DP1 receptor signaling prevents the onset of intrinsic apoptosis in eosinophils and functions as a transcriptional modulator

Abstract

Prostaglandin (PG) D₂ is the ligand for the G-protein coupled receptors DP1 (D-type prostanoid receptor 1) and DP2 (also known as chemoattractant receptor homologous molecule, expressed on Th2 cells; CRTH2). Both, DP1 and DP2 are expressed on the cellular surface of eosinophils; although it has become quite clear that PGD₂ induces eosinophil migration mainly via DP2 receptors, the role of DP1 in eosinophil responses has remained elusive. In this study, we addressed how DP1 receptor signaling complements the pro-inflammatory effects of DP2. We found that PGD₂ prolongs the survival of eosinophils via a DP1 receptor-mediated mechanism that inhibits the onset of the intrinsic apoptotic cascade. The DP1 agonist BW245c prevented the activation of effector caspases in eosinophils and protected mitochondrial membranes from depolarization which-as a consequence-sustained viability of eosinophils. DP1 activation in eosinophils enhanced the expression of the anti-apoptotic gene BCL-X_L , but also induced pro-inflammatory genes, such as VLA-4 and CCR3. In HEK293 cells that overexpress recombinant DP1 and/or DP2 receptors, activation of DP1, but not DP2, delayed cell death and stimulated proliferation, along with induction of serum response element (SRE), a regulator of anti-apoptotic, early-response genes. We conclude that DP1 receptors promote the survival via SRE induction and induction of pro-inflammatory genes. Therefore, targeting DP1 receptors, along with DP2, may contribute to anti-inflammatory therapy in eosinophilic diseases.

Keywords: allergy; apoptosis; inflammation; prostanoids.

Figure 1

DP1 receptor activation promotes survival of eosinophils. Isolated eosinophils were cultured with or without 1 μM of PGD₂, DK‐PGD₂, BW245c, or IL‐5 [100 pM] for 18 h. BW245c, PGD₂, and IL‐5 significantly enhanced the portion of annexin V⁻/PI⁻ eosinophils (A) and PGD₂, BW245c, DK‐PGD₂, and IL‐5 decreased the annexin V+ population (B) as compared to vehicle‐treated cells. (C) Shows the percentage of annexin V⁻/PI⁻, annexin V⁺/PI⁻, annexin V⁺/PI⁺, annexin V⁻/PI⁺ populations of total eosinophils at 18 h. Data show mean ± sem of 5 individual experiments using eosinophils from different donors

Figure 2

Activation of the DP1 receptor inhibits the intrinsic apoptotic pathway in eosinophils. Caspase 3/7 activity in eosinophils was assessed in a luminescent assay after 18 h incubation with vehicle, 1 μM of PGD₂, BW245c, DK‐PGD₂, or 15d‐PGJ₂, Rosiglitazone (Rosi), or 100 pM of IL‐5. Cells were pretreated with 1 μM of vehicle, Cay10471 or T0070907 or increasing concentrations of MK0524 prior to agonist treatment. (A) BW245c protected from effector caspase 3/7 activity in eosinophils cultured for 18 h in serum‐reduced media (RPMI 1% FBS, 1% PenStrep). Data show means ± SEM of 5–6 individual experiments. (B) DP1 receptor antagonist MK0524 reversed the BW245c‐induced protection from caspase 3/7 activation. Data show mean ± sem of 9 individual experiments. (C) Blockade of DP2 (Cay10471) unmasks PGD₂ as a potent inhibitor of caspase 3/7 activation. Data show mean ± sem of 4 individual experiments. (D) 15dPGJ₂, but not rosiglitazone (Rosi), increased caspase 3/7 activity, which was not altered by the PPAR‐α antagonist T0070907 (n = 7)

Figure 3

Activation of the DP1 receptor maintains mitochondrial function in aging eosinophils. (A, B) Flow cytometric dot plot analysis of eosinophils stained with JC1 after 18 h incubation with 1 μM of vehicle, PGD₂, BW245c, DK‐PGD₂, or 50 pM of IL‐5. Depolarized mitochondrial membrane potential (MMP) is shown by the loss in yellow fluorescence (FL2). (A) Shows representative dot plots and (B) means of n = 3–4 individual experiments ± sem. **P ≤ 0.01. (C) MitoTracker Red CMXRos staining of eosinophils incubated with 1 μM of vehicle, PGD₂, BW245c, and DK‐PGD₂, and 100 pM of IL‐5 up to 18 h. Representative images of 3 independent experiments are shown

Figure 4

Bcl‐2 family protein inhibitor BH3I‐1 reverses the anti‐apoptotic effect of DP1 receptor activation. Eosinophils isolated from peripheral blood were incubated with BW245c [1 μM] in combination with or without Bcl‐X_L inhibitors (A and C) BH3l‐1 [50 μM] for 5h or (B) HA14.1 [10 μM] for 3 h; (C) 18 h. (A) BH3l‐1 prevented the reduction of annexin V⁺ apoptotic cells induced by BW245c or IL‐5. (B) HA14.1 induced apoptosis of veh‐ and BW245c‐treated eosinophils. (C) In contrast to BH3I‐1, HA14.1 reversed the BW245c‐induced inhibition of caspase 3/7 activity. Data show mean ± sem of 5 individual experiments

Figure 5

PGD₂ enhances the viability of HEK‐DP2+DP1 and HEK‐DP1 but not of HEK‐DP2 cells. (A) HEK293 cell lines were starved in OptiMEM for 4 h and incubated with increasing concentrations of PGD₂ for 24 of 48 h. Viability was detected by MTS assay (n = 5). (B) MK0524 but not Cay10471 antagonized the viability‐enhancing action of PGD₂ on HEK‐DP2+DP1. (n = 3). Data show means of 3–5 independent experiments ± sem

Figure 6

PGD₂ treatment causes a growth advantage of HEK‐DP2+DP1 and HEK‐DP1 but not of HEK‐DP2 cells. (A) PGD₂ treatment of DP1‐expressing cells led to morphologically visible growth advantage compared to HEK‐DP2 cells. Data show representative images of 4 independent experiments. (B) Cultures of serum‐starved HEK‐DP2+DP1, HEK‐DP1, or HEK‐DP2 cells were treated with increasing concentrations of PGD₂ or 10% FBS and their electrical resistance was monitored for more than 60 h on an ECIS device. Data show means of 5 independent experiments + sem

Figure 7

DP1 induces SRE activation. (A) PGD₂ activates SRE in HEK‐DP2 + DP1 and HEK‐DP1 but not in HEK‐DP2. (B) DP1 agonist BW245c induces SRE activation in HEK‐DP2 + DP1 and HEK‐DP1. (C) DP2 agonist DK‐PGD₂ does not induce SRE activation. (D) Antagonists of DP1 (BWA868c) and DP2 (Cay10471) block PGD₂‐ and (E) BW245c induced SRE activation in HEK‐DP2 + DP1. Data are shown as mean ± sem of 3–6 independent experiments

Figure 8

DP1 receptor agonist BW245c enhances (A) mRNA expression of CCR3 and VLA‐4 and (B) upregulates surface expression of CCR3 and VLA‐4. Isolated eosinophils (5 × 10⁶/ml) were incubated with vehicle (EtOH), DK‐PGD₂ [1 μM], or BW245c [1 μM] for 3 h (A) or 18h (B) (RPMI, 1% FBS, 1%, PenStrep, 37°C). mRNA expression was measures by qRT PCR (A). Surface expression was determined by flow cytometric analysis (B). Data show mean ± sem (n = 5–10)