one of the two types of PGDS, expression was not changed in the diseased BSM tissues in the microarray analysis (GSE116504).

The current time-course study also revealed a sustained increase in PGD₂ in the airways: the PGD₂ levels in BALFs were already increased just after the antigen exposure (Fig. 2). The findings indicate that smooth muscle cells of the airways are continually exposed to PGD₂, and remind us of an idea that the sustained increase in PGD₂ might be a cause of augmented BSM contractility at the AHR. Indeed, consistently with the previous report (8), the 24-h incubation with PGD₂ induced a hyperresponsiveness to ACh in the BSM tissues isolated from naive animals (Fig. 3A). PGD₂ had no effect on contractile responsiveness to high K⁺ depolarization (in the presence of 10⁻⁶ M atropine, to block the effect of endogenously released ACh: Fig. 3B). In the antigen-induced AHR mice, an augmented BSM contractility to ACh without alteration of high K⁺ responsiveness has also been observed (14, 18). It is thus possible that PGD₂ is one of the factors that contribute to the altered property of BSMs in allergic asthma.

To identify the signaling molecule(s) activated by PGD₂ in BSM cells, cultured human BSM cells (hBSMCs) were stimulated with PGD₂. Because the mouse BSM tissue contains various types of cells other than the BSM cells, the commercially available hBSMCs were used in the present study. Reportedly, PGD₂ has an ability to activate various signaling molecules, such as ERK1/2, p38, JNK, and STAT3 (7, 20,21,22,23). Among these molecules, the current immunoblot analyses revealed that PGD₂ caused a distinct activation of ERK1/2 and p38, i.e., phosphorylation of these proteins, in the hBSMCs (Fig. 4). The findings are consistent with the previous report, in which phosphorylation of these molecules induced by PGD₂ were demonstrated in a human BSM cell line, BSM2146 cells (7). To date, two distinct receptor subtypes for PGD₂, termed DP₁ and DP₂ receptors, have been identified (24,25,26). To identify receptor subtype responsible for the phosphorylation of ERK1/2 and p38 induced by PGD₂, the hBSMCs were pre-incubated with a DP₁ antagonist laropiprant (27) or a DP₂ antagonist fevipiprant (28) during the PGD₂ stimulation. As a result, both the PGD₂-induced phosphorylation of ERK1/2 and p38 were abolished by laropiprant, whereas fevipiprant had no effect on their phosphorylation (Fig. 4). Thus, PGD₂ acts on DP₁ receptors to activate ERK1/2 and p38 in BSM cells.

The results that PGD₂ activated ERK1/2 and p38 in the BSM cells suggest an involvement of these signaling molecules in the augmented BSM responsiveness to ACh. Indeed, activation of these molecules could cause an augmentation of the contraction in airway smooth muscles (29,30,31,32). To determine the roles of ERK1/2 and p38 on the BSM hyperresponsiveness induced by PGD₂, the BSM tissues isolated from naive mice were co-incubated with an ERK1/2 inhibitor U0126 (15) or a p38 inhibitor SB203580 (16) during the 24-h PGD₂ incubation period. As shown in Fig. 5, although U0126 had no significant effect (Fig. 5A), the BSM hyperresponsiveness to ACh induced by PGD₂ was attenuated by the co-incubation with SB203580 (Fig. 5B): the ACh concentration-response curve was significantly shifted downward when the BSM tissues were incubated in the presence of SB203580 (Fig. 5B). The findings strongly suggest that the PGD₂-induced BSM hyperresponsiveness is mediated by an activation of p38.

