Point of
VIEW Small Molecule Signaling in Caenorhabditis elegans Frank C. Schroeder* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
A B S T R A C T Whereas the C. elegans genome was sequenced many years ago, the role of small molecule signals in its biology is still poorly understood. A recent publication reports the identification of two steroidal signaling molecules that regulate C. elegans reproductive development and dauer diapause via the nuclear receptor DAF-12. The two compounds, named dafachronic acids, represent the first endogenous ligands identified for any of the 284 nuclear receptors in C. elegans.
*To whom correspondence should be addressed. E-mail: Frank_Schroeder@hms. harvard.edu.
Published online May 19, 2006 10.1021/cb600173t CCC: $33.50 © 2006 by American Chemical Society
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ecause of its short life span and genetic tractability, the nematode Caenorhabditis elegans has long been a pet organism of geneticists and molecular biologists. As one of the first complex organisms whose genomes were sequenced, many of its physiological pathways show analogies to corresponding pathways in higher animals, with interesting implications for research related to human disease. Of particular interest are signaling pathways regulating development that control growth, reproductive maturation, and ultimately life span in C. elegans and thus might hold cues for the regulation of analogous endocrine signaling in higher organisms. Surprisingly, very little is known about the role that small molecules play in C. elegans endocrine signaling. In a beautiful example for the use of genetic information to deduce structure and function of a small molecule signal, Motola et al. (1) have now identified two steroidal hormones, the dafachronic acids 1 and 2 (Figure 1), as the long-anticipated endogenous ligands of the nuclear receptor DAF-12. The life cycle of C. elegans, which normally develops through four larval stages before reaching adulthood, can include a unique phase of metabolic diapause, which seemingly allows the larvae to put aging on hold. Under unfavorable environmental conditions, development arrests prior to reaching reproductive maturity, and the larvae enter the so-called “dauer” stage (from the German “dauer” for durable), an alternative, nonfeeding larval stage which
can persist for up to 2 months, compared to a normal life span for C. elegans of 14 days (2). Upon return to favorable conditions, dauer larvae resume feeding and continue normal development into adulthood. The discovery of such a well-defined phase of metabolic diapause suggested the presence of an elaborate circuitry for its control and for coordination of cellular programs required for the associated stage transitions throughout the organism (3). In fact, studies of the genes involved in dauer formation have provided tantalizing insights in regulation of metabolism, reproductive development, and life span of C. elegans. Genetic screens for mutants that either cannot attain the dauer stage or form dauer larvae constitutively have identified about three dozen genes directly involved in dauer stage control (2). A main switch in the signaling pathway controlling dauer formation appears to be the nuclear receptor DAF-12 (DAuer Formation). Loss of daf-12 as well as certain daf-12 mutations result in larvae that cannot attain the dauer stage and further develop into heterochronic phenotypes, indicating that daf-12 is required for dauer formation as well as for proper developmental timing (3). DAF-12 is one of at least 284 nuclear receptors in C. elegans, many of which have been shown to serve important roles in reproductive development and metabolism (4); however, prior to the identification of the dafachronic acids by Motola et al., not a single ligand that regulates their functions had been identified. In the case of DAF-12,
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Point of
VIEW
Figure 1. Regulation of reproductive growth in C. elegans via DAF-12. Under favorable environmental conditions, signaling through TGF and insulin/IGF-1 pathways activates genes including daf-9 required for the synthesis of ⌬4- and ⌬7-dafachronic acids (1 and 2), which bind to DAF-12 promoting reproductive growth. Under unfavorable conditions, daf-9 is inactive; thus synthesis of the dafachronic acids ceases and the resulting unliganded DAF-12 induces dauer diapause (not shown). DAF-36 functions as a Rieske-like oxygenase, which introduces the ⌬7 double bond in the synthesis of 2 (8).
