MHR3 is an ecdysone-inducible transcription factor whose expression in both Manduca sexta epidermis and the Manduca GV1 cell line is induced by 20-hydroxyecdysone (20E) in vitro. There are four putative ecdysone response elements (EcRE) in the 2.6-kb flanking region of the MHR3 promoter. The most proximal, EcRE1, is necessary for activation of the promoter by 20E in the GV1 cells because the mutation of EcRE1 caused the loss of responsiveness to 20E. Previous studies showed that EcR-B1/USP-1 bound only to EcRE1 and high levels of this complex increased the 20E-induced activation, whereas the presence of high USP-2 prevented this increased activation. When we expressed EcR-A alone or in combination with USP-1 under the control of Autographa californica baculovirus promoter (pIE1hr), the activation of the 2.6-kb promoter by 20E was reduced by about 50%. Moreover, when EcR-A was expressed together with both EcR-B1 and USP-1, it reduced the normal activation caused by EcR-B1 and USP-1 by 50%. Gel mobility shift assays showed no binding of EcR-A/USP-1 to EcRE1. The presence of EcR-A, however, reduced the binding of EcR-B1/USP-1 by about 50%. These findings suggest that EcR-A competes with EcR-B1 for binding of USP-1, leading to a decline in activity of the promoter. In addition, E75A, another ecdysone-induced transcription factor, and MHR3 itself suppressed MHR3 promoter activity by binding to the monomeric response element (MRE2). Therefore, MHR3 can be down-regulated both by itself and by E75A.

Insect molting is triggered by ecdysteroids, which are produced in the prothoracic glands (PG). The broad (br) gene is one of the ’early genes’ directly regulated by ecdysteroids. Ectopic expression of the BR-Z3 isoform in early second instar Drosophila larvae (L2) before the rise of the ecdysteroid titer prevented molting to the third instar, but the larvae subsequently formed L2 prepupae after prolonged feeding. When these larvae were fed on diet containing 20-hydroxyecdysone (20E), they formed pharate third instar larvae. The critical weight for normal L3 pupariation of w(1118) larvae was found to be 0.8 mg and that for L2 pupariation was 0.45 mg. We also defined a threshold weight for metamorphosis of 0.3 mg, above which L2 larvae will metamorphose when provided with 20E. BR-Z3 apparently works through the PG cells of the ring gland but not the putative neurosecretory cells that drive ecdysone secretion, because ectopic expression of BR-Z3 specifically in the ring gland caused 53% of the larvae to become permanent first instar larvae. Driving other BR isoforms in the ring gland prevented larval molting or pupariation to varying degrees. These molting defects were rescued by feeding 20E. Overexpression of each of the BR isoforms caused degeneration of the PG cells but on different time courses, indicating that BR is a signal for the degeneration of the PG cells that normally occurs during the pupal-adult transition.