To investigate the possibility that Hr38 may function in an ecdysteroid-mediated transcriptional pathway, a screening assay was developed in which Hr38 was heterodimerized with ligand-activated RXR. The feasibility of this approach was based on the finding that RXR can substitute for Usp as a productive heterodimeric partner for EcR. A distinct advantage of substituting RXR for Usp is that although ligands for Usp are not known, several potent RXR ligands (i.e., rexinoids) have been characterized. It was reasoned that assaying Hr38 activity in the presence of rexinoid-activated RXR may be important because previous work has shown that some RXR heterodimers require sensitization with ligand for one receptor before the partner receptor can become ligand responsive (Baker, 2003 and references therein).
To screen for a Hr38 ecdysteroid response, transient cotransfections were performed in the Drosophila SL2 cell line using chimeric GAL4-receptor proteins (hormone receptor proteins with an inserted GAL4 transcriptional activation domain) and a GAL4-responsive luciferase reporter gene (Baker, 2000). In this assay, GAL4-EcR and GAL4-Hr38 were screened in the presence of RXR, the synthetic rexinoid LG268, and the potent plant ecdysteroid muristerone A. Similar to previous work (Baker, 2000), the GAL4-EcR/RXR heterodimer responds to 100 nM muristerone A as expected but is not activated significantly by the RXR-specific ligand LG268 alone. Addition of both ligands results in only a modest increase in EcR/RXR activity. In contrast, GAL4-Hr38/RXR, which is known to have a potent basal activity (Baker, 2000), is not induced by the addition of ecdysteroid alone but, instead, exhibits a strong, dose-dependent response to LG268. Rexinoid activation of the Hr38/RXR heterodimer is consistent with the rexinoid response seen with other NGFI-B family members when paired with RXR (Perlmann, 1995). Surprisingly, however, there is a significant, 3- to 4-fold response to muristerone A when it is added together with LG268. A similar response is obtained with the endogenous insect ecdysteroid 20E. Both rexinoid and ecdysteroid responses are abolished when a GAL4-Hr38 construct was utilized that lacks the ligand-dependent activation function-2 (AF-2) domain. Identical results are obtained (i.e., loss of rexinoid and ecdysteroid response) when the AF-2 domain of RXR is also deleted. These data suggest that the Hr38 heterodimeric complex is responsive to ecdysteroid but, like other RXR heterodimers, it requires transactivation of both receptor partners for full agonist activity (Baker, 2003).
The results above reveal the possible existence of two ecdysteroid signaling pathways, one mediated by EcR and the other by Hr38. To begin to delineate the specificity of the Hr38 pathway and show that it functions independently of the EcR pathway, double-stranded RNA (dsRNA) directed against the coding region of the EcR ligand binding domain was used to reduce the expression of endogenous EcR in the SL2 cell assay. Treatment of SL2 cells with increasing amounts of EcR dsRNA completely eliminates muristerone-A-dependent transcription when tested using endogenous EcR/Usp heterodimers on an hsp27-EcRE reporter gene. This RNAi-mediated repression of the EcR-dependent response also completely blocks the activity of exogenously transfected EcR and GAL4-EcR. In contrast, under the same experimental conditions, where the EcR response is abolished, the GAL4-Hr38/RXR heterodimer is fully responsive to ecdysteroid and LG268. These results demonstrate that the Hr38 response to ecdysteroids is independent of EcR (Baker, 2003).
To define the spectrum of potential Hr38 agonists and further delineate the differential ecdysteroidal response of Hr38 and EcR, a panel of naturally occurring Drosophila ecdysteroids, phytoecdysteroids, synthetic ecdysteroids, and a synthetic juvenile hormone (methoprene acid) were tested for activity using the reporter gene assay described above. SL2 cells were transfected with either GAL4-EcR or GAL4-Hr38 plus RXR and tested for agonist activity in the presence of 10 nM LG268. Consistent with previous results (Baker, 2000), GAL4-EcR responds selectively to the endogenous ecdysteroids 20E and makisterone A and the plant ecdysteroids muristerone A, ponasterone A, and cyasterone. In marked contrast, the GAL4-Hr38/RXR response is promiscuous for several different ecdysteroids when LG268 is included as a coagonist. In addition to the compounds that activated EcR, at least six other ecdysteroids (α-ecdysone, 3-epi-20E, 2-deoxy-20E, 3-dehydromakisterone A, 3-epimakisterone A, and 3-dehydro-20-deoxyponsterone) also exhibit significant Hr38-dependent activity. Dose-response profiles demonstrated that all of these compounds are more potent agonists for Hr38 than for EcR. In fact, 20E, which is believed to be the endogenous hormone agonist for EcR, exhibits a 100-fold greater potency for Hr38-dependent transcription. These data suggest that the Hr