Opposing effects of prostaglandin E2 receptors EP3 and EP4 on mouse and human β-cell survival and proliferation

Abstract

Objective: Hyperglycemia and systemic inflammation, hallmarks of Type 2 Diabetes (T2D), can induce the production of the inflammatory signaling molecule Prostaglandin E₂ (PGE₂) in islets. The effects of PGE₂ are mediated by its four receptors, E-Prostanoid Receptors 1-4 (EP1-4). EP3 and EP4 play opposing roles in many cell types due to signaling through different G proteins, G_i and G_S, respectively. We previously found that EP3 and EP4 expression are reciprocally regulated by activation of the FoxM1 transcription factor, which promotes β-cell proliferation and survival. Our goal was to determine if EP3 and EP4 regulate β-cell proliferation and survival and, if so, to elucidate the downstream signaling mechanisms.

Methods: β-cell proliferation was assessed in mouse and human islets ex vivo treated with selective agonists and antagonists for EP3 (sulprostone and DG-041, respectively) and EP4 (CAY10598 and L-161,982, respectively). β-cell survival was measured in mouse and human islets treated with the EP3- and EP4-selective ligands in conjunction with a cytokine cocktail to induce cell death. Changes in gene expression and protein phosphorylation were analyzed in response to modulation of EP3 and EP4 activity in mouse islets.

Results: Blockade of EP3 enhanced β-cell proliferation in young, but not old, mouse islets in part through phospholipase C (PLC)-γ1 activity. Blocking EP3 also increased human β-cell proliferation. EP4 modulation had no effect on ex vivo proliferation alone. However, blockade of EP3 in combination with activation of EP4 enhanced human, but not mouse, β-cell proliferation. In both mouse and human islets, EP3 blockade or EP4 activation enhanced β-cell survival in the presence of cytokines. EP4 acts in a protein kinase A (PKA)-dependent manner to increase mouse β-cell survival. In addition, the positive effects of FoxM1 activation on β-cell survival are inhibited by EP3 and dependent on EP4 signaling.

Conclusions: Our results identify EP3 and EP4 as novel regulators of β-cell proliferation and survival in mouse and human islets ex vivo.

Keywords: COX-2, cyclooxygenase-2; Cell death; DAG, diacylglycerol; EP1-4, E-Prostanoid Receptors 1-4; GPCR, G protein-coupled receptor; IP3, inositol 1,4,5-trisphosphate; PGE2, prostaglandin E2; PKA, protein kinase A; PL, placental lactogen; PLC, phospholipase C; PT, pertussis toxin; Pancreatic β-cell; Proliferation; Prostaglandin E2.

Figure 1

Placental lactogen decreases Ptger3γ in mouse islets. Wild type mouse islets (8–10 weeks old) were cultured for four days in the presence of vehicle (n = 4) or placental lactogen (n = 4). qRT-PCR was performed for PGE₂ synthesis genes (Ptgs2, Ptges1-3) and receptor genes (Ptger1-4). *p = 0.0147 vs vehicle. All data are represented as 2^−ΔΔCt relative to vehicle. Data were analyzed using a Student's t test.

Figure 2

EP3 inhibits, while EP4 does not affect, mouse β-cell proliferation and displays increased expression with age. (A) Young mouse islets (8–10 weeks of age) were treated with vehicle, PL, sulprostone +/− PL, or DG-041 +/− PL for 4 days before being immunolabeled for insulin, Ki67, and DAPI. *versus vehicle; ^versus PL; ⁺versus DG-041 alone. (B) Young mouse islets were treated with vehicle, PL, CAY10598 +/− PL, or L-161,982 +/− PL as described in A. *versus vehicle; ^versus PL. (C) Aged mouse islets (1 year old) were treated with vehicle, PL, DG-041 +/− PL, or CAY10598 as described in A. *versus vehicle; ^versus PL. (D) RNA was isolated from mouse islets at 2 (n = 3), 4 (n = 4), 8 (n = 4), or 12 (n = 5) months of age. qRT-PCR was performed for PGE₂ receptor expression. Data are expressed as 2^−ΔΔCt relative to 2 months old. All data were analyzed using a One-way ANOVA with Bonferroni post hoc analysis. For A–C, one symbol represents p < 0.05, two symbols indicate p < 0.01, four symbols represent p < 0.0001. For D, *p < 0.05.