strong evidence had accumulated suggesting the presence of a small molecule ligand that would inhibit its dauerpromoting activity and trigger its functions relating to reproductive development. Two important signaling pathways converge on DAF-12, the insulin/IGF-1 (Insulin-like Growth Factor 1) pathway acting through DAF-2 and the transcription factor DAF-16, a worm-ortholog of FOXO, and the TGF- (Transforming Growth Factor ) pathway, acting through DAF-4 and the Smad transcription factor DAF-3 (Figure 1). These pathways had been shown to act cell nonautonomously, suggesting that they directly or indirectly induce the production of a smallmolecule ligand of DAF-12. Of particular www.acschemicalbiology.org
significance was the finding that a CYP2 cytochrome-P450 enzyme, DAF-9, which is expressed only in a few specific cell types, acts cell nonautonomously directly upstream from DAF-12 (5). Furthermore, daf-12 mutants mapping to the predicted binding sites of the presumed hormone closely mimic phenotypes of daf-9 mutants (6). As mammalian CYP2 enzymes can metabolize steroid hormones and the vertebrate DAF-12 orthologs represent steroid hormone receptors (3), it seemed likely that DAF-9 would participate in the synthesis of a steroidal ligand of DAF-12. Additional evidence showed that lipophilic extracts from wildtype worms can rescue worms that are dauer-constitutive as a result of a daf-9
defect (7), suggesting the presence of the suspected steroid hormone(s) in these extracts. In order to identify the suspected small molecule hormone, a traditional natural products chemist might have resorted to activity-guided fractionation of the lipophilic worm extract, with the intention of isolating the active component(s) in pure form to determine their structure via spectroscopic methods. However, Motola et al. pursued a different approach. As a first step, they screened a variety of commercially available steroids for DAF-12 binding using a GAL4DAF-12 cotransfection assay, which identified a 3-keto sterol, 3-ketolithocholic acid, as a weak activator of DAF-12. As the corresponding alcohol, lithocholic acid, was not active, it was concluded that a 3-keto functionality was essential. To confirm the relevance of a 3-keto functionalization and to further explore the role of DAF-9, Motola et al. incubated a series of 3-keto-sterols with Sf9 microsomes containing DAF-9 and screened the resulting extracts for their ability to rescue daf-9(⫺) worms. Extracts from the DAF-9 microsomes incubated with 4-cholesten-3-one or lathosterone, two cholesterol metabolites known to occur in C. elegans, resulted in 100% rescue of the daf-9(⫺) mutants, producing a phenotype indistinguishable from wild-type adults, whereas controls exposing daf-9(⫺) mutants to unmetabolized 4-cholesten3-one or lathosterone did not recover. These results suggested that daf-9 encodes an enzyme converting the 3-ketosterols 4-cholesten-3-one and lathosterone into steroidal hormones which then activate DAF-12. Additional chemical characterization of the 4-cholesten-3-one or lathosterone metabolites obtained from DAF-9 microsomes revealed that DAF-9 hydroxylates and then further oxidized these substrates in a non-stereoselective manner at C-26/C-27 in the side chain, producing two diastereomers of the dafachronic acids 1 and 2 (from dauer formation and heteroVOL.1 NO.4 • 198–200 • 2006
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chronic). Motola et al. confirmed the presence of the dafachronic acids in lipophilic wild-type worm extracts via a short fractionation scheme. Using synthetic samples of 1, the authors showed that the (25S)-diastereomers of 1 and 2 in fact constitute the ligands that activate DAF-12 in cotransfection assays and rescue dauer-constitutive daf-9 mutants. Both 1 and 2 are potently active. (25S)⌬4-Dafachronic acid (1) completely rescued daf-9(⫺) worms at concentrations of 250 nM, and it appears that (25S)-⌬7dafachronic acid (2), which has not yet been synthesized, might exhibit even higher specific activity. As expected, the dafachronic acids act downstream of the insulin/ IGF-1 and TGF pathways; however, insulin/ IGF-1 signaling might also act downstream of the dafachronic acids, or via a parallel pathway, as (25S)-⌬4-dafachronic acid (1) did not fully rescue certain daf-2 mutants. It seems likely that the two dafachronic acids have somewhat distinct biological function, which will have to be explored in more detail once (25S)-⌬7-dafachronic acid (2) becomes synthetically available. Furthermore, the question remains whether the much less active (25R)-diastereomers of 1 and 2 are indeed produced in vivo and, if so, what possible function they might serve. There is strong evidence that DAF-12 not only is involved in the critical decision between dauer phase and reproductive growth but also plays an important role in the control of adult life span via DAF-16 (8). Future synthetic availability of both ligands and possibly of derivatives that act as DAF-12 agonists or antagonists will greatly aid further exploration of DAF-12 biological function. The identification of the dafachronic acids represents only the beginning of molecular endocrinology in C. elegans, and a chemist might well ask, why did it take so long? As one of the best-studied higher life forms on earth, next to fruit flies and Arabidopsis, our understanding of C. elegans 200
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O H 3C HO
O
O OH
Figure 2. Structure of daumone, 3.
