Giripladib

Sonic Hedgehog (SHH) is a driver of embryonic patterning that, when corrupted, triggers developmental disorders and cancers. SHH effector responses are organized through primary cilia (PC) that grow and retract with the cell cycle and in response to extracellular cues. Disruption of PC homeostasis corrupts SHH regulation, placing significant pressure on the pathway to maintain ciliary fitness. Mechanisms by which ciliary robustness is ensured in SHH-stimulated cells are not yet known. Herein, we reveal a crosstalk circuit induced by SHH activation of Phospholipase A2α that drives ciliary E-type prostanoid receptor 4 (EP4) signaling to ensure PC function and stabilize ciliary length. We demonstrate that blockade of SHH-EP4 crosstalk destabilizes PC cyclic AMP (cAMP) equilibrium, slows ciliary transport, reduces ciliary length, and attenuates SHH pathway induction. Accordingly, Ep4−/− mice display shortened neuroepithelial PC and altered SHH-dependent neuronal cell fate specification. Thus, SHH initiates coordination between distinct ciliary receptors to maintain PC function and length homeostasis for robust downstream signaling.

Cyclooxygenase-2 converts arachidonic acid to prostaglandins (PGs) and the endocannabinoid, 2-arachidonoylglycerol (2-AG), to PG glyceryl esters (PG-Gs). The physiological function of PG biosynthesis has been extensively studied, but the importance of the more recently discovered PG-G synthetic pathway remains incompletely defined. This disparity is due in part to a lack of knowledge of the physiological conditions under which PG-G biosynthesis occurs. We have discovered that RAW264.7 macrophages stimulated with Kdo2-lipid A (KLA) produce primarily PGs within the first 12 h followed by robust PG-G synthesis between 12 h and 24 h. We suggest that the amount of PG-Gs quantified is less than actually synthesized, because PG-Gs are subject to a significant level of hydrolysis during the time course of synthesis. Inhibition of cytosolic phospholipase A2 by giripladib does not accelerate PG-G synthesis, suggesting the differential time course of PG and PG-G synthesis is not due to the competition between arachidonic acid and 2-AG. The late-phase PG-G formation is accompanied by an increase in the level of 2-AG and a concomitant decrease in 18:0-20:4 diacylglycerol (DAG). Inhibition of DAG lipases by KT-172 decreases the levels of 2-AG and PG-Gs, indicating that the DAG-lipase pathway is involved in delayed 2-AG metabolism/PG-G synthesis. These results demonstrate that physiologically significant levels of PG-Gs are produced by activated RAW264.7 macrophages well after the production of PGs plateaus.