Figure 3

EP3 and EP4 have opposing effects on human β-cell proliferation. (A) Human islets were treated with vehicle, PL, DG-041, or CAY10598 and immunolabeled for insulin (green), Ki67 (red), and DAPI (blue). *p = 0.0177 vs vehicle; ****p < 0.0001 vs vehicle; ⁺⁺p = 0.0038 vs DG-041; ###p < 0.001 vs CAY10598. (B) Representative images of vehicle- and DG-041-treated human islets (donor H1588). Scale bars represent 100 μM. Red arrows point to proliferating β-cells; white arrowhead indicates non-specific immunolabeling. (C) Human islets were treated with vehicle, PL, DG-041, or CAY10598 and immunolabeled for glucagon, Ki67, and DAPI (not shown). Each distinct human donor is represented by a different colored symbol. All data are represented as fold change in β- or α-cell proliferation compared to vehicle-treated islets. Data were analyzed using a One-way ANOVA with Bonferroni post hoc analysis.

Figure 4

Increased PTGER3 expression is correlated with lower BMI in T2D human islets. RNA was isolated from human islets and qRT-PCR was performed for EP3 (A) and EP4 (B) expression in non-diabetic (A, B) and T2D (A’, B’) islets. Data are represented as ΔCt relative to TBP and were analyzed using a linear regression.

Figure 5

EP3 increases, while EP4 protects against, β-cell death in mouse and human islets. (A) Wild type (WT) or β-FoxM1* islets (8–10 weeks) or (B) human islets were treated for 48 h with a species-specific cytokine cocktail plus one of the following compounds: vehicle, sulprostone, DG-041, CAY10598, or L-161,982. Following treatment, islets were immunolabeled for insulin, TUNEL, and DAPI. Each distinct human donor is represented by a different colored symbol. (A) *significant versus WT Vehicle + Cytokines; ^significant vs β-FoxM1* Vehicle + Cytokines. (B) *Significant vs Vehicle + Cytokines. Data were analyzed using a One-way ANOVA with Bonferroni post hoc analysis. Three symbols indicate p < 0.001, four symbols represent p < 0.0001.

Figure 6

Changes in cell cycle and apoptosis genes with inhibition of EP3 or activation of EP4. (A and A’) Wild type mouse islets (8–10 weeks old; n = 4) were cultured for two days in the presence of vehicle, DG-041, or CAY10598. Following treatment, qRT-PCR was performed for cell cycle (A) and apoptosis (A’) genes. (B and B’) Wild type mouse islets (8–10 weeks old; n = 4) were cultured for four days as described in A and qRT-PCR was performed for cell cycle (B) and apoptosis (B’) genes. *p < 0.05 vs vehicle. (C, D) Human islets (n = 5–7) were cultured for one (C) or four (D) days in the presence of vehicle, DG-041, or CAY10598. Following treatment, qRT-PCR was performed for cell cycle genes. *p < 0.05 vs vehicle. (E) Human islets (n = 4) from donors with T2D were cultured for one day as described in C. *p < 0.05 vs vehicle; **p < 0.001 vs vehicle. All data are represented as 2^−ΔΔCt relative to vehicle. Data were analyzed using a One-way ANOVA with Bonferroni post hoc analysis.

Figure 7

EP3 modulation of β-cell proliferation involves PLC-γ1 while EP4-induced protection against β-cell death involves PKA. (A) Young WT mouse islets (8–10 weeks old) were treated with vehicle, PL, DG-041 + PL, rapamycin (mTOR inhibitor) +/− DG-041 + PL, or U-73122 (PLC-γ1 inhibitor) +/− DG-041 + PL for 4 days before being immunolabeled for insulin, Ki67, and DAPI. *versus vehicle; ^versus PL; ⁺versus DG-041 + PL. (B) WT mouse islets (8–10 weeks old) were treated for 48 with a species-specific cytokine cocktail plus one or more of the following compounds: vehicle, CAY10598, or Rp-CAMPS (PKA inhibitor). Following treatment, islets were immunolabeled for insulin, TUNEL, and DAPI. *versus vehicle + cytokines; ⁺versus CAY10598. All data were analyzed using a One-way ANOVA with Bonferroni post hoc analysis. One symbol represents p < 0.05, two symbols indicate p < 0.01, three symbols denote p < 0.001, four symbols represent p < 0.0001.