Reporter gene assays for screening and identification of novel molting hormone- and juvenile hormone-like chemicals

Abstract

A reporter gene assay (RGA) is used to investigate the activity of synthetic chemicals mimicking the molting hormones (MHs) and juvenile hormones (JHs) of insects, so-called insect growth regulators (IGRs). The MH receptor, a heterodimer of the ecdysone receptor (EcR) and ultraspiracle (USP), and the JH receptor Methoprene-tolerant (Met) are ligand-dependent transcription factors. Ligand-bound EcR-USP and Met bind to specific cis-acting DNA elements, referred to as the ecdysone-responsive element (EcRE) and the JH-responsive element (JHRE), respectively, in order to transactivate target genes. Insect hormone-induced transactivation systems have been reconstituted by the introduction of reporter genes under the control of EcRE and JHRE, or two-hybrid reporter genes, into insect, mammalian, and yeast cells expressing receptor proteins. RGA is easy to use and convenient for examining the MH- and JH-like activities of synthetic chemicals and is suitable for the high-throughput screening of novel structural classes of chemicals targeting EcR-USP and Met.

Introduction

Insect species that belong to a large class of Arthropoda utilize unique endocrine systems consisting of two peripheral hormones, molting hormones (MHs) and juvenile hormones (JHs). These hormones are important for regulating the growth and development of insects. The steroid 20-hydroxyecdysone (20E) and sesquiterpenoid JH III are a representative MH and JH, respectively. Synthetic nonsteroidal ecdysone agonists and juvenile hormone agonists (JHAs) have recently been developed as insecticides and categorized as insect growth regulators (IGRs).^1,2)

Since these synthetic insect hormones interfere with insect-specific hormone responses, they are considered to be safer for mammals than other classes of insecticides.¹⁾ Nonsteroidal ecdysone agonists exhibit insect-selective toxicity for certain taxonomic orders. These IGRs may be ideal compounds for pest control.^1,3,4)

The activities of synthetic MHs and JHs were previously analyzed in the whole body using classical in vivo bioassay methods.^5–11) Experimental procedures to examine hormone-dependent morphological changes and the stimulation/inhibition of proliferation using cultured insect cell lines have also been developed as in vitro bioassays.^12–18) The measurement of ligand-receptor binding affinity using insect cell extracts or in vitro translated receptor proteins is another procedure for detecting natural and synthetic hormones.^19–28) In the 1990s, genes encoding the ecdysone receptor (EcR) and its partner protein ultraspiracle (USP), a homolog of mammalian retinoid X receptor (RXR), and the JH receptor Methoprene-tolerant (Met) were initially identified in the fruit fly Drosophila melanogaster.^29–32) EcR-USP and Met are ligand-dependent transcription factors belonging to the nuclear receptor (NR) superfamily and the basic helix-loop-helix Per/Arnt/Sim (bHLH-PAS) family, respectively (Fig. 1a and Fig. 4a). The genes encoding EcR-USP and Met are conserved among various insect species and have been functionally characterized.^33–35)

Fig. 1. MH-receptor EcR. (a) Domain structure of EcR. NR superfamily proteins including EcR are comprised of six domains (A–F) with conserved functions and sequences. AF: activation function; DBD: DNA-binding domain; H: hinge domain; LBD: ligand-binding domain.¹³⁶⁾ (b) Nucleotide sequence of D. melanogaster hsp27 EcRE.⁴³⁾ The arrows indicate half-site sequences forming a putative palindromic structure. (c) Patterns of typical REs for vertebrate NRs. Half sites with a canonical sequence (AGGTCA) are aligned in three different orientations: inverted repeat (IR), direct repeat (DR), and everted repeat (ER). Two half sites are zero to several nucleotides apart.^137,138) (d) Schematic representation of the molecular mechanism underlying the MH-dependent transactivation of target genes by EcR-USP in D. melanogaster. See the text for details.

The reporter gene assay (RGA) is a versatile experimental method for monitoring gene expression associated with signal transduction cascades in response to intra- and extracellular stimuli. RGA has been employed to elucidate the molecular mechanisms of signal transduction via EcR-USP and Met, and it has also played important roles in the development of high-throughput screening systems for IGRs with MH- and JH-like activities. In this review, we summarized the application of RGA to the study of IGRs.

