Prostaglandin E2 regulates pancreatic stellate cell activity via the EP4 receptor

Abstract

Objectives: Pancreatic stellate cells are source of dense fibrotic stroma, a constant pathological feature of chronic pancreatitis and pancreatic adenocarcinoma. We observed correlation between levels of cyclooxygenase 2 (COX-2) and its product prostaglandin E2 (PGE2) and the extent of pancreatic fibrosis. The aims of this study were to delineate the effects of PGE2 on immortalized human pancreatic stellate cells (HPSCs) and to identify the receptor involved.

Methods: Immunohistochemistry, reverse transcription-polymerase chain reaction and quantitative reverse transcription-polymerase chain reaction were used to assess COX-2, extracellular matrix, and matrix metalloproteinase gene expression. Eicosanoid profile was determined by liquid chromatography-tandem mass spectrometry. Human pancreatic stellate cell proliferation was assessed by MTS assay, migration by Boyden chamber assay, and invasion using an invasion chamber. Transient silencing was obtained by small interfering RNA.

Results: Human pancreatic stellate cells express COX-2 and synthesize PGE2. Prostaglandin E2 stimulated HPSC proliferation, migration, and invasion and stimulated expression of both extracellular matrix and matrix metalloproteinase genes. Human pancreatic stellate cells expressed all 4 EP receptors. Only blocking the EP4 receptor resulted in abrogation of PGE2-mediated HPSC activation. Specificity of EP4 for the effects of PGE2 on stellate cells was confirmed using specific antagonists.

Conclusions: Our data indicate that PGE2 regulates pancreatic stellate cell profibrotic activities via EP4 receptor, thus suggesting EP4 receptor as useful therapeutic target for pancreatic cancer to reduce desmoplasia.

Fig.1. COX-2 expression and eicosanoid levels in PDAC and the stroma

(A) Immunohistochemistry showing the levels of COX-2 in normal and PDAC tissue sections from human samples. (B) RT-PCR showing the expression of COX-2 in HPSC. Liquid chromatography tandem mass spectrometry (LC/MS/MS) was performed to determine the profile of eicosanoids present in HPSC cellular extracts (C) or HSPC conditioned medium (D).

Fig.2. PGE₂ stimulates migration, invasion and gene expression

Cell migration and invasion were assessed by counting the number of stained cells that penetrated the migration membrane after 24h following PGE₂ (100nM) stimulation. (A) membrane alone (right, stained cells; left migration numbers p<0.05 versus control) (B) matrigel coated membrane (right, stained cells; left migration numbers p<0.05 versus control) (C) RT-PCR showing the expression of HSPG2, COL1A1, elastin, fibronectin, vimentin and (D) the expression of MMP-2, 3, 9, 11 and TIMP-1, 2 before and after stimulation with PGE₂ (100nM). β-actin served as control.

Fig.3. EP receptor differential expression by HPSC and silencing by siRNA

(A) EP1, 2, 3, and 4 receptor mRNA are differentially expressed by HPSC as determined by RT-PCR (B) silencing of mRNA expression was achieved by transient transfection with human siControl and siEP2 (B) or siEP4 (C) β-actin served as control.

Fig.4. Effects of EP2 and EP4 silencing on PGE₂ mediated HPSC functions

HPSC were transiently transfected with siControl, siEP2 or siEP4. After 48 hours, cell migration and invasion were assessed by counting the number of stained cells that penetrated the migration membrane after 24h following PGE₂ (100nM) stimulation. (A) membrane alone (right, stained cells; left migration numbers p<0.05 versus control) (B) matrigel coated membrane (right, stained cells; left migration numbers p<0.05 versus control) (C) RT-PCR shows the effects of PGE₂ mediated stimulation of COL1A1, MMP-2 and 9 after the silencing of EP4 receptor. β-actin served as control. (D) Q-RT-PCR shows the effects of PGE₂ mediated stimulation of COL1A1, MMP-2 and 9 and decrease after the silencing of EP4 receptor. β-actin served as control.

Fig.5. Effects of specific EP receptor antagonists on PGE₂ mediated HPSC functions

HPSC were plated and allowed to settle overnight. Serum containing media was re-plated with serum free media for 24 hours before addition of EP1, EP2 or EP4 antagonists at a dose of 10μM. 1 hour after the antagonist addition, cell migration and invasion were assessed by counting the number of DAPI stained cells that penetrated the migration membrane after 24 h following PGE₂ (100nM) stimulation. (A) membrane alone (right, stained cells; left migration numbers p<0.05 versus control) (B) matrigel coated membrane (right, stained cells; left migration numbers p<0.05 versus control). Addition of EP4 antagonist showed a significant reduction of PGE₂ mediated HPSC migration and invasion. (C) RT-PCR showing the reduction in collagen 1A1 expression on treatment with EP4 antagonist (10μM) as compared to increase on treatment with PGE₂ (100nM) alone.