PPARγ as an E3 Ubiquitin-Ligase Impedes Phosphate-Stat6 Stability and Promotes Prostaglandins E2-Mediated Inhibition of IgE Production in Asthma

Abstract

Increased serum IgE level is one of the features of allergic asthma. It is reported that IgE production can be enhanced by E-prostanoid 2 (EP2) receptor of prostaglandin E₂ (PGE₂); however, whether E-prostanoid 4 (EP4) receptor (encoded by Ptger4) has a unique or redundant role is still unclear. Here, we demonstrated the mice with B cell-specific deletion of the EP4 receptor (Ptger4^fl/flMb1^cre+/-) showed their serum levels of IgE were markedly increased. A much more severe airway allergic inflammation was observed in the absence of EP4 signal using the OVA-induced asthma model. Mechanistic studies demonstrated that the transcription levels of AID, GLTε, and PSTε in EP4-deficient B cells were found to be significantly increased, implying an enhanced IgE class switch. In addition, we saw higher levels of phosphorylated STAT6, a vital factor for IgE class switch. Biochemical analyses indicated that inhibitory effect of EP4 signal on IgE depended on the activation of the PI3K-AKT pathway. Further downstream, PPARγ expression was up-regulated. Independent of its activity as a transcription factor, PPARγ here primarily functioned as an E3 ubiquitin-ligase, which bound the phosphorylated STAT6 to initiate its degradation. In support of PPARγ as a key mediator downstream of the EP4 signal, PPARγ agonist induced the down-regulation of phospho-STAT6, whereas its antagonist was able to rescue the EP4-mediated inhibition of STAT6 activation and IgE production. Thus, our findings highlight a role for the PGE₂-EP4-AKT-PPARγ-STAT6 signaling in IgE response, highlighting the therapeutic potential of combined application of EP4 and PPARγ agonists in asthma.

Keywords: AKT; EP4; IgE class switching; PGE2; PPARγ; STAT6; asthma.

Figure 1

Exaggerated asthmatic responses in EP4-deficiency mice. (A) Serum level of total IgE of Ptger4^fl/fl (EP4^f/f) and Ptger4^fl/flMb1^cre+/− (EP4^f/f Mb1^cre) were detected by ELISA (n = 6). (B) EP4^f/f and EP4^f/f Mb1^cre were immunized with OVA following a protocol as described in the methods. (C,D) Serum levels of total (C) and OVA-specific (D) IgE were determined by ELISA 24 h after the last challenge (n = 5). (E) Total cell number in the BALF (n = 5). (F,G) Representative images showing hemotoxylin and eosin (HE) (F) and Periodic Acid-Schiff (PAS) (G) staining of the lung tissue. Scale bar equals 100 μm (F) or 50 μm (G). Data are presented as mean ± SD, representing one of three independent experiments. Statistical differences were determined by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test (C–E) or by unpaired two-tailed Student's t-test (A). ***P < 0.001.

Figure 2

Anti-CD40 + IL4 amplifies the effect of PGE₂ on EP4. (A) Splenic B cells from EP4^f/f or EP4^f/f Mb1^cre were stimulated with anti-CD40 (1 μg/ml) + IL4 (50 ng/ml). IgE concentrations in the culture supernatant at day 7 were measured by ELISA (n = 6). (B) ELISA of IgE levels from WT B cells treated with anti-CD40+ IL4 with or without concomitant administration of PGE₂ (10 nM), PGE₁-alcohol (1 μM), or ONO-AE3-208 (10 μM) for 7 days (n = 5). (C,D) Mice were treated with OVA with or without concomitant administration of PGE₂ (300 μg/kg), PGE₁-alcohol (500 μg/kg), or ONO-AE3-208 (1,000 μg/kg) following a protocol as described in the methods. Airway inflammatory responses were analyzed 24 h after the last challenge. Serum levels of total (C) and OVA-specific (D) IgE were determined by ELISA (n = 7). Each symbol represents an individual mouse. (E) Confocal microscopy of the expression of Rab5, EP2, and EP4 immunostaining in the indicated colors in WT B cells under different stimulations for 15 min. Scale bars, 2.5 μm. Data in (E) are representative data of three independent experiments. (F,G) Surface expression of EP4 was determined by flow cytometry at 15 min. Representative histogram (F) and the percentage of decreased EP4 expression (G) are shown (n = 3). Control means unstimulated B cells. Data in (C–F) are representing one of three independent experiments. Data in (A,B,G) are pooled from three independent experiments. Statistical differences were determined by one-way ANOVA with Tukey's multiple comparisons test (A–G). Data are presented as mean ± SD. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001, ns, not significant.

