The morphological response of Kc-H cells to ecdysteroids: Hormonal specificity (original) (raw)
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Journal of Insect Physiology, 2008
During insect development, ecdysteroids and juvenile hormones (JHs) interact to regulate larval growth, metamorphosis and reproduction but the molecular mechanisms by which both hormones influence each other's activity remain unknown. Because of their ease of use and straightforward genetic manipulation, insect cell lines often have been used to clarify the actions and interactions of hormones at the molecular level. Here we report on the use of two insect culture cell lines, Drosophila melanogaster S2 and Bombyx mori Bm5 cells, to investigate two molecular processes in which ecdysteroids and JH have been shown to interact: (1) direct modulation of the activity of the ecdysteroid receptor transcription complex and (2) interference at the level of induction of the primary gene E75. Our data do not support JH analogs (JHAs) acting through the above processes: 'antagonism' of ecdysteroid receptor activity by JHAs correlated with cytotoxicity and induction of E75 expression by JHAs was not demonstrated. However, we confirm previous studies in which it was observed that methoprene can partially reverse the growth inhibition by 20E in S2 cells (but not Bm5 cells). Therefore, the molecular mechanism by which both hormones influence each other's activity to regulate cell growth in S2 cells remains unknown. r
Demonstration of an ecdysteroid receptor in a cultured cell line of Drosophila melanogaster
Proceedings of the National Academy of Sciences, 1978
ABSTRACr Incubation of the high-speed supernatant from the KC cell line of Drosophila melanogaster with [3lJponasterone A results in significant binding of the ligand as determined by gel filtration and dextran-coated charcoal binding assays. In vivo exposure of the KC line to [3Hjponasterone A for 30 min results in a marked binding of the ligand by a KCI-soluble nuclear extract. With both the cytosol and nuclear preparations the binding has specificity and a low dissociation constant (3 X 10-9 M). In addition the labeling of the nuclear preparation exhibits a saturation at approximately 7 X 10-10 M which probably reflects the molar concentration of cytoplasmic receptors.
Ecdysteroid receptors in Drosophila melanogaster adult females
Molecular and Cellular Endocrinology, 1989
Ecdysteroid receptors were identified and partially characterized from total cell extracts of whole animals and dissected tissues from Drosophila melunogaster adult females. Binding studies indicated the presence of two ecdysteroid binding components having high affinity and specificity consistent with receptors previously identified in embryos and larvae. The highest affinity binding component in 3-to 4-day females had a dissociation constant of 9.2 x lo-lo M and a maximal binding concentration of approximately 90 pmol/g protein, with a lower affinity component having a dissociation constant of 2.94 X 10e9 M. Receptors at similar concentrations were also observed in abdominal walls containing adult fat body, with relatively lower receptor levels observed in ovaries. These results indicate that the observed ecdysteroid hormone concentrations in adult females can account for a physiological stimulator-y effect on yolk protein synthesis in adult fat body.
Embryonic ecdysteroids of Drosophila melanogaster
Journal of Insect Physiology, 1988
Titration by RIA of ecdysteroids during embryogenesis of Drosophilu shows one single peak at 8 h, i.e. the time of germ-band shortening. An analysis of the ecdysteroids reveals a general shift from low polarity material at 6 h to free ecdysone and (less) 20-hydroxyecdysone at 8 h, and finally to ecdysteroids of mostly higher polarity at 12 h. Adult body walls, when incubated with tritiated 20-hydroxyecdysone, can produce the 22-acetate of this ecdysteroid which then has similar chromatographic properties as ecdysone. We have isolated the putative ecdysone fraction from 8 h embryonic extracts and showed by enzymatic hydrolysis and HPLC that the embryo does not contain 20-hydroxyecdysone-22-acetate, but rather free ecdysone.
Ecdysteroids in adult males and females of Drosophila melanogaster
Journal of Insect Physiology, 1984
Ecdysteroid titres in whole flies and different tissues of adult male and female Drosophila were determined at various times after eclosion using a radioimmunoassay. The ecdysteroid titre decreased as the flies matured after eclosion. The differences in titre between males and females can be accounted for by their difference in body weight. The ecdysteroids were found to be distributed throughout several tissues. At eclosion not all of the ecdysteroid complement present could be accounted for by that found locahsed in tissues. After maturation of the flies the ecdysteroids in various tissues can account for the majority of that detected in whole-fly extracts. Ecdysteroids were produced during in uirro culture of various tissues, but the quantities detected were low by comparison with ring glands of wandering 3rd-instar larvae. Neither the ovaries nor the abdominal body walls (fat body) seem to be a major source of hormone, and they are only able to convert minute quantities of ecdysone to the biologically active form, 20-hydroxyecdysone, in vitro. The amounts of 20-hydroxyecdysone present were measured using high performance liquid chromatography and radioimmunoassay. We tentatively suggest that the differential expression of the yolk-protein-genes in the fat bodies of males and females does not result from differences in hormone titres between them.
Ecdysteroid-inducible polypeptides in a Drosophila cell line
Cell, 1980
In the Drosophila melanogaster cell line Kc-H, ecdysteroid hormone treatment causes increased relative synthesis of three ecdysteroid-inducible polypeptides (EIPs), named according to their molecular weights (in kilodaltons) EIP 40, EIP 29 and EIP 28. Increased synthesis of the ElPs is detectable within 45 min (EIP 28) or 75 min (EIPs 40 and 29), is maximal at 4-8 hr and continues for almost 2 days. During this period no other major changes in protein synthesis are discernible using one-dimensional gels. At maximum, EIP 28 synthesis is elevated at least 10 fold above its basal level, and ElPs 40 and 29 somewhat less. EIP induction is ecdysteroidspecific and is detectable in the presence of lo-' M 20-hydroxyecdysone. It does not occur in hormone-resistant cells. Apparently identical polypeptides are inducible in another ecdysteroid-responsive cell line, Schneider's line 3. Because EIP synthesis is an early and substantial response to ecdysteroids, this is a promising system for the study of steroid hormone action.
ECDYSTEROID CONJUGATION BY TISSUES OF ADULT FEMALES OF DROSOPHILA MELANOGASTER
Ecdysone, 1986
In order to determine whether female adults of Drosophila can synthesize enzymatically cleavable ecdysteroid conjugates, we injected radiolabelled ecdysone into flies and cultured various organs m vitro in the presence of labelled ecdysteroids. Within 1 hr most of the injected ecdysone was metabolized to 20-hydroxyecdysone, high polarity products (HPP), low polarity products (LPP) and some highly apolar compounds (AP).
Ecdysone-induced changes in morphology and protein synthesis in Drosophila cell cultures
Developmental Biology, 1978
Permanent cell lines derived from embryonic Drosophilu melanogaster show pronounced morphological and biochemical changes in response to the addition of &ecdysone, the moulting hormone, or two of its analogs. Schneider's line 3 cells, and the French line Kc, morphologically differentiate into what appear to be neural-like cells. During this period levels of acetylcholinesterase activity increase lo-to 30-fold, and DNA synthesis ceases. The level of choline acetyltransferase activity remains unchanged in line 3, and is very low. Schneider's line 2 cells show no morphological transformation in response to fi-ecdysone, but undergo a marked chase in the nattern of nrotein synthesis. The six major ecdysone proteins are not "heat shock" proteins.
Development, 1994
In insects, the ecdysteroids act to transform the CNS from its larval to its adult form. A key gene in this response is the ecdysone receptor (EcR), which has been shown in Drosophila to code for 3 protein isoforms. Two of these isoforms, EcR-A and EcR-B1, are prominently expressed in the CNS and we have used isoform-specific antibodies to examine their fluctuations through postembryonic life. EcR expression at the onset of metamorphosis is extremely diverse but specific patterns of EcR expression correlate with distinct patterns of steroid response. Most larval neurons show high levels of EcR-B1 at the start of meta-morphosis, a time when they lose larval features in response to ecdysteroids. Earlier, during the larval molts, the same cells have no detectable receptors and show no response to circulating ecdysteroids; later, during the pupal-adult transformation, they switch to EcR-A expression and respond by maturing to their adult form. During the latter period, a subset of the l...