Decreased survival of mosquito cells after stable transfection with a Drosophila ecdysteroid response element: possible involvement of a 40 kDa DNA binding protein (original) (raw)
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Archives of Insect Biochemistry and Physiology, 2000
The reverse transcriptase polymerase chain reaction (RT-PCR) was used to examine whether the C7-10 cell line from the mosquito, Aedes albopictus, expresses transcripts encoding 20hydroxyecdysone receptor (EcR) and ultraspiracle (USP) isoforms known to constitute a functional 20-hydroxyecdysone receptor. Here we describe recovery and analysis of products with high similarity to the EcR and to the USP isoform "a" that have been reported from the related mosquito, Aedes aegypti. The C7-10 EcR was 97% identical to Aedes aegypti EcR in amino acid sequence. Key features of the nuclear/steroid hormone receptor superfamily, including the zinc fingers, proximal (P)-box, and distal (D)-box were well conserved. However, the C7-10 EcR contained 5 additional amino acids in the Cterminal domain F, which required introduction of gaps to maximize alignment. The 5¢-untranslated regions of the two mosquito EcRs were 98% identical, but the function of this region remains unknown. The C7-10 USP was 95% identical in amino acid sequence to the longer Aedes aegypti isoform "a." Although only the C7-10 EcR was detected on Northern blots using total RNA from the cell line, transcripts for both EcR and USP were detected using the RT-PCR procedure. These transcripts appeared to be expressed constitutively and expression levels were not affected by treatment of cells with 20hydroxyecdysone. Arch. Insect Biochem. Physiol. 43:87-96, 2000.
2001
The insect steroid hormone 20-hydroxyecdysone initiates a cascade of regulatory events in a temporal and tissuespecific manner by first binding to a complex of an ecdysone receptor (EcR) protein and a ultraspiracle protein. Using an antisense (As) ribonucleic acid approach, we show that disruption of EcR expression in transfected C7-10 cells from the mosquito Aedes albopictus affects survival and growth. From stably transfected cells, we recovered a new isoform of A. albopictus AalEcRa, which is named AalEcRb. The deduced amino acid sequence of AalEcRb was almost identical to that of AalEcRa, with the exception of a seven amino acid sequence near the C-terminus. Using polymerase chain reaction followed by restriction enzyme analysis, we found that AalEcRa is the predominant species expressed by wildtype C7-10 cells, while cells transfected with As-EcR expressed both isoforms at approximately equal levels.
Mosquito ecdysteroid receptor: Analysis of the cDNA and expression during vitellogenesis
Insect Biochemistry and Molecular Biology, 1995
An insect steroid hormone, 20-hydroxyecdysone (20E), plays an important role in regulating egg maturation in mosquitoes. To better understand its role, we cloned the cDNA coding for the putative ecdysteroid receptor from the mosquito, Aedes aegypti (AaEcR). The 4158 hp AaEcR cDNA has an open reading frame of 675 amino acids with 10 potential glycosylation sites and a putative phosphorylation polyserine domain. The AaEcR has a DNA binding domain with two zinc fingers and a ligand binding domain characteristic of members of the steroid hormone receptor superfamily. These AaEcR domains share 97 and 87% identities with the respective domains of the Drosophila ecdysteroid receptor (DmEcR). However, the A/B region of the AaEcR shares 35% identity with that of DmEcR-Bl isoform. The F region, located at the carboxyl-terminal of the AaEcR, has only 9% identity with the corresponding region of DmEcR. Potential nuclear targeting and dimerization signals are also present in the AaEcR sequence. There are three AaEcR transcripts of 4.2 kb, 6 kb and 11 kb in adult mosquitoes. 4.2 kb mRNA is predominantly expressed in female mosquitoes during vitellogenesis. In both the fat body and ovaries of the female mosquito, the level of AaEcR mRNA is high at the previtellogenic period and after the onset of vitellogenesis (6 h post blood meal, PBM). Aedes aegypti Fat body Ovary Steroid receptor Ecdysone 1NTRODUCTION Steroid hormones and their receptors are important regulatory molecules controlling a wide array of essential functions in both lower and higher eukaryotes. For vertebrates, a wealth of information has been accumulated concerning the mode of action of steroid hormones, their interaction with receptors and target genes. Along with receptors for thyroid hormone, retinoic acid, and vitamin D, steroid hormone receptors form a family of hormoneregulated transcription factors, known as the steroid receptor superfamily (Evans, 1988; Green and Chambon, 1988; Beato, 1989). The key steroid hormone in arthropods is 20-hydroxyecdysone (20E). It regulates developmental processes such as molting, metamorphosis, and reproduction (Riddiford,
The vitellogenin gene of the mosquito Aedes aegypti is a direct target of ecdysteroid receptor
Molecular and Cellular Endocrinology, 2001
In the female mosquito Aedes aegypti, vitellogenin (Vg), the major YPP, is activated by 20-hydroxyecdysone (20E) at the transcriptional level. We used cell transfection assays in the Drosophila S2 cells to investigate whether 20E acts directly on the Vg gene via its functional receptor, the heterodimer composed of the ecdysteroid receptor (EcR) and the ultraspiracle (USP) proteins. We demonstrated that the Vg 5%-regulatory region contains a functional ecdysteroid-responsive element (VgEcRE1) that is necessary to confer responsiveness to 20E. VgEcRE binds directly to EcR-USP produced in vitro and extracted from the vitellogenic fat body nuclei. The binding intensity of the EcR-USP-EcRE1 complex from nuclear extracts corresponds to the levels of ecdysteroids and of the Vg transcript during the vitellogenic cycle. Given the modest level of 20E-dependent activation, it is likely that the EcR-USP receptor acts synergistically with other transcription factors to bring about the high level of Vg gene expression.
