The Radioprotective Effects of 16, 16 dimethyl Prostaglandin E2 on Survival and Hematopoiesis are Mediated Through Co-Stimulation of the EP3 and EP4 Receptors

16, 16 dimethyl-Prostaglandin E2 (dmPGE2) administered prior to lethal irradiation protects against mortality from the hematopoietic acute radiation syndrome (H-ARS). It protects hematopoietic stem (HSC) and progenitor (HPC) cells and accelerates hematopoietic recovery by attenuating mitochondrial compromise, epigenetic downregulation of p53, and inhibition of histone acetylation at the promoters of genes involved in cell cycle, DNA repair and apoptosis. Since PGE2 mediates it effects through 4 conserved G-protein coupled receptors (EP1-4) we utilized highly selective EP receptor agonists to identify the EP receptors mediating radioprotection in H-ARS and evaluated the genes, cellular pathways and biological functions downstream of the EP receptors involved in HSC radioprotection. Radioprotection of mice from lethal radiation exposure was observed for the EP3 agonist sulprostone (65% survival) and the EP4 agonist rivenprost (50% survival), with the combination of EP3 + EP4 agonists providing 100% survival. Misoprostol, a PGE1 analog with similar EP receptor affinities as dmPGE2 also provided >90% survival. The combination of EP3 and EP4 agonists was highly efficacious in accelerating recovery of all peripheral blood cell counts. Analysis of bone marrow HSPC populations from lethally irradiated mice by flow cytometry indicated that the EP3 + EP4 agonist combination trended closest to dmPGE2 in protecting total HSC and HPC, preventing early entry of these cells into cell cycle, and attenuating radiation-induced upregulation of the proapoptotic death receptor Fas, with similar activity also shown by misoprostol. Several genes involved in cell cycle and/or apoptosis control were upregulated (s1pr1, arrdc3, osm) or downregulated (hcar2 and cxcl10) in HSCs by all efficacious agonist treatments. Analysis of gene expression profiles and functional pathway analysis in HSC suggests that the EP4 receptor signals primarily through cAMP/PKA/CREB1, while EP3 signals primarily through a PI3K/Akt pathway initiated through activation of the Ras/Rho GTPases. In the combination setting, EP4 signaling appears dominant. Co-stimulation of EP3 and EP4 gave a stronger z-score for CREB1 activation with EP3 signaling augmenting/enhancing gene expression downstream of EP4 predominantly through CREB1. Comparison of KEGG pathways regulated by dmPGE2 and those regulated by the combination of EP3 + EP4 agonists indicate that both groups' TNF signaling pathways may be key functional components for radioprotection by dmPGE2 in HSC. Differentially expressed genes (DEG) associated with GTPase activity were observed in HSCs from mice treated with both EP3 and EP4 agonists likely contributing to their enhanced radioprotective effect mediated through the PI3K/Akt pathways downstream of both receptors. Some upstream regulators most strongly activated by dmPGE2 in bone marrow stromal cells overlapped with those observed in HSCs, with the most striking similarity being inhibition of p53 activation. In summary, these studies show that signaling through both the EP3 and EP4 PGE receptors recapitulates maximal radioprotection of mice from H-ARS by dmPGE2 via inhibition of cell cycle progression and apoptosis.