Nongenomic action of an insect steroid hormone in steroid-induced programmed cell death
Introduction
In animal development, tissues that possess stage-specific functions are eliminated through programmed cell death (PCD) after completing their roles (Vaux and Korsmeyer, 1999). In insects, the larval specific tissues degenerate at or shortly after the larval–pupal transformation in response to the steroid hormone, 20-hydroxyecdysone (20E). Such tissues include the intersegmental muscles (Schwartz, 1992) and motoneurons (Streichert et al., 1997) of Manduca sexta, the salivary glands of Drosophila melanogaster (von Gaudecker and Schmale, 1974) and the silk glands of Bombyx mori (Chinzei, 1975, Terashima et al., 2000). The roles of a number of molecules and their interactions in PCD have been elucidated mostly in mammalian cells in which PCD is triggered by blood-born factors that bind to membrane receptors and activate intercellular signaling pathways (Ashkenazi and Dixit, 1998). The molecular genetic regulatory hierarchies underling the 20E-induced cell death have been extensively studied in Drosophila (Jiang et al., 2000, Yin and Thummel, 2004), in which the hormonal triggers direct the gene activation through a nuclear heterodimeric receptor, ecdysone receptor (EcR) and ultraspiracle (Usp), the insect homologue of the vertebrate nuclear hormone receptor partner RXR (Yao et al., 1992). Execution of the cell death program by 20E in B. mori anterior silk glands (ASGs), however, cannot be accounted for simply by 20E-regulated gene expressions through the nuclear receptor (Terashima et al., 2000).
The B. mori ASG is the nozzle-like organ to spin silk thread from liquid silk proteins produced in the middle and posterior silk glands. After completion of spinning a cocoon, the entire gland degenerates through PCD in response to a large increase in 20E in hemolymph, that induces pupal metamorphosis (Sakurai et al., 1998, Terashima et al., 2000). Since 20E acts through binding to EcR/USP, which serves as a transcription factor, 20E-induced PCD has been considered to begin with de novo gene expression (Huet et al., 1993), although there was no experimental confirmation. This belief was called into question by the results obtained using α-amanitin, a potent inhibitor of RNA polymerase II. α-Amanitin prevents PCD by 20E when added to the culture of ASGs with 20E from the beginning of the culture, while its addition 8 h after the exposure to 20E does not do so, indicating that the gene transcription needed for the PCD is accomplished by 8 h. Nevertheless, withdrawal of 20E from the culture medium between 8 and 42 h of the culture interferes the progression of PCD sequence (Terashima et al., 2000). Based on these previous results, we supposed an involvement of nongenomic action of 20E until 42 h in addition to its genomic action by 8 h.
Cell death is classified into caspase dependent and independent ones (Saelens et al., 2004). In the caspase dependent pathways, caspase activation is crucial for inducing DNA fragmentation (Liu et al., 1997, Sakahira et al., 1998, Enari et al., 1998), nuclear fragmentation (Woo et al., 1998) and chromatin condensation (Sahara et al., 1999). 20E-induced PCD of Drosophila midgut and salivary gland is accompanied by DNA fragmentation and the PCD pathway requires caspase activation (Jiang et al., 1997). The PCD of B. mori ASG proceeds through sequential cellular events including nuclear condensation, DNA fragmentation and nuclear fragmentation (Terashima et al., 2000), an indication of an involvement of caspase in the PCD.
Protein synthesis inhibitors are common tool to inhibit gene translation. In addition, these inhibitors, such as cycloheximide (CHX), can act as apoptosis inducers in Drosophila (Manaka et al., 2004) and mammalian cells (Tang et al., 1999), and CHX activates drICE, a caspase-3 like protease, in Drosophila cells (Fraser et al., 1997). In order to use the inhibitors to block protein synthesis while not inducing PCD, we cultured the ASGs with various concentrations of three protein synthesis inhibitors, CHX, emetine and anisomycin, in the presence or absence of 20E. We found inhibitor concentrations that did not induce the PCD, when added alone, but partly induced PCD, if 20E was present, with retaining the inhibitory activity towards protein synthesis. These culture conditions made it possible to discriminate the nongenomic action of 20E from the genomic one.
Here we show that the cell shrinkage and apoptotic body formation are under the control of the genomic pathway of 20E, while the nongenomic pathway is involved in the DNA fragmentation and nuclear fragmentation via Ca2+–PKC–caspase-3 like protease signaling pathway.
Section snippets
Animals
Larvae of the silkworm, B. mori, were reared and staged as previously described (Sakurai et al., 1998). Last (fifth) instar larvae enter the prepupal period in the scotophase of day 6 or 7 of the instar as indicated by a behavior to purge out their gut contents. Only the larvae in the photophase following gut purge were used in experiments.
Hormones and chemicals
20E (Sigma, St. Louis, MO) was dissolved in water (1 mg/ml) and stored at −20 °C. Protein synthesis inhibitors, cycloheximide (CHX), emetine, anisomycin, and
PCD is induced by 20E and protein synthesis inhibitors
PCD in the ASGs triggered by 20E takes 144 h to be completed and is accompanied by serial changes in cellular and nuclear morphology (Fig. 1). In intact ASGs, the cells are irregularly hexagonal and nearly transparent (Fig. 1A). The nuclei are highly branched, and individual branches are filamentous (Fig. 1a and a′). In 20E-induced PCD, the cells shrink to be a short hexagonal and become opaque (Fig. 1B). In these cells, the nuclear branches are thick (Fig. 1b and b′), an indication of nuclear
Discussion
The classic view of steroid hormones is that they exert their effects primarily through nuclear receptors that regulate transcription. In addition to the genomic action, a non-classical steroid hormone signaling mechanism has been proposed to explain various rapid responses to steroids that are unlikely to involve their cognates nuclear receptors (Falkenstein et al., 2000, Lösel and Wehling, 2003). Nevertheless, there is little information on steroid-dependent responses in which both genomic
Acknowledgements
We are grateful to Dr. Robert Rybczynski, University of North Carolina at Chapel Hill, for his critical reading of the manuscript. This work was supported by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science to S.S. (14300033 and 17380035).
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