Influence of cell cycle on ecdysteroid receptor in CHO‐K1 cells

Abstract

CHO-K1 cells are routinely used for characterization of ecdysone receptor (EcR) function, because these vertebrate cells are devoid of endogenous ecdysone receptor protein. Moreover, the endogenous expression of RXR, the vertebrate orthologue of Ultraspiracle (Usp), the most important heterodimerization partner, is neglectable. In contrast to insect cells, there is also no influence of moulting hormone on CHO-K1 cells on cell proliferation either in the absence or presence of transiently expressed EcR. In contrast to Usp, which is exclusively found in nuclei, EcR is heterogeneously distributed between cytoplasm and nuclei in non-synchronized cells. Synchronization of CHO-K1 cells by nocodazole revealed that the cell cycle influences receptor concentration with lowest amounts in late S-phase and G2/M phase and intracellular distribution of the receptor protein showing a minimum of receptors present in nuclei during S-phase. EcR, but not Usp reduces cyclin D1 expression and cyclin D1 concentration is impaired by cyclin D1. Coimmunoprecipitation studies reveal physical interaction of EcR and cyclin D1.

Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila

“…Ecdysone activates key cell cycle genes, including Cyclin B, Cdc2, and Cyclin D in Drosophila midgut metamorphosis in an EcR-dependent manner (Li and White 2003), and EcR can bind Cyclin D1, thereby influencing progression of the cell cycle, in a mammalian cell expression system (Betanska et al 2009). Furthermore, ecdysone signaling may regulate S-phase progression by modulating Myc levels in Drosophila (Cranna and Quinn 2009; Delanoue et al 2010).…”

Transcriptional activity of ecdysone receptor isoforms is regulated by modulation of receptor stability and interaction with Ab‐ and C‐domains of the heterodimerization partner ultraspiracle

“…The same mechanism may also result in isoform-specific differences in the stability of EcR. The stability of the receptor protein is influenced by many factors, such as interaction with comodulators and additional proteins like heat shock proteins (Cronauer, unpublished observations), or cyclins (Betanska et al, 2009). Stabilisation, which is enhanced additionally by heterodimerization and ligand binding, is clearly regulated in a complex cooperative manner.…”

“…The RXR content of the CHO-K1 subline used for our experiments is below the detection limit and does not cause any measurable physiological effect (Nieva et al, 2008;). Therefore, these cells were routinely used for the characterization of the molecular properties of EcR and Usp (Henrich et al, 2003(Henrich et al, , 2009Beatty et al, 2006;Betanska et al, 2007Betanska et al, , 2009Dutko-Gwóźdź et al, 2008;Nieva et al, 2007Nieva et al, , 2008. At least some accessorial proteins like comodulators, which are required for receptor action, seem to be sufficiently conserved to allow structurefunction studies (Tsai et al, 1999;Tran et al, 2001b;Henrich et al, 2009).…”

Smad nuclear interacting protein, SNIP1, mediates the ecdysteroid signal transduction in red crayfish Procambarus clarkii

“…Ecdysteroids play an important role in molting process of crustacean and controlled by complex mechanisms (Huberman, 2000; Nakatsuji et al, 2006, Covi et al, 2009). In other animals, SNIP1 has been reported as a modulator of target genes participating in the control of ecdysteroid signaling such as c‐Myc (Fujii et al, ; Bracken et al, ; Chen and Xu, ), TGF‐β (Kim et al, ; Gibbens et al, ) and cyclin D1 (Roche et al, ; Betanska et al, ). To investigate whether the SNIP1 mediates the ecdysteroid signal transduction in P. clarkii or not, we describe the cloning, expression, and function analysis of SNIP1 gene in the present research.…”