1. RGA

The term reporter gene refers to a gene encoding a protein product with unique and readily measurable enzymatic activity or that is distinguishable from a large amount of various proteins expressed in cells.^36–38) The following reporter genes are frequently and commonly used: genes for bacterial chloramphenicol acetyltransferase (CAT), β-galactosidase (β-gal), and β-glucuronidase (GUS); luciferase from bacteria, firefly, or Renilla; green fluorescent protein (GFP) of jellyfish; and the secretable form of alkaline phosphatase (SEAP) derived from the human placenta.^36–40) These reporter gene products have been characterized in detail, and their advantages and disadvantages as well as detection methods have been reviewed elsewhere.^36,39,40)

Reporter genes are placed downstream of promoters that function in host cells. RGA is a versatile experimental method for examining various cellular events. The reporter gene may be used as a marker of gene transfer or in promoter analysis, the identification of cis- and trans-acting DNA elements of genes of interest, the spatiotemporal imaging of gene expression patterns, the characterization of receptors and their ligands, and analyses of signaling pathways.⁴¹⁾

2. Molecular mechanisms underlying the activation of EcR-USP by MHs

The RGA procedure has been applied in many studies to investigate the molecular mechanisms underlying signal transduction initiated by MHs. Riddihough et al. reported that the transcription of the D. melanogaster heat shock protein 27 (hsp27) gene was induced by ecdysone as well as heat shock. A deletion analysis of the hsp27 promoter identified ecdysone-specific cis-acting DNA elements, referred to as the ecdysone-responsive element (EcRE), with a putative palindrome structure (Fig. 1b).^42,43) A gene for the MH receptor EcR was cloned as the first member of the NR superfamily in D. melanogaster. The EcR protein was shown to bind to active MH 20E as well as DNA with high specificity at EcRE.²⁹⁾ MH-bound EcR forms a heterodimer with its partner protein, USP, on EcRE.^30,31,44) Furthermore, the steroid receptor coactivator (SRC), Taiman of D. melanogaster (DmTai), was identified as an important component in the transduction of MH signals to downstream effectors.⁴⁵⁾

EcREs with distinct sequences were subsequently identified in the regulatory region of D. melanogaster Fbp1, Eip28/29, and Aedes aegypti vitellogenin (Vg) genes.^46–49) Previous studies also indicated that EcR-USP induced reporter gene expression via asymmetric RE, a direct repeat of the core sequence (half site) in African green monkey CV-1 cells.^50,51) The heterodimerization of EcR and USP on DR-RE (Fig. 1c) was confirmed using a biochemical approach, the electrophoretic mobility shift assay (EMSA).⁵²⁾ The activation mechanism of EcR-USP revealed in D. melanogaster was conserved in other insect species (Fig. 1d).^33,50,53,54)

3. RGA for detecting Synthetic ecdysone agonists by surveying gene expression mediated by EcR-USP

The identification of EcRE drove researchers to develop RGA systems with the capacity to detect synthetic ecdysone agonists by examining EcR-USP-mediated gene expression that enables activity-based high-throughput screening. RGA systems previously developed for EcR-USP in various host cells are summarized in Table 1. The basis for using RGA to monitor the ligand-dependent activation of EcR-USP-mediated gene expression is the construction of a reporter plasmid that results in the expression of reporter genes, including CAT, β-gal, Luc, and GFP, under the control of EcRE (Fig. 2a). Dm hsp27 EcRE is recognized as an effective EcRE, and it is used in the development of a large number of RGA systems, regardless of host cell species (Table 1). The Dm hsp27 promoter (∼650 bps), or the fusion of a short oligonucleotide containing Dm hsp27 EcRE with the basal promoters of insect genes, such as D. melanogaster alcohol dehydrogenase (ADH) and Bombyx mori actin A3 (ACT3), has frequently been used to express reporter genes in insect cell lines.^29,55–58) In mammalian RGA, Dm hsp27 EcRE was connected to minimal promoters of thymidine kinase (TK) or mouse mammary tumor virus (MMTV).^30,31,59) Ligand-bound EcR and USP expressed from the insect genome (endogenous EcR-USP) or transfected genes (in mammalian or yeast cells) bind to the EcRE of the reporter plasmid and then induce the transcription of reporter genes instead of native target genes (Fig. 2a). Two-hybrid-based RGAs are often used to examine ligand-dependent interactions between EcRs and USPs in heterologous hosts (Fig. 2b and Table 1). The heterodimerization of ligand-bound EcR and USP enables the transactivation of two-hybrid reporter genes.^3,4,60–64)