The current study clearly demonstrated that the 24-h incubation with PGD₂ caused an augmented BSM contractility to ACh (Fig. 3). The results indicate that PGD₂ acts on DP₁ receptor on the BSMs to activate p38 (Figs. 4 and 5), resulting in the induction of BSM hyperresponsiveness. On the other hand, some of the PGD₂ metabolites may also be involved in the BSM hyperresponsiveness. PGD₂ is known to convert to PGJ₂ family of prostanoids (33), and their physiological and pathophysiological roles have also been suggested. For example, a PGD₂ metabolite 15-deoxy-Δ^12,14-prostaglandin J₂ (15-deoxy PGJ₂) has an ability to activate signaling molecules, such as peroxisome proliferator-activated receptor-γ (PPAR-γ) (34) and phosphoinositide 3-kinase (PI3 K)-Akt (35). Interestingly, exogenously applied 15-deoxy PGJ₂ could inhibit airway inflammation and hyperresponsiveness in murine models of allergic asthma (36). Further studies are needed to make clear the exact role of PGD₂ on the development of BSM hyperresponsiveness in allergic asthma.

Again, the current study revealed that PGD₂ caused a phosphorylation of p38 in the BSM cells via an activation of DP₁ receptor (Fig. 4B). The BSM hyperresponsiveness induced by the 24-h PGD₂ incubation was inhibited by a p38 inhibitor SB203580 (Fig. 5B). These findings indicate that the DP₁-mediated activation of p38 is responsible for the BSM hyperresponsiveness induced by PGD₂. However, it is well known that the DP₁ is a Gs protein-coupled receptor. In smooth muscle cells including the airways, the Gs protein activation causes an inhibition of the contraction via an increase in cAMP level (e.g., 37,38,39). Indeed, PGD₂ reportedly induced a relaxation via an activation of DP₁ receptor in the tracheal smooth muscles pre-contracted with carbachol (11). In contrast, PGD₂ also elicited a contraction of the airway smooth muscle (8, 40,41,42) probably via an activation of DP₁ receptor (42). Although the contrariety is not explainable currently, the DP₁ receptor stimulation with PGD₂ may primarily activate signaling molecules other than the cAMP-dependent relaxing ones, resulting in an augmentation of the BSM contraction.

A small GTPase RhoA is a key protein that mediates sensitization of smooth muscle contraction including the airways (43, 44). In the BSMs of experimental asthma, an augmentation of agonist-induced RhoA-mediated Ca²⁺ sensitization has been demonstrated (14, 18, 45). No change in the contraction mediated solely by cytosolic Ca²⁺, such as the contraction induced by high K⁺ depolarization, has also been demonstrated in the diseased BSMs (14, 18). The current findings that PGD₂ augmented the contraction induced by ACh, but not by high K⁺ depolarization (Fig. 3), also support an idea that PGD₂ may augment the RhoA-mediated Ca²⁺ sensitization of contraction. Indeed, our previous study demonstrated that PGD₂ has an ability to activate RhoA in the BSMs (42). However, in our preliminary study, the 24-h incubation with PGD₂ did not cause the RhoA up-regulation, that is often observed in BSMs of experimental asthma (14, 18, 45).

As described above, our previous study demonstrated that PGD₂ has an ability to activate RhoA/Rho-kinase signaling: the Ca²⁺ sensitization of contraction was augmented in the presence of PGD₂ (42). Currently, the BSM tissues were incubated with PGD₂ for 24 h, and their contractile responses were then measured after washing out PGD₂. Nevertheless, in the ABSENCE of PGD₂, the BSM responsiveness to ACh, but not to high K⁺ depolarization, was significantly augmented (Fig. 3). On the other hand, the 24-h PGD₂ incubation did not cause the RhoA up-regulation (see above). Taken together, the sustained exposure to PGD₂ may up-regulate signaling molecule(s) other than RhoA activated by the muscarinic receptor stimulation. Further studies are needed to identify the mechanism of BSM hyperresponsiveness induced by the sustained exposure to PGD₂.

In conclusion, the current study demonstrated that the 24-h incubation with PGD₂ caused an augmentation of contraction in BSMs isolated from naive mice. The pharmacological study also revealed an involvement of DP₁ receptor-mediated activation of p38 in the BSM hyperresponsiveness induced by PGD₂. Thus, a sustained increase in PGD₂ in the airways might be a cause of the AHR in allergic asthmatics.

Funding

This work was partly supported by Grant-in-Aid for Scientific Research (C) Grant Number 15K08248 (YC) from the Japan Society for the Promotion of Science (JSPS).

Conflict of Interest

None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.