20-Hydroxyecdysone

Dev Biol. 1991 Aug;146(2):569-82.

20-Hydroxyecdysone is required for, and negatively regulates, transcription of Drosophila pupal cuticle protein genes.[Pubmed: 1713868]

METHODS AND RESULTS:
Transcripts of ecdysone-dependent genes (EDGs) accumulate in isolated imaginal discs with 8 hr after exposure to a pulse of the steroid hormone 20-Hydroxyecdysone (20-HE; 1 microgram/ml for 6 hr) but not in discs cultured in the continuous presence or absence of the hormone. Sequence analyses show that two of the EDGs are members of gene families encoding insect cuticle proteins. We conclude that a third EDG encodes a cuticle protein because the conceptual glycine-rich protein contains sequence motifs similar to those found in insect egg shell proteins and vertebrate cytokeratins and because expression of this gene is limited to tissues that deposit the pupal cuticle. Nuclear run-on assays show that the hormone-dependent expression of each of these EDGs is due to transcriptional regulation. Readdition of hormone to imaginal discs actively synthesizing the EDG messages causes rapid repression of EDG transcription.
CONCLUSIONS:
Thus, 20-HE acts as both a positive and a negative regulator of EDG transcription. Sequences in the promoter regions of two of the EDGs are similar to an ecdysone response element and may play a role in negative regulation.

Insect Mol Biol. 2014 Aug;23(4):407-16.

20-hydroxyecdysone mediates non-canonical regulation of mosquito vitellogenins through alternative splicing.[Pubmed: 24720618]

Vitellogenesis is one of the most well-studied physiological processes in mosquitoes. Expression of mosquito vitellogenin genes is classically described as being restricted to female adult reproduction.
METHODS AND RESULTS:
We report premature vitellogenin transcript expression in three vector mosquitoes: Culex tarsalis, Aedes aegypti and Anopheles gambiae. Vitellogenins expressed during non-reproductive stages are alternatively spliced to retain their first intron and encode premature termination codons. We show that intron retention results in transcript degradation by translation-dependent nonsense-mediated mRNA decay. This is probably an example of regulated unproductive splicing and translation (RUST), a mechanism known to regulate gene expression in numerous organisms but which has never been described in mosquitoes. We demonstrate that the hormone 20-Hydroxyecdysone (20E) is responsible for regulating post-transcriptional splicing of vitellogenin. After exposure of previtellogenic fat bodies to 20E, vitellogenin expression switches from a non-productive intron-retaining transcript to a spliced protein-coding transcript. This effect is independent of factors classically known to influence transcription, such as juvenile hormone-mediated competence and amino acid signalling through the target of rapamycin pathway.
CONCLUSIONS:
Non-canonical regulation of vitellogenesis through RUST is a novel role for the multifunctional hormone 20E, and may have important implications for general patterns of gene regulation in mosquitoes.

Insect Biochem Mol Biol. 2014 Feb;45:30-9.

E93 predominantly transduces 20-hydroxyecdysone signaling to induce autophagy and caspase activity in Drosophila fat body.[Pubmed: 24316411]

During the larval-prepupal transition in Drosophila, a balancing crosstalk occurs between autophagy and caspase activity in the remodeling fat body: the inhibition of autophagy induces caspase activity and the inhibition of caspases induces autophagy. Both autophagy and caspase activity are induced by a pulse of molting hormone (20-Hydroxyecdysone, 20E) via the 20E nuclear receptor complex, EcR-USP.
METHODS AND RESULTS:
We here demonstrate that E93, a 20E primary-response gene encoding an HTH transcription factor, predominantly transduces 20E signaling to induce autophagy and caspase activity in the remodeling fat body. RNAi knockdown or mutation of E93 blocks autophagy and caspase activity, E93 overexpression induces them both, while E93 overexpression has a better rescuing effect on the inhibition of autophagy than caspase activity caused by EcR(DN) overexpression. At the transcriptional level, E93 not only greatly impacts the 20E-triggered transcriptional cascade, but also upregulates essential autophagy and apoptosis genes. Meanwhile, at the phosphorylational level, E93 blocks the PI3K-TORC1 signaling to initiate autophagy.
CONCLUSIONS:
Taken together, we conclude that autophagy and caspase activity are induced by 20E and predominantly transduced by E93 in the remodeling fat body of Drosophila.

J Biol Chem. 2015 Mar 27;290(13):8469-81.

The steroid hormone 20-hydroxyecdysone via nongenomic pathway activates Ca2+/calmodulin-dependent protein kinase II to regulate gene expression.[Pubmed: 25670853 ]

The steroid hormone 20-Hydroxyecdysone (20E) triggers calcium signaling pathway to regulate 20E response gene expression, but the mechanism underlying this process remains unclear.
METHODS AND RESULTS:
We propose that the 20E-induced phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) serves an important function in 20E response gene transcription in the lepidopteran insect Helicoverpa armigera. CaMKII showed increased expression and phosphorylation during metamorphosis. 20E elevated CaMKII phosphorylation. However, the G protein-coupled receptor (GPCR) and ryanodine receptor inhibitor suramin, the phospholipase C inhibitor U73122, and the inositol 1,4,5-triphosphate receptor inhibitor xestospongin C suppressed 20E-induced CaMKII phosphorylation. Two ecdysone-responsible GPCRs and Gαq protein were involved in 20E-induced CaMKII phosphorylation by RNA interference analysis. 20E regulated CaMKII threonine phosphorylation at amino acid 290, thereby inducing CaMKII nuclear translocation. CaMKII knockdown by dsCaMKII injection into the larvae prevented the occurrence of larval-pupal transition and suppressed 20E response gene expression. CaMKII phosphorylation and nuclear translocation maintained USP1 lysine acetylation at amino acid 303 by inducing histone deacetylase 3 phosphorylation and nuclear export. The lysine acetylation of USP1 was necessary for the interaction of USP1 with EcRB1 and their binding to the ecdysone response element.
CONCLUSIONS:
Results suggest that 20E (via GPCR activation and calcium signaling) activates CaMKII phosphorylation and nuclear translocation, which regulate USP1 lysine acetylation to form an EcRB1-USP1 complex for 20E response gene transcription.

J Insect Physiol. 2010 Oct;56(10):1436-44.

Transcriptional regulation of the insulin signaling pathway genes by starvation and 20-hydroxyecdysone in the Bombyx fat body.[Pubmed: 20197069 ]

Genetic studies in the fruitfly, Drosophila melanogaster, have uncovered a conserved insulin/insulin growth factor signaling (IIS) pathway that regulates nutrition-dependent growth rates of insects.
METHODS AND RESULTS:
From the silkworm, Bombyx mori, we have identified and characterized several key genes involved in the IIS pathway, including InR, IRS, PI3K110, PI3K60, PTEN, PDK, and Akt. Tissue distribution analysis showed that most of these genes were highly expressed in the fat body implying that the IIS pathway is functionally important within insect adipose tissue. Developmental profile studies revealed that the expression levels of InR, IRS, PI3K110, and PDK were elevated in the fat body during molting and pupation, periods when animals ceased feeding and hemolymph levels of 20-Hydroxyecdysone (20E) were high. Starvation rapidly up-regulated the mRNA levels of these same genes in the fat body, while 20E slowly induced their transcription.
CONCLUSIONS:
We conclude that 20E slowly reduces food consumption and then indirectly induces a state of starvation resulting in the elevation of the mRNA levels of InR, IRS, PI3K110, and PDK in the Bombyx fat body during molting and pupation.