Table 1. In vitro RGA systems for the screening of EcR-USP ligands

Host cells	Organism and cell lines	Transfected receptor genes	EcRE	Reporter gene	Types of test compounds	Ref.
Insect cells*	Drosophila melanogaster Kc	—	Dm hsp27 promoter	Luc	20E, α-ecdysone	55)
	D. melanogaster Kc	—	Dm hsp27 promoter	Luc	3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide (DTBHIB)	56)
	D. melanogaster Kc	—	Dm hsp27 promoter	Luc	Diacylhydrazine (DAH) compounds	57)
	D. melanogaster Schneider 2 (S2)	—	Dm hsp27	lacZ	Cucurbitacins	134)
			EcRE
	D. melanogaster Schneider SL2	—	Dm hsp27	Luc	Insect and plant ecdysteroids	139)
			EcRE
	Spodoptera frugiperda Sf9	—	Dm hsp27	Luc	20E, ponasterone A (pon A), chromafenozide	67)
			EcRE
	D. melanogaster Kc	—	Dm hsp27	Luc	20E, pon A, chromafenozide	67)
			EcRE
	Bombyx mori Bm5	—	Dm hsp27	CAT	Plant compounds	58)
			EcRE	GFP	DAH library
	S. frugiperda Sf9	CfEcR	Dm hsp27	CAT	RG-102240	68)
			EcRE
	Lymantria dispar (Ld652Y)	CfEcR	GAL4 RE (two-hybrid)	CAT	RG-102240	68)
		MmRXR		GFP
	B. mori Bm5	—	Dm hsp27	GFP	DAHs	70)
			EcRE
	D. melanogaster S2	—	Dm hsp27	Luc	Chromafenozide	69)
			EcRE		Methoxyfenozide
	B. mori Bm5	—	Dm hsp27	Luc	Chromafenozide	69)
			EcRE		Methoxyfenozide
	Anthonomus grandis BRL-AG-3A	—	Dm hsp27	Luc	DAHs	76)
	BRL-AG-3C		EcRE	GFP
	Leptinotarsa decemlineata BCRL-Lepd-SL1	—	Dm hsp27	Luc	DAHs	76)
			EcRE	GFP
	D. melanogaster S2	—	Dm hsp27	CAT	DAHs, acylaminoketone (AAK) analogs, tetrahydroquinoline (THQ) compounds, piperidiamine analogs	80)
			EcRE	Luc
	B. mori Bm5	—	Dm hsp27	GFP	DAHs, AAK analogs, THQs	71)
			EcRE
	S. littoralis Sl2, Slj2b	—	Dm hsp27	Luc	DAHs, AAK analogs, THQs	71)
			EcRE
	S. frugiperda Sf9	—	Dm hsp27	Luc	Non-steroidal compounds	78)
			EcRE
	L. decemlineata	—	Dm hsp27	Luc	DAHs	77)
	BCRL-Lepd-SL1		EcRE
	D. melanogaster S2	—	Dm hsp27	Luc	Ecdysteroids	135)
			EcRE
	B. mori Bm5	—	Dm hsp27	Luc	Ecdysteroids	135)
			EcRE
	S. frugiperda Sf9	—	Dm hsp27	Luc	N-tert-butylphenyl thenoylhydrazide compounds	140)
			EcRE
	D. melanogaster S2	—	Dm hsp27	Luc	Compounds registered in Maybridge database	79)
			EcRE
	B. mori Bm5	—	Dm hsp27	Luc	Compounds registered in Maybridge database	79)
			EcRE
Mammalian cells	Cricetulus griseus	VgEcR	E/GRE	lacZ	Phytochemicals	90)
	CHO	HsRXR
	Mus musculus	AaEcR	GAL4 RE (two-hybrid)	Luc	THQs	3)
	NIH 3T3	MmRXR
Mammalian cells	Homo sapiens	BmEcR	Dm hsp27 EcRE	β-gal	AAKs	72)
	HEK293
	H. sapiens	BmEcR	Dm hsp27 EcRE	β-gal	AAKs	60)
	HEK293
	C. griseus	CfEcR	GAL4 RE (two-hybrid)	Luc	AAKs	60)
	CHO	HsRXR-LmRXR chimera
	M. musculus	CfEcR	GAL4 RE (two-hybrid)	Luc	DAHs, THQs	61)
	NIH 3T3	LmRXR
		DmTai
	M. musculus	AaEcR	GAL4 RE (two-hybrid)	Luc	THQs	4)
	NIH 3T3	CfEcR
		DmEcR
		MsEcR
		BmEcR
		LmEcR
		TmEcR
		AmaEcR
		HsRXR
	M. musculus	AaEcR	GAL4 RE (two-hybrid)	Luc	Natural and semi-synthetic ecdysteroids (hydroxylation)	62)
	NIH 3T3	CfEcR
		DmEcR
		MsEcR
		BmEcR
		TmEcR
		AmaEcR
		BaEcR
		NcEcR
		HsRXR
	M. musculus	VgEcR	E/GRE	Luc	Natural and semi-synthetic ecdysteroids (hydroxylation)	62)
	NIH 3T3	HsRXR
	M. musculus	AaEcR	GAL4 RE (two-hybrid)	Luc	Natural and semi-synthetic ecdysteroids (alkylation)	63)
	NIH 3T3	CfEcR
		DmEcR HsRXR-LmRXR chimera
	M. musculus	VgEcR	E/GRE	Luc	Natural and semi-synthetic ecdysteroids (alkylation)	63)
	NIH 3T3	HsRXR
	H. sapiens	BmaEcR	GAL4 RE (two-hybrid)	Luc	Steroidal ecdysones, DAHs	64)
	HEK293	HsRXR
Yeast	Saccharomyces cerevisiae	CfEcR-CfUSP	Dm hsp27 EcRE	β-gal	Nonsteroidal ecdysone agonists	85)
		HsRXR
		MmGRIP1
	S. cerevisiae	AaEcR-AaUSP	Dm hsp27 EcRE	β-gal	Ecdysteroids, nonsteroidal ecdysone agonists	84)
		DmUSP
		CfUSP
		HsRXR
		MmGRIP1
	S. cerevisiae	DmEcR-DmUSP	IR0	β-gal	Ecdysteroids, DAHs, THQ	87)
		CsEcR-CsUSP	DR2
		LdEcR-LdUSP	DR4
		DmTai

