Identification of Ligands for the Orphan Nuclear Receptor DAF-12 That Govern Dauer Formation and Reproduction in C. Elegans
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The orphan nuclear hormone receptor, DAF-12, plays a central role in the physiology of free-living nematode, C. elegans. DAF-12 is best known for its role in regulating dauer formation, a non-reproductive larval state entered in harsh environments and marked by developmental arrest, stress resistance, and extended life-span. Genetic screens for genes controlling dauer formation have identified conserved endocrine signaling pathways that converge on DAF-12 to influence the choice between dauer formation and reproductive development. Detailed genetic analysis of these signaling pathways suggests that they promote reproductive development in favorable environments by influencing the production of a ligand for DAF-12 by the cytochrome p450, DAF-9. Despite abundant evidence for hormonal control, the identity of the DAF-12 ligand has remained elusive. Using a cell-based ligand screening assay I initially identified a group of 3-keto-containing sterols that potently activated DAF-12 in a DAF-9-dependent manner. Subsequent analysis using a variety of techniques showed that DAF-9 catalyzes the non-stereo-selective addition of a carboxylic acid to the terminal side-chain methyl groups of 3-keto-sterols, producing 3-keto-cholestenoic acids. In collaboration with the laboratory of Dr. Eric Xu, we demonstrated that 3-keto-cholestenoic acids, referred to as dafachronic acids, directly bind to DAF-12 as bona fide ligands. In collaboration with Dr. Adam Antebi we found that these ligands also potently rescued the phenotypes resulting from mutations in daf-9 or its upstream activating genes. Dafachronic acids are also shown to be endogenous hormones, as they could be detected in DAF-12 activating lipid fractions from wild-type but not daf-9 null worms. Taken together, this work defines 3-keto-cholestenoic acids as the first hormonal ligands for an invertebrate orphan nuclear receptor and the first endogenous steroid hormones in nematodes. In addition, these findings demonstrate that steroid hormone control of reproduction is conserved from worms to humans. Finally, given the existence of DAF-12 homologs in parasitic nematodes, this work raises the possibility of targeting DAF-12 in parasitic nematodes as a means for controlling their growth.