Molecular cloning and induction of nuclear receptors from insect cell lines

Fragments of EcR and USP were cloned from two insect cell lines, Sf21 and High Five cells (derived respectively from Spodoptera frugiperda and Trichoplusia ni), using a PCR-based approach employing degenerate primers designed on the basis of conserved regions of nuclear receptors, together with 5'- and 3'-RACE. An additional orphan nuclear receptor, HR4 fragment, was cloned from High Five cells. Comparison of these fragments with Manduca sexta counterparts showed that the cloned SfEcR [ecdysone receptor (EcR) from Sf21 cells] had high similarity to MsEcR-B1, whereas the cloned SfUSP [ultraspiracle (USP) from Sf21 cells] and TnUSP (USP from High Five cells) matched more closely to MsUSP-2 than to MsUSP-1. The TnHR4 showed most similarity to a recently cloned Bombyx mori GRF. While EcR and USP were constitutively expressed in both cell lines, HR4 was barely detectable by Northern blot analysis in High Five cells. Treatment with 20-hydroxyecdysone (20E) and agonist RH-5992 enhanced transcription of EcR in both cell lines, while the transcription of USP was suppressed in High Five cells. Such suppressed USP transcription was not observed in Sf21 cells. Transcription of TnEcR could also be enhanced by ecdysone and 3-dehydroecdysone, whereas transcription of SfEcR was unchanged with these two ecdysteroid compounds. Induction of HR4 transcription was also observed with 20E, RH-5992, ecdysone and 3-dehydroecdysone. The protein synthesis inhibitor, cycloheximide, superinduced expression of EcR and HR4 and restored the 20E/RH-5992-suppressed expression of TnUSP in the cells. Northern blot analysis also revealed that PCR, using degenerate USP primers, was able to amplify some other orphan nuclear receptors and their expression was inducible by 20E and RH-5992 and some of them were superinducible by cycloheximide.