Abbreviations Aa: Aedes aegypti; Ama: Amblyomma americanum; Ba: Bemisia argentifolii; Bm: Bombyx mori; Bma: Brugia malayi; Cf: Choristoneura fumiferana; Cs: Chilo suppressalis; Dm: Drosophila melanogaster; E/GRE: ecdysone/glucocorticoid responsive element (hybrid RE); Hs: Homo sapiens; Lm: Locusta migratoria; Ms: Manduca sexta; Nc: Nephotettix cincticeps; Tc: Tribolium castaneum; Tm: Tenebrio molitor; Mm: Mus musculus; CHO; Chinese hamster ovary; GAL4 RE: DNA element for binding of the GAL4 DNA-binding domain (DBD); VgEcR: hybrid of D. melanogaster EcR carrying modified DBD and VP16 AD that recognizes an E/GRE *Reporter gene assays in insect cells expressing endogenous EcR and USP. Only the reporter plasmid was introduced.

Fig. 2. RGA for EcR-USP. (a) Schematic summary of RGA for EcR-USP. RGA using a reporter gene under the control of EcRE, in which multiple copies of EcRE are integrated upstream of the promoter (EcRE×n). Ligand-bound EcR and USP form a heterodimer on EcRE. The interaction of transcriptional coactivator SRC enhances reporter gene expression by recruiting RNA pol II general transcription factors of host cells. (b) Two-hybrid-based RGA for EcR-USP. EcR and USP (LBD or full length) fused to Gal4-DBD and transcriptional activation domain (AD) of Gal4 or VP16, respectively (and vice versa), interact in an MH-dependent manner and induce reporter gene expression in host cells.

Pioneering studies using RGA on insect cell lines were conducted by Mikitani, who introduced a Luc reporter plasmid connected to a Dm hsp27 promoter into D. melanogaster Kc cells that were highly sensitive to ecdysteroids. The ecdysteroid-dependent expression of luciferase by endogenous EcR-USP was reproducibly observed. This procedure was very sensitive, rapid, and simple, suggesting that RGA of insect cells is a valuable tool in the search for new ecdysteroid compounds.⁵⁵⁾ The findings obtained using RGA correlated with the effects of synthetic ecdysteroid compounds on ligand-binding affinity, efficacy toward cellular morphological changes, and larvicidal activity.^56–58) To date, a number of RGA systems for EcR-USP have been developed in several cell lines from insect species in distinct phylogenetic orders.