CulturedAedes albopictus mosquito cells synthesize hormone-inducible proteins
In Vitro Cell Dev Biol Animal, 1993
To provide a framework for biochemical investigation of ecdysteroid action in Aedes albopictus mosquito cells, we examined the effect of 20-hydroxyecdysone on cell growth and morphology, synthesis of inducible proteins (EIPs), and expression of a transfected gene regulated by a synthetic ecdysteroid response element. When cells were cultured in the continuous presence of 10-6 M 20-hydroxyecdysone, the rate of growth decreased and subtle changes in cell morphology were observed. In both Aeries aegypti and A. albopictus cells, synthesis of a small number of radiolabeled proteins, which appeared as minor bands on sodium dodecyl sulfate-polyacrylamide gels, was induced by treatment with 20-hydroxyecdysone. On two-dimensional polyacrylamide gels, 11 EIPs, ranging in size from approximately 22 to 52 kDa, were identified in A. albopictus C7-10 cells. Ten inducible proteins were localized in the cytoplasmic fraction; EIP28 and EIP31 were detected in both cytoplasmic and nuclear extracts, and EIP29 was detected only in the nucleus, at a very low level. None of these proteins corresponded to small heat shock proteins, whose genes are 20-hydroxyecdysone-inducible in some Drosophila cell lines. The juvenile hormone analog, methoprene, induced expression of a 25 kDa protein in C7-10 ceils. Although 20-hydroxyecdysone sustained the synthesis of this methoprene-inducible protein, synthesis did not occur in the presence of 20-hydroxyecdysone alone. In transfected A. albopictus cells, expression of a recombinant DNA construct containing two tandem synthetic ecdysteroid regulatory elements based on a D. melanogaster small heat shock protein gene was modestly induced by 20-hydroxyecdysone.
Differential Expression and Regulation by 20-Hydroxyecdysone of Mosquito Ultraspiracle Isoforms
Developmental Biology, 2000
Ultraspiracle (USP), the insect homologue of the vertebrate retinoid X receptor, is an obligatory dimerization partner for the ecdysteroid receptor (EcR). Two USP isoforms, USP-A and USP-B, with distinct N-termini, occur in the mosquito Aedes aegypti. In the fat body and ovary, USP-A mRNA is highly expressed during the pre-and late vitellogenic stages, corresponding to a period of low ecdysteroid titer, while USP-B mRNA exhibits its highest levels during the vitellogenic period, correlating with a high ecdysteroid titer. Remarkably, 20-hydroxyecdysone (20E) has opposite effects on USP isoform transcripts in in vitro fat body culture. This steroid hormone upregulates USP-B transcription and its presence is required to sustain a high level of USP-B expression. In contrast, 20E inhibits activation of USP-A transcription. Although EcR ⅐ USP-A recognizes the same ecdysteroid-responsive elements, EcR ⅐ USP-B binds them with an affinity twofold higher than that of EcR ⅐ USP-A. Likewise, EcR ⅐ USP-B transactivates a reporter gene in CV-1 cells twofold more strongly than EcR ⅐ USP-A. These results suggest that USP-B functions as a major heterodimerization partner for EcR during the vitellogenic response to 20E in the mosquito.
