Prostaglandin E2 promotes human CD34+ cells homing through EP2 and EP4 in vitro

Abstract

Recently, certain studies have demonstrated in vitro that prostaglandin E2 (PGE2) promotes human cluster of differentiation (CD)34+ cell homing. However, the sub‑type receptors activated by PGE2 are unknown, as the PGE2 receptor EP1-4 subtypes (EP1-4) are expressed on the membrane of human CD34+ cells. Based on the above, the present study aimed to screen the receptor subtype activity by PGE2 to promote human CD34+ cell homing. It was observed that human CD34+ cells expressed the four PGE2 sub‑receptors, particularly EP2 and 4. PGE2 increased EP2 and 4 mRNA expression significantly, while EP1 and 3 mRNA exhibited no significant alteration. PGE2, EP2 agonist (EP2A), and EP4A upregulated C‑X‑C chemokine receptor 4 mRNA and protein expression in human CD34+ cells, and promoted stromal cell‑derived factor 1α (SDF‑1α) expression in bone marrow mesenchymal stem cells (BMMSCs). These phenomena were inhibited by the associated receptor antagonists. PGE2, EP2A, and EP4A facilitated human CD34+ cell migration towards SDF‑1α and BMMSCs. The results of the present study suggested that PGE2 promoted human CD34+ cell homing through EP2 and 4 receptors in vitro.

Figure 1.

CD34⁺ cell purity flow cytometry assay. CD, cluster of differentiation; FITC, fluorescein isothiocyanate.

Figure 2.

In vitro morphology, osteogenic and adipogenic differentiation, and immunophenotypic characterization of BMMSCs. (A) The morphology of BMMSCs (magnification, ×10). (B) Osteogenic differentiation of BMMSCs (magnification, ×4). (C) Adipogenic differentiation of BMMSCs (magnification, ×10). (D) Adipogenic differentiation of BMMSCs (magnification, ×4). (E) Immunophenotypic characterization of BMMSCs using flow cytometry. The expression of CD73, CD90 and CD105 is high in BMMSCs, while expression of CD14, CD34 and CD45 is absent. BMMSC, bone marrow mesenchymal stem cell; CD, cluster of differentiation; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PC5, phycoerythrincyanin 5.

Figure 3.

Expression of EP mRNA in human CD34⁺ cells and BMMSCs. (A) The expression of EP mRNA in human CD34⁺ cells. (B) The expression of EP mRNA in human BMMSCs. (C) The CD34⁺ cells were treated with PGE2 and EP mRNA expression was evaluated. Data are from three independent experiments *P<0.05. CD, cluster of differentiation; BMMSC, bone marrow mesenchymal stem cell; EP1, prostaglandin E2 receptor EP1 subtype; PGE2, prostaglandin E2.

Figure 4.

Expression of CXCR4 following treatment with EP2A and EP4A in CD34⁺ cells. (A) CXCR4 mRNA expression was evaluated in the six groups of CD34⁺ cells. (B) Western blotting of CXCR4 protein expression in the six groups of CD34⁺ cells. Data are from three independent experiments *P<0.05, **P<0.01. CXCR4, C-X-C chemokine receptor type 4; EP2A, prostaglandin E2 receptor EP2 subtype agonist; CD, cluster of differentiation; PGE2, prostaglandin E2; EP2AA, prostaglandin E2 receptor EP2 subtype antagonist.

Figure 5.

Expression of SDF-1a following treatment with EP2A and EP4A treatment in BMMSCs. (A) SDF-1a mRNA expression was evaluated in the six groups of human BMMSCs. (B) SDF-1a protein expression was evaluated in the six groups of human BMMSCs. Data are from three independent experiments. *P<0.05, **P<0.01, ***P<0.001. SDF-1a, stromal cell-derived factor 1α; EP2A, prostaglandin E2 receptor EP2 subtype agonist; BMMSC, bone marrow mesenchymal stem cell; PGE2, prostaglandin E2; EP2AA, prostaglandin E2 receptor EP2 subtype antagonist.

Figure 6.

Migratory rate of the six groups of human cluster of differentiation 34⁺ cells towards (A) SDF-1a and (B) BMMSCs. Data are from three independent experiments. *P<0.05, **P<0.01. SDF-1a, stromal cell-derived factor 1α; EP2A, prostaglandin E2 receptor EP2 subtype agonist; BMMSC, bone marrow mesenchymal stem cell; PGE2, prostaglandin E2; EP2AA, prostaglandin E2 receptor EP2 subtype antagonist.