RH5849, a diacylhydrazine (DAH)-type lead compound exhibiting MH-like activity, was identified as the first nonsteroidal ecdysone agonist.^65,66) DAHs exhibited in vivo toxicity in a lepidopteran insect–selective manner, which correlated with the transactivation activity observed in RGA.^58,65–71) α-Acylaminoketone and its analogs were identified as novel types of nonsteroidal ecdysone agonists with potent selectivity on lepidopteran EcR-USP, while tetrahydroquinoline (THQ) compounds were shown to strongly activate dipteran EcR-USP.^{3,4,60,61,71,72)} The insect order-selective activity of nonsteroidal ecdysone agonists was associated with the divergence of the primary sequence of the ligand-binding domain (LBD) of EcR. Differences in the three-dimensional (3D) structure of the ligand-binding pocket affected the binding potency of nonsteroidal ecdysone agonists; however, the binding mode of natural ecdysteroids was similar among different phylogenetic orders of insects.^73–75)

To discover new classes of ecdysone agonists for non-lepidopteran insect pests, RGA was established in coleopteran cell lines from Leptinotarsa decemlineata and Anthonomus grandis, and various nonsteroidal ecdysone agonists were tested by methods combined with (quantitative) structure–activity relationship ((Q)SAR) studies and/or the 3D-modeling of the ligand-binding pocket and ligand docking studies (virtual screening).^71,76,77) Similarly, the agonist/antagonist activities for dipteran and lepidopteran EcR-USP of several compounds identified by ligand-based virtual screening were confirmed by RGA.^78,79) Approaches combining (Q)SAR and RGA were also used to examine the ligand potencies of semi-synthetic ecdysteroids for EcR-USP of various insect species.^62,63)

In mammalian cell-based RGA, EcR-USP derived from several different orders of insects was expressed to examine the MH-like activity of test compounds.^4,62,63) RGA was also utilized to compare the species-selective in vivo toxicities and in vitro transactivation activities of synthetic compounds in the lepidopteran insects B. mori and Spodoptera littoralis.⁸⁰⁾

4. Reconstitution of MH-induced transactivation systems in yeast

RGA has been established in the yeast Saccharomyces cerevisiae as another heterologous host system. S. cerevisiae is the simplest eukaryote possessing highly conserved gene expression mechanisms of higher eukaryotes.⁸¹⁾ Previous studies reported that the ligand-dependent transactivation activity of D. melanogaster EcR-USP via hsp27 EcRE was not confirmed, whereas the EcR protein expressed in yeast was capable of binding to ecdysteroid ligands with the co-expression of USP.^82,83) Tran et al. showed that the expression of mouse SRC GRIP1 was required to reconstitute the ligand-dependent transactivation of EcR-USP from Choristoneura fumiferana and Aedes aegypti.^84,85) The ligand-induced heterodimerization of the LBDs of EcR and USP from D. melanogaster was also demonstrated by a two-hybrid method and EMSA in yeast.⁸⁶⁾ Based on these findings, we recently established a new yeast RGA system for detecting MHs.⁸⁷⁾ EcR and USP derived from three insect species belonging to different taxonomic orders—the dipteran D. melanogaster, lepidopteran Chilo suppressalis, and coleopteran L. decemlineata—were expressed in yeast in conjunction with the SRC DmTai (Fig. 3). By optimizing response elements, yeast RGA exhibited marked responses and detected EcR ligands in dose-dependent and ecdysteroid-specific manners. The insect order-selective ligand activities of the nonsteroidal ecdysone agonists, DAHs and THQ, for lepidopteran and dipteran EcRs, respectively, were also detectable. The potencies of these ligands in yeast RGA linearly correlated with their binding activities.⁸⁷⁾

Fig. 3. Principle of RGA in yeast strains expressing insect ecdysone receptors EcR and USP. In response to ligands, EcR and USP expressed in yeast cells form a heterodimer and bind to response elements upstream of the minimal promoter of the iso-cytochrome c (CYC) gene. D. melanogaster Tai, a steroid receptor coactivator (SRC), cooperates with EcR-USP to enhance the expression of the lacZ reporter gene by recruiting the RNA pol II general transcription factors of yeast. The expression of β-galactosidase may be visualized and quantified by the development of a yellow color due to the accumulation of ONP in the assay buffer. ONPG: o-nitrophenyl-β-D-galactopyranoside; ONP: o-nitrophenol; RNA pol II: RNA polymerase II; P_CYC: minimal promoter of the CYC gene.⁸⁷⁾

