We report that methoprene and its derivatives can stimulate gene transcription in vertebrates by acting through the retinoic acid-responsive transcription factors, the retinoid X receptors (RXRs). Methoprene is an insect growth regulator in domestic and agricultural use as a pesticide. At least one metabolite of methoprene, methoprene acid, directly binds to RXR and is a transcriptional activator in both insect and mammalian cells. Unlike the endogenous RXR ligand, 9-cis-retinoic acid, this activity is RXR-specific; the methoprene derivatives do not activate the retinoic acid receptor pathway. Methoprene is a juvenile hormone analog that acts to retain juvenile characteristics during insect growth, preventing metamorphosis into an adult, and it has been shown to have ovicidal properties in some insects. Thus, a pesticide that mimics the action ofjuvenile hormone in insects can also activate a mammalian retinoid-responsive pathway. This finding provides a basis through which the potential bioactivity of substances exposed to the environment may be reexamined and points the way for discovery of new receptor ligands in both insects and vertebrates.With the exception of their role in vision, the manner in which the retinoids exert their biological effects resides in their ability to regulate gene expression. Vitamin A metabolites-i.e., retinoids-play essential roles in many aspects of development, metabolism, and reproduction in vertebrates (1). Some of the end products of vitamin A metabolism have been identified as the molecules responsible for the action of retinoids. Retinol, the major circulating form of retinoid, is converted within cells to all-trans-retinoic acid and 9-cis-retinoic acid (9cRA) (2-4). The retinoic acids function through two classes of receptors: the retinoic acid receptors (RARs), which bind to both atRA and 9cRA, and the retinoid X receptors (RXRs), which bind only to 9cRA. These receptors modulate ligand-dependent gene expression by interacting as RXR/RAR heterodimers or RXR homodimers on specific target-gene DNA sequences known as hormone response elements. In addition to their role in retinoid signaling, RXRs also serve as heterodimeric partners of nuclear receptors for vitamin D, thyroid hormone, and peroxisome proliferators (reviewed in ref. 5).Although both RXR and RAR bind and respond to 9cRA, evolutionarily these receptors are quite distinct. RXR and RAR share only 27% amino acid identity in their ligandbinding domains (6). In addition, at least one homolog of RXR has been identified in insects, called ultraspiracle (7). Like RXR, ultraspiracle serves as a heterodimeric partner to other receptors. For example, the ecdysone receptor requires ultraspiracle as its coreceptor to bind and respond to its ligand, 20-hydroxyecdysone (8). Significantly, however, ultraspiracle does not respond to any of the known retinoids, including 9cRA. These results are consistent with the finding that insectsThe publication costs of this article were defrayed in part by page charge payment. This a...

The parasitic protozoa Trypanosoma brucei utilizes a novel cofactor (trypanothione, T(SH) 2 ), which is a conjugate of GSH and spermidine, to maintain cellular redox balance. ␥-Glutamylcysteine synthetase (␥-GCS) catalyzes the first step in the biosynthesis of GSH. To evaluate the importance of thiol metabolism to the parasite, RNA i methods were used to knock down gene expression of ␥-GCS in procyclic T. brucei cells. Induction of ␥-GCS RNA i with tetracycline led to cell death within 4 -6 days post-induction. Cell death was preceded by the depletion of the ␥-GCS protein and RNA and by the loss of the cellular pools of GSH and T(SH) 2 . The addition of GSH (80 M) to cell cultures rescued the RNA i cell death phenotype and restored the intracellular thiol pools to wild-type levels. Treatment of cells with buthionine sulfoximine (BSO), an enzyme-activated inhibitor of ␥-GCS, also resulted in cell death. However, the toxicity of the inhibitor was not reversed by GSH, suggesting that BSO has more than one cellular target. BSO depletes intracellular thiols to a similar extent as ␥-GCS RNA i ; however, addition of GSH did not restore the pools of GSH and T(SH) 2 . These data suggest that BSO also acts to inhibit the transport of GSH or its peptide metabolites into the cell. The ability of BSO to inhibit both synthesis and transport of GSH likely makes it a more effective cytotoxic agent than an inhibitor with a single mode of action. Finally the potential for the T(SH) 2 biosynthetic enzymes to be regulated in response to reduced thiol levels was studied. The expression levels of ornithine decarboxylase and of S-adenosylmethionine decarboxylase, two essential enzymes in spermidine biosynthesis, remained constant in induced ␥-GCS RNA i cell lines.

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