Figure 3

Modulation of the pSTAT6 signaling by EP4-mediated signal. (A) Proliferation assays were identified by Brdu incorporation with the stimulation of anti-IgM or anti-CD40. Representative histogram and the percentage of BrdU⁺ B cells are shown (n = 3). (B) Apoptosis assays were determined by flow cytometry after the stimulation of anti-IgM 24 h. Representative histogram and the percentage of Annexin V⁺ 7-AAD⁺ B cells are shown (n = 3). (C,D) RNA was prepared from day 3 cultures of EP4^f/f and EP4^f/f Mb1^cre B cells in the presence of CD40+IL4 and examined for AID (C), GLTε and PSTε (D) mRNA expression by quantitative PCR (n = 5). (E,F,G) RT-qPCR analysis of AID (E), GLTε (F), and PSTε (G) on WT B cells from day 3 cultures with indicated groups (n = 6). (H,I) Splenic B cells from EP4^f/f or EP4^f/f Mb1^cre mice were stimulated with anti-CD40+ IL-4 in the presence or absence of PGE₂ and PGE₁-alcohol. Cells were harvested at 30 min after stimulation. Phosphorylated and total STAT6 was detected by Western blotting. Representative blots are shown of three independent experiments. (J) Expression of IL4R was determined by flow cytometry. Representative histogram and the percentage of B220⁺IL4R⁺ B cells are shown. Data in (A,B,H,J) are representative data of three independent experiments. Data are pooled from two or three independent experiments. Statistical differences were determined by one-way (E,F,G) ANOVA with Tukey's multiple comparisons test or by unpaired two-tailed Student's t-test (A–D,J). Data are presented as mean ± SD. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001, ns, not significant.

Figure 4

PGE₂-EP4 Signaling Promotes the Phosphorylation of Akt to Regulate pSTAT6. (A) Immunoblot analysis of p-PKA C, PKA C-α, p-CREB, CREB treated with anti-CD40+ IL-4 for 30 min. (B,C) B cells were stimulated with anti-CD40+ IL-4 in the presence or absence of dibutyryl cAMP (db-cAMP) (4 μM) and the cultures were analyzed on day 7. (D) PI3 Kinase p110 δ in EP4^f/f and EP4^f/f Mb1^cre B cells treated with anti-CD40+ IL-4 for 30 min. (E) Immunoblot analysis of PI3 Kinase p110 δ in WT B cells treated with anti-CD40+ IL-4 in the presence or absence of PGE₁-alcohol (1 μM) for 30 min. (F) Immunoblot analysis of p-Akt (Ser473), Akt, p-FoxO1 (Ser256), and FoxO1in EP4^f/f and EP4^f/f Mb1^cre B cells treated with anti-CD40+ IL-4 for 30 min. (G) Immunoblot analysis of p-Akt (Ser473) and Akt in WT B cells treated with anti-CD40+ IL-4 in the presence or absence of PGE₁-alcohol (1 μM) for 30 min. (H) Immunoblot analysis of p-STAT6, STAT6, p-Akt (Ser473), and Akt in WT B cells treated with anti-CD40+ IL-4 in the presence or absence of PGE₂ (10 nM), PGE₁-alcohol (1 μM), and MK2206 (0.5 μM) for 30 min. (I) ELISA of IgE levels from WT B cells treated with anti-CD40+ IL4 with or without concomitant administration of PGE₂ (10 nM), PGE₁-alcohol (1 μM), or ONO-AE3-208 (10 μM), MK2206 (0.5 μM) for 7 days (n = 3). Data in (A,D,E,F,G,H) are representative data of three independent experiments. Data are pooled from three independent experiments. Statistical differences were determined by one-way ANOVA with Tukey's multiple comparisons test. Data are presented as mean ± SD. ^**p < 0.01 and ^***p < 0.001, ns, not significant.

