The LPA2 receptor agonist Radioprotectin-1 spares Lgr5-positive intestinal stem cells from radiation injury in murine enteroids

Abstract

Rapidly proliferating cells are highly sensitive to ionizing radiation and can undergo apoptosis if the oxidative and genotoxic injury exceed the defensive and regenerative capacity of the cell. Our earlier work has established the antiapoptotic action of the growth factor-like lipid mediator lysophosphatidic acid (LPA). Activation of the LPA GPCR has been hypothesized to elicit antiapoptotic and regenerative actions of LPA. Based on this hypothesis we developed a novel nonlipid agonist of LPA, which we designated Radioprotectin-1 (RP-1). We tested RP-1 at the six murine LPA GPCR subtypes using the transforming growth factor alpha shedding assay and found that it had a 25 nM EC that is similar to that of LPA18:1 at 32 nM. RP-1 effectively reduced apoptosis induced by γ-irradiation and the radiomimetic drug Adriamycin only in cells that expressed LPA either endogenously or after transfection. RP-1 reduced γ-H2AX levels in irradiated mouse embryonic fibroblasts transduced with the human LPA GPCR but was ineffective in vector transduced MEF control cells and significantly increased clonogenic survival after γ-irradiation. γ-Irradiation induced the expression of lpar2 transcripts that was further enhanced by RP-1 exposure within 30 min after irradiation. RP-1 decreased the mortality of C57BL/6 mice in models of the hematopoietic and gastrointestinal acute radiation syndromes. Using Lgr5-EGFP-CreER;Tdtomato transgenic mice, we found that RP-1 increased the survival and growth of intestinal enteroids via the enhanced survival of Lgr5 intestinal stem cells. Taken together, our results suggest that the LPA-specific agonist RP-1 exerts its radioprotective and radiomitigative action through specific activation of the upregulated LPA GPCR in Lgr5 stem cells.

Lysophosphatidic acid type 2 receptor agonists in targeted drug development offer broad therapeutic potential

“…by guest, on July 11, 2020 www.jlr.org Downloaded from a 25 nM EC 50 at the murine LPA 2 and a 5 pM EC 50 at the human LPA 2 (14,37). RP-1 significantly enhanced enteroid growth and survival of LGR-5-positive ISCs after exposure to 5 Gy -irradiation (Fig.…”

“…3C). Furthermore, RP-1 decreased mortality in murine models of the HEM-ARS and the GI-ARS after a 16 Gy -irradiation dose (14). In summary, the follow-on new generation SBA analogs are specific LPA 2 agonists and effectively mitigate irradiation-induced apoptosis and enhance the regeneration of LGR-5-positive ISCs, indicating that the LPA 2 expressed in this ISC subset represents the molecular target of SBA drugs.…”

“…To further enhance the stability of the ligands, we inserted a thiophosphate in place of the phosphate group, as thiophosphates also show increased resistance to cleavage by many lipases. Octadecyl thiophosphate (OTP; licensed under the trade name Rx100) showed full agonist activity at LPA 2 and LPA 5 , with reasonable submicromolar potency at all LPA GPCRs ( Table 2) (14). Resistance to degradation by phospholipases and lipid phosphatases is the likely cause for the long 10.5 h plasma half-life of OTP in Rhesus monkey plasma (15).…”

“…The synthetic improvements using the sulfamoyl benzoic acid (SBA) scaffold have identified multiple new compounds with subnanomolar agonist activity specific to the LPA 2 without any effect at the other LPA receptors ( Fig. 3A) (14,37). We chose one of the SBA analogs {2-[4-(1,3-dioxo-1H,3H-benzoisoquinolin-2-yl) butylsulfamoyl]benzoic acid (DBIBB)} for further testing.…”

“…Double labeling of the LGR-5-positive ISC allowed us not only to identify this stem cell population but also enabled us to induce the expression of the red protein, which would only be synthesized in surviving cells capable of protein synthesis. These enteroid cultures were irradiated and treated with the most potent SBA analog, designated radioprotectin (RP)-1 (14). RP-1 has Fig.…”