Identification of steroid hormone signaling pathway in insect cell differentiation

Abstract

To dissect the steroid hormone signaling pathway involved in insect cell morphological differentiation, we extended the application of the double-stranded RNA-mediated interference (dsRNAi) method to the epidermal IAL-PID2 cell line from Plodia interpunctella Lepidoptera. We first demonstrated that dsRNA was capable of efficiently blocking the steroid hormone 20-hydroxyecdysone (20E) inducibility of proteins that belong to the nuclear receptor superfamily, including the ecdysone receptor (EcR), its partner Ultraspiracle (USP), the insect homolog of the vertebrate retinoid X receptor and the HR3 transcription factor. We then showed that inhibiting the 20E induction of EcR, USP or HR3 proteins prevented the increased synthesis of beta tubulin and consequently the morphological transformation of cells. Thanks to this functional approach, we have shown, for the first time, the participation of EcR, USP and HR3 in a 20E signaling pathway that directs morphological differentiation in insect cells by regulating beta tubulin expression.

The 20-hydroxyecdysone-induced signalling pathway in G2/M arrest of Plodia interpunctella imaginal wing cells

“…Therefore, our RNAi results provided evidence for the involvement of EcR-B1, USP-2 and HR3 in a 20E signalling pathway that induces G2/M arrest of imaginal cells by regulating B cylin expression. It is known that the G2/M arrest of IAL-PID2 cells is followed by a morphological differentiation with the formation of cytoplasmic extensions accompanied by an increased synthesis of b tubulin (Siaussat et al, 2007). Other recent RNAi experiments have demonstrated that the silencing of EcR-B1, USP-2 and HR3 prevented the 20E effects on the b tubulin and the shape of IAL-PID2 cells (Siaussat et al, 2007).…”

“…The dsRNAs were stored at À20 1C for several months. For RNAi experiments, the preparation of dsRNA-Lipofectamine 2000 complexes and the transfection into IAL-PID2 cells are described by Siaussat et al (2007).…”

“…Only descriptive information exists for the regulation of the cyclin gene by ecdysteroids in correlation with G2/M arrest, and there are no functional data on the 20E signalling transduction pathway responsible for these genetic and cellular responses. Recent experiments have highlighted the efficacy of doublestranded RNA-mediated interference (dsRNAi) on the steroid hormone-inducible gene expression in IAL-PID2 cells (Siaussat et al, 2007). Therefore, this 20E responsive cell line seemed to be an ideal system to undertake the identification of the signalling pathway responsible for G2/M arrest of imaginal cells by dsRNAi.…”

Cell-Based Screening Systems for Insecticides

“…It is proposed that the resistance mechanism exists at the junction between the conserved ecdysone regulatory cascade and the differentiation program in the cell line. RNAi studies in IAL-PID2 cells have con fi rmed the involvement of the ecdysone regulatory cascade in cell cycle arrest and morphological transformation (Siaussat et al 2007(Siaussat et al , 2008 .…”

“…Other assays employed organ cultures to evaluate molting hormone activity such as those based on the evagination of imaginal discs (Smagghe et al 2000 ) and the promotion of chitin synthesis by cuticle fragments (Nakagawa et al 1998 ;review by Nakagawa 2005 ) . Insect cell lines generally respond to ecdysteroids and non-steroidal ecdysone agonists by cessation of cell proliferation and formation of fi lamentous extensions (Wing 1988 ;Sohi et al 1995 ;Siaussat et al 2007 ;Mosallanejad et al 2008 ) and this property has indeed been exploited for development of screening systems for ecdysone analogs (e.g. the B II bioassay; Dinan et al 2001 ;Harmatha et al 2002 ) .…”