Figure 5

The Phosphorylation of STAT6 is Mediated by PPARγ. (A) Protein–protein interaction network (PPI) of Ptger4, Akt, FoxO3, and PPARγ, PPI enrichment p-value: 0.000834. (B,C) RT-qPCR analysis of the Hsp90aa1, Nos3, Mtor, and Pparg gene expression in EP4^f/f and EP4^f/f Mb1^cre B cells for 3 days [n = 4(B), 3(C)]. (mean ± SEM) (D) Immunoblot analysis of the Pparg gene expression in EP4^f/f and EP4^f/f Mb1^cre B cells for 3 days. (E) Immunoblot analysis of PPARγ in WT B cells treated with anti-CD40+ IL-4 in the presence or absence of PGE₂ (10 nM) and PGE₁-alcohol (1 μM) for 30 min. (F,G) Immunoblot analysis of PPARγ, p-STAT6, STAT6, p-Akt (Ser473), and Akt in WT B cells treated with anti-CD40+ IL-4 in the presence or absence of PGE₂ (10 nM), PGE₁-alcohol (1 μM), Pioglitazone (2 μM), T0070907 (100 nM) and MK2206 (0.5 μM) for 30 min. (H) ELISA of IgE levels from WT B cells treated with anti-CD40+ IL4 with or without concomitant administration of PGE₁-alcohol (1 μM) and T0070907 (100 nM) for 7 days (n = 3). (mean ± SEM) (I), (J) Mice were treated with OVA with or without concomitant administration of PGE₁-alcohol (500 μg/kg) and T0070907 (500 μg/kg) following a protocol as described in the methods. Serum levels of total (H) and OVA-specific (I) IgE were determined by ELISA (n = 5). (mean ± SD) Data in (D–G) are representative data of three independent experiments. Data are pooled from three (B,C,H–J) independent experiments. Statistical differences were determined by one-way (H–J) ANOVA with Tukey's multiple comparisons test or by unpaired two-tailed Student's t-test (B,C). Data are presented as mean ± SD, unless specifically noted. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001, ns, not significant.

Figure 6

PPARγ induces the ubiquitination of pSTAT6. (A) Whole-cell extracts were immunoprecipitated (IP) with anti-PPARγ followed by immunoblotting (IB) with anti-p-STAT6 antibody. Rabbit IgG represents a control antibody used for IP. (B) Confocal microscopy of the expression of STAT6 and PPARγ in WT B cells treated with or without anti-CD40+ IL4 for 30 min. Scale bars, 2.5 μm. (C) Confocal microscopy of the expression of p-STAT6 and PPARγ immunostaining in the indicated colors in WT B cells treated with or without anti-CD40+ IL4 for 30 min. Scale bars, 2.5 μm. (D) EP4^f/f and EP4^f/f Mb1^cre B cells were treated with 20 μM MG132 or DMSO for 10 h before 30 min stimulation of anti-CD40+ IL4 and immunoblotted with indicated antibodies. (E) EP4^f/f and EP4^f/f Mb1^cre B cells were treated with 20 μM MG132 for 10 h before 30 min stimulation of anti-CD40+ IL4. IP with anti-p-STAT6 followed by IB with anti-Ubiquitin and anti-p-STAT6 antibody. (F) WT B cells were treated with 20 μM MG132 for 10 h before 30 min stimulation of anti-CD40+ IL4 with or without T0070907 (100 nM). IP with anti-p-STAT6 followed by IB with anti-Ubiquitin and anti-p-STAT6 antibody. Each experiment was independently repeated at least three times.