Heterologous RGAs established in mammalian or yeast cells tended to exhibit lower ligand responses than those in insect cells.^85,87,88) In mammalian RGAs, chimeric EcR (VgEcR), in which D. melanogaster EcR carrying modified DBD was fused to VP16 AD, was constructed in order to achieve robust responses.⁸⁹⁾ VgEcR recognizes hybrid response elements containing ecdysone and glucocorticoid responsive element (E/GRE)⁸⁹⁾ and has been used in some mammalian RGAs.^62,63,90) In yeast RGA, the genetic manipulation of the host yeast strain was effective for improvement; the deletion of plasma membrane-localized efflux pumps and/or cell wall mannoproteins resulted in increased ligand responses.^85,91,92) Heterologous host cells that lack ecdysteroid-regulated gene expression systems, however, are considered to be suitable for examining EcR-USP-ligand interactions without perturbation by endogenously expressed EcR-USP.⁹³⁾ Unlike mammalian cells that express RXRs, functional homologs of insect USPs,^30,31,84,85) yeast does not possess NR homologs.⁹⁴⁾ Yeast RGA, which is a rapid, easy, and cost-effective procedure, may be advantageous for directly measuring the MH-like activities of synthetic compounds on EcR-USP.

5. Molecular mechanisms underlying JH-dependent transactivation in arthropods

JH receptor Met proteins are members of the bHLH-PAS family that function as ligand-dependent transcription factors as well as EcR-USP (Fig. 4a).^34,35) The Met gene was originally cloned as a gene that complemented the methoprene resistance of a mutant fly.³²⁾ The Met protein was shown to bind to JH III with high affinity. The GAL4-DBD-Met fusion protein directed to GAL4 RE transactivated the Luc reporter in a JH/JHA-dependent manner.²⁴⁾ Met homologs have since been identified in various insects, such as holo-, hemi-, and ametabolous species.^26,95–97) The paralogous gene germ cell–expressed (Gce), which is conserved in Drosophila species, is also involved in signal transduction for the exertion of JH effects.^98–100) Met-Met and Met-Gce dimer complexes are formed as inactive states in the absence of JHs. Upon ligand binding to the PAS-B domain of Met and Gce,²⁶⁾ Met-Met and Met-Gce complexes are rapidly dissociated.¹⁰¹⁾ Met/Gce proteins then form a heterodimer with other bHLH-PAS protein SRCs, such as DmTai, A. aegypti FISC, and Tribolium castaneum TcSRC.^102,103) The Met-SRC and Gce-SRC heterodimer complex binds to a specific DNA element (JHRE) in a JH-dependent manner and activates the transcription of early JH-inducible genes, such as Krüppel homolog 1 (Kr-h1) and early trypsin (ET) (Fig. 4b).^102–104) Previous studies on the expression of anti-metamorphic gene Kr-h1 identified a 13-nucleotide motif containing an E-box (CAC GTG) as JHRE, which was essential for the binding of Met-SRC to mediate the effects of JH. This motif is highly conserved in the Kr-h1 regulatory region of a wide range of insect species (Fig. 4c).^105–107) In A. aegypti, Met and Cycle (Cyc) have been shown to form a heterodimer on response elements containing an imperfect E-box-like (CAC GCG) sequence in female mosquitos. The Met-Cyc complex induces the JH-dependent expression of the Kr-h1 gene, as well as the Met-FISC heterodimer.¹⁰⁸⁾

Fig. 4. JH-receptor Met. (a) Domain structures of Met and Gce, members of the bHLH-PAS domain protein family. Met and Gce are comprised of three functional domains—bHLH: a basic helix-loop-helix domain involved in DNA binding; PAS-A: a domain for transcriptional activation; PAS-B: a domain for ligand binding.^26,116) (b) Schematic representation of the JH-dependent transactivation of a target gene by Met/Gce in D. melanogaster. See the text for details. (c) Conservation of the JHRE motif including the E-box in the regulatory regions of the Kr-h1 gene of various insects.^105–107)

JH-regulated gene expression is shared in microcrustacea Daphnia pulex and D. magna, in which Met and SRC form a heterodimer in response to various juvenoids.¹⁰⁹⁾ Juvenoids are involved in environmental sex determination and induce male offspring production in female populations of daphnids.^110–112) Although Met and SRC play essential roles in normal embryogenesis,¹⁰⁹⁾ the expression of D. pulex Kr-h1 is not regulated by the JH-Met signaling cascade, as it is in insects.¹¹³⁾

