ACTION OF DIFFERENT ECDYSTEROIDS ON THE REGULATION OF MESSENGER-RNAS FOR THE ECDYSONE RECEPTOR, MHR3, DOPA DECARBOXYLASE, AND A LARVAL CUTICLE PROTEIN IN THE LARVAL EPIDERMIS OF THE TOBACCO HORNWORM, MANDUCA-SEXTA

To determine which ecdysteroids may be biologically active in the larv al epidermis of the tobacco hornworm, Manduca sexta, we studied the ac tion of several known ecdysteroids and metabolites on the expression o f the genes encoding the ecdysone receptor (EcR), Manduca hormone rece ptor 3 (MHR3), dopa decarboxylase (DDC), and a larval cuticle protein (LCP-14). Both Day 2 fourth- and Day 2 fifth-instar larval epidermis c ontained significant 3 beta-reductase activity which metabolized 3-deh ydroecdysone (3DE) and 3-dehydro-20-hydroxyecdysone (3D20E) to ecdyson e (E) and 20-hydroxyecdysone (20E), respectively, but had only very lo w amounts of ecdysone oxidase activity (E to 3DE) and no detectable ec dysone 20-monooxygenase activity (E to 20E). When the expression of th e various genes was studied in the epidermis in vitro, 20E and 3D20E h ad similar effects, whereas E, 3DE, 26-hydroxyecdysone and 20,26-dihyd roxyecdysone were ineffective. Exposure of Day 2 fifth-instar epidermi s to 500 ng/ml of either 20E or 3D20E for 24 hr caused a rapid, biphas ic increase in EcR-B1 mRNA. By contrast, EcR-A mRNA showed a less rapi d initial increase followed by a slow steady rise and was less respons ive to 3D20E. Ecdysone in a 1:1 mixture with 20E effectively; halved t he concentration of 20E needed to induce EcR-B1 mRNA but showed no syn ergism in the induction of EcR-A mRNA. The induction of MHR3 mRNA and of DDC mRNA in Day 2 fourth-instar epidermis as well as the suppressio n of DDC and LCP-14 gene expression by 3D20E was indistinguishable fro m that of 20E. Therefore, for Manduca larval epidermis, only 20E and 3 D20E are biologically active ecdysteroids. Since the 3D20E can be conv erted to 20E by the epidermis, its effects are likely mediated by 20E. (C) 1997 Academic Press.