small molecule chemistry seems highly underdeveloped. Just recently work by Jeong et al. (9) addressed another long-standing question in C. elegans signaling: What are the environmental cues that trigger entry and exit from the dauer stage? Over 20 years ago, Riddle and Golden (10) had shown that a group of unknown small molecules released from C. elegans constitute one primary cue for dauer entry. Jeong et al. now identified a glycoside of the dideoxysugar ascarylose, which they termed “daumone” (3), as the dauer pheromone; however, the exact mechanism of daumone signaling is not yet understood. Still, there are many more observations in C. elegans biology that suggest the presence of small molecule signals, whose identification would contribute greatly to the understanding of corresponding pathways. For example, germline control of adult longevity via DAF-12 and DAF-16 might involve additional small molecule signals (8). And, as Motola et al. point out, the identification of the dafachronic acids as ligands of the nuclear receptor DAF-12 still leaves 283 of 284 nuclear receptors in C. elegans as orphans, providing plenty of opportunity for small molecule chemists.
3. Antebi, A., Yeh, W. H., Tait, D., Hedgecock, E. M., and Riddle, D. L. (2000) daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age in C. elgans, Genes Dev. 14, 1512–1527. 4. Gissendanner, C. R., Crossgrove, K., Kraus, K. A., Maina, C. V., and Sluder, A. E. (2004) Expression and function of conserved nuclear receptor genes in Caenorhabditis elegans, Dev. Biol. 266, 399–416. 5. Mak, H. Y., and Ruvkin, G. (2004) Intercellular signaling of reproductive development by the C. elegans DAF-9 cytochrome P450, Development 131, 1777–1786. 6. Jia, K., Albert, P. S., and Riddle, D. L. (2002) DAF-9, a cytochrome P450 regulating C. elegans larval development and adult longevity, Development 129, 221–231. 7. Gill, M. S., Held, J. M., Fisher, A. L., Gibson, B. W., and Lithgow, G. J. (2004) Lipophilic regulator of a developmental switch in Caenorhabditis elegans, Aging Cell 3, 413–421. 8. Berman, J. R., and Kenyon, C. (2006) Germ-cell loss extends C. elegans life span through regulation of DAF-16 by kri-1 and lipophilic hormone signaling, Cell 124, 1055–1068. 9. Jeong, P. Y., Jung, M., Yim, Y. H., Kim, H., Park, M., Hong, E., Lee, W., Kim, Y. H., Kim, K., and Paik, Y. K. (2005) Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone, Nature 433, 541–545. 10. Golden, J. W., and Riddle, D. L. (1984) A Caenorhabditis elegans dauer-inducing pheromone and an antagonistic component of the food supply, J. Chem. Ecol. 10, 1265–1280.
REFERENCES 1. Motola, D. L., Cummins, C. L., Rottiers, V., Sharma, K. K., Li, T., Li, Y., Suino-Powell, K., Xu, H. E., Auchus, R. J., Antebi, A., and Mangelsdorf, D. J. (2006) Identification of Ligands fore DAF-12 that govern dauer formation and reproduction in C. elegans, Cell 124, 1209–1223. 2. Riddle, D. L., and Albert, P. S. (1997) in C. elegans II (Riddle, D. L., Blumenthal, T., Meyer, B. J., and Priess, J. R., Eds.), pp 739–768, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. SCHROEDER
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