6. RGA systems for detecting JHs and JHAs

Table 2 summarizes RGAs previously established in studies on JH receptors. The strategy of constructing reporter plasmids is similar to that of constructing RGAs for EcR-USP: DNA fragments containing JHRE identified in genes such as Kr-h1, juvenile hormone esterase (jhe), and early trypsin (ET) were integrated upstream of basal promoters (Table 2).^{27,28,105–107,114–117)} Ligand-bound Met heterodimerizes with SRC and binds to JHRE in order to transactivate reporter gene expression (Fig. 5a). The JH-dependent expression of reporter genes by Met and/or Gce has been measured in various insect cell lines (Table 2).^{27,102,104–107,114–116)} Although two-hybrid-based RGAs have been used frequently to examine JH-dependent heterodimer formation between Met and SRC in heterologous host systems (Fig. 5b), insect Kr-h1 JHRE-regulated transactivation was successfully reconstituted using mammalian cells in three studies (Table 2).^105,117,118) Some of these RGAs indicated does-dependent responses against various juvenoids, such as JH III, methyl farnesoate, methoprene, pyriproxyfen, and fenoxycarb.^{27,105,109,117,119,120)}

Table 2. RGA systems for arthropod JHRs

Host cells	Organisms and cell lines	Transfected receptor genes	Response element	Reporter gene	Ref.
Insect cells	C. fumiferana CF-203*	—	Cfjhe JHRE	Luc	114)
	D. melanogaster S2	DmMet	GAL4 RE (one-hybrid)	Luc	24)
	D. melanogaster L57	AaMet-AaFISC	GAL4 RE (two-hybrid)	Luc	102)
	D. melanogaster L57*	—	AaET JHRE	Luc	102)
	B. mori NIAS-Bm-aff3*	—	BmKr-h1 JHRE	Luc	105)
	Tribolium castaneum Tc81*	—	TcKr-h1 JHRE	Luc	106)
	D. melanogaster S2	TcMet-TcSRC	TcKr-h1 JHRE	Luc	106)
	Aedes aedypti Aga2*	—	Synthetic AaMFBS	Luc	104)
	Aedes aegypti Aga2*	—	AaKr-h1 JHRE	Luc	107)
	D. melanogaster Kc*	—	DmKr-h1 JHRE	Luc	115)
	D. melanogaster S2	BmMet-BmSRC	BmKr-h1 JHRE	Luc	116)
	D. melanogaster S2	DmMet/Gce-DmTai	AaET JHRE	Luc	27)
	D. melanogaster S2*	—	AaET JHRE	Luc	28)
Mammalian cells	M. musculus	TcMet-TcSRC	GAL4 RE (two-hybrid)	Luc	103)
	NIH 3T3
	H. sapiens HEK293	BmMet2-BmSRC	GAL4 RE (two-hybrid)	Luc	105)
	H. sapiens HEK293	BmMet2-BmSRC	BmKr-h1 JHRE	Luc	105)
	H. sapiens HEK293	TcMet-TcSRC	GAL4 RE (two-hybrid)	Luc	106)
	C. griseus CHO	DapmaMet-DapmaSRC	GAL4 RE (two-hybrid)	Luc	109)
		DappuMet-DappuSRC
	H. sapiens HEK293	BmMet2-BmSRC	BmKr-h1 JHRE	Luc	116)
		BmMet1-BmSRC
	H. sapiens HEK293	BmMet1-BmSRC	GAL4 RE (two-hybrid)	Luc	116)
	C. griseus CHO	DapmaMet-DapmaSRC	GAL4 RE (two-hybrid)	Luc	119)
	C. griseus CHO	DappuMet-DappuSRC	GAL4 RE (two-hybrid)	Luc	120)
		TcMet-TcSRC
		AaMET-AaFISC
		DmMet-DmTai
		DmGCE-DmTai
	C. griseus CHO	DappuMet-DappuSRC	TcKr-h1 JHRE	Luc	118)
	H. sapiens HEK293T	DmMet-DmTai	GAL4 RE (two-hybrid)	Luc	28)
		DmGCE-DmTai
	H. sapiens HEK293T	DmMet-DmTai	BmKr-h1 JHRE	Luc	117)
Yeast	S. cerevisiae	AaMet-AaCYC	GAL4 RE (two-hybrid)	β-gal	122)
		AaMet-AaFISC
	S. cerevisiae	AaMet-AaFISC	GAL4 RE (two-hybrid)	β-gal	123)