Developmental Biology, 2000
In the mosquito Aedes aegypti, the adult female becomes competent for a vitellogenic response to ecdysone after previtellogenic development. Here, we show that FTZ-F1, the nuclear receptor implicated as a competence factor for stage-specific responses to ecdysone during Drosophila metamorphosis, serves a similar function during mosquito vitellogenesis. AaFTZ-F1 is expressed highly in the mosquito fat body during pre-and postvitellogenic periods when ecdysteroid titers are low. The mosquito AaFTZ-F1 transcript nearly disappears in mid-vitellogenesis when ecdysteroid titers are high. An expression peak of HR3, a nuclear receptor implicated in the activation of FTZ-F1 in Drosophila, precedes each rise in mosquito FTZ-F1 expression. In in vitro fat body culture, AaFTZ-F1 expression is inhibited by 20-hydroxyecdysone (20E) and superactivated by its withdrawal. Following in vitro AaFTZ-F1 superactivation, a secondary 20E challenge results in superinduction of the early AaE75 gene and the late target VCP gene. Electrophoretic mobility-shift assays show that the onset of ecdysone-response competence in the mosquito fat body is correlated with the appearance of the functional AaFTZ-F1 protein at the end of the previtellogenic development. These findings suggest that a conserved molecular mechanism for controlling stage specificity is reiteratively used during metamorphic and reproductive responses to ecdysone.
Journal of Molecular Endocrinology, 2004
The broad (br ) gene, encoding a family of C2H2 type zinc-finger DNA-binding proteins, has been shown to act as a crucial member of the 20-hydroxyecdysone (20E) regulatory hierarchy in the fruitfly, Drosophila melanogaster and the moth, Manduca sexta. In this study, we have shown that the br gene is involved in the 20E-regulatory hierarchy controlling vitellogenesis in the mosquito, Aedes aegypti. Unlike E74 and E75 early genes, expression of br was activated in previtellogenic females, during a juvenile hormone (JH)-dependent period. The levels of Z1, Z2 and Z4 isoform mRNA were elevated in the fat body of 2-day-old females after in vitro exposure to JH III. However, JH III repressed 20E activation of br in 3-to 5-day-old females, indicating a switch in hormonal commitment. Expression of Z1, Z2 and Z4 was stimulated after blood feeding in both vitellogenic tissues, the fat body and the ovary, corresponding to peaks of ecdysteroid titers. In the fat body, the mRNA profiles of these ...
Journal of Insect Physiology, 2006
Ecdysteroid titers and expression profiles of ecdysone-regulated genes were determined during the last instar larval and during the pupal stages of Aedes aegypti (Diptera: Culicidae). Three peaks of ecdysteroids occurring at approximately 24, 30-33 and 45-48 h after ecdysis to the fourth instar larval stage were detected. In the pupa, a large peak of ecdysteroids occurred between 6 and 12 h after ecdysis to the pupal stage. A small rise in ecdysteroids was also detected at the end of the pupal stage. Quantitative reverse transcriptase polymerase chain reaction analyses of the expression of ecdysone receptors and ecdysone-regulated genes showed that the peaks of expression of most of these genes coincided with the rise in ecdysteroid levels during the last larval and pupal stages. In the last larval stage, ecdysteroid titers and mRNA expression profiles of ecdysone-regulated genes are similar to those observed for Drosophila melanogaster. However, in the early pupal stage, both ecdysteroid titers and the expression of ecdysone-regulated genes are somewhat different from those observed in D. melanogaster, probably because the duration of the pupal stage in D. melanogaster is 84 h while in Ae. aeqypti the duration is only 48 h. These data which describe the relationship between ecdysteroid titers and mRNA levels of Ae. aegypti ecdysteroid-regulated genes lay a solid foundation for future studies on the hormonal regulation of development in mosquitoes.
Molecular and Cellular Endocrinology, 2007
We investigated the role of the mosquito broad (br) gene in regulating the 20-hydroxyecdysone (20E) effector Vitellogenin (Vg) gene. Injection of double-stranded RNA corresponding to the BR isoform Z2 led to a significant decrease in expression of the Vg gene at 8 and 24 h post-blood meal. Knockdown of Z1 or Z4 resulted in enhanced Vg expression beyond its normal expression time. In vitro studies suggested that the effects of BR require its direct binding to the Vg promoter, as well as protein-protein interaction between BR and the ecdysone receptor complex. The BR isoforms are therefore essential for a proper stage-specific biological response to 20E in the adult female mosquito. In particular, the isoform Z2 is required for 20E-mediated activation of Vg, while isoforms Z1 and Z4 serve as repressors to ensure appropriate termination of Vg expression.