Abbreviations JHR: juvenile hormone receptor, Aa: Aedes aegypti; Bm: Bombyx mori; Cf: Choristoneura fumiferana; Dm: Drosophila melanogaster; Tc: Tribolium castaneum; Dapma: Daphnia magna; Dappu: Daphnia pulex; jhe: juvenile hormone esterase; ET: early trypsin; MFBS: Met-FISC binding site * Reporter gene assays in insect cells expressing endogenous JHRs and a transcriptional coactivator. Only the reporter plasmid was introduced.

Fig. 5. RGA for Met. (a) Schematic summary of RGA for Met. RGA using a reporter gene under the control of JHRE, in which multiple copies of JHRE are integrated upstream of the promoter (JHRE×n). Ligand-bound Met and SRC form a heterodimer on JHRE, recruiting RNA pol II general transcription factors of host cells for the induction of the reporter gene. (b) Two-hybrid-based RGA for Met. Met and SRC (partial or full length) fused to Gal4-DBD and AD, respectively (and vice versa), interact in a JH-dependent manner and induce reporter gene expression in host cells.

RGAs for Met from Daphnia species also have been established.^109,119,120) D. pulex Met exhibited dose-dependent responses against several juvenoids via T. castaneum Kr-h1 JHRE,¹¹⁸⁾ while the Kr-h1 homolog of D. pulex was not regulated by JH-Met signaling.¹¹³⁾ This finding suggests that the DNA-binding properties of Met proteins are conserved in insects and daphnids. RGA for Met derived from non-insect species will be useful for examining the influence of JHAs in environmental assessments.

RGA is valuable for revealing structural features in the binding between JHs/JHAs and Met. Bittova et al. examined the stereoselectivity and binding mechanisms of D. melanogaster Gce by RGA combined with a molecular modeling approach.²⁸⁾ Yokoi et al. investigated the SAR of 14 natural and synthetic JHAs using their newly established RGA in HEK293T cells.¹¹⁷⁾ Unlike synthetic EcR ligands, JHAs with potent insect selectivities have not yet been developed.¹²¹⁾ However, a mutational analysis of the PAS-B domain identified two amino acid residues crucial for the ligand selectivity of Met proteins between insects and daphnids.^109,120) An analysis of structural aspects of the ligand-binding pocket of Met may facilitate the development of JHAs specific to insect pests.

Two-hybrid-based yeast RGAs expressing A. aegypti Met and FISC/CYC were also established. These RGAs were used to screen for plant-derived compounds with agonist or antagonist activities.^122,123)

Conclusion

Since synthetic ecdysone agonists and JHAs act on insect-specific endocrine systems, extensive efforts have been made to obtain novel compounds.^1,121) Regarding ecdysone agonists, derivatives of imidazothiadiazole,¹²⁴⁾ 1-phenyl-4-cyano-5-aminopyrazoles,¹²⁵⁾ heptacyclic pyrazolamide,¹²⁶⁾ methylene lactams,¹²⁷⁾ oxadiazolines,¹²⁸⁾ and imidazoles¹²⁹⁾ have been lined up by (Q)SAR studies and/or virtual screening methods. These compounds have potential as candidates of new classes of EcR-targeting insecticides. An in silico screening method was also used to obtain novel bioactive juvenoids.¹³⁰⁾ Previous studies have reported several thousand synthetic compounds with JH-like effects, and chemically divergent compounds act as juvenoids.^131–133) Therefore, a novel class of JHAs, ideally insect-selective juvenoids, may be obtained in the future. Furthermore, RGA can confirm the antagonist activity of chemicals of interest.^{58,78,79,90,122,123,134,135)} In comparison with agonist screening, however, the general cytotoxicity of test chemicals that may repress reporter gene expression in a receptor-independent manner should be carefully considered in antagonist assays.⁸⁰⁾ RGA using a specifically engineered reporter gene construct to positively select for a decrease in receptor activity may allow for the effective screening of antagonists from a large pool of chemical compounds. RGA systems are suitable and advantageous for the high-throughput screening of chemicals that affect the activity of EcR and Met.

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (KAKENHI #19K06092) in 2019–2021 (S. I.-H.), and by an A-STEP Grant from the Japan Science and Technology Agency (JST) in 2014–2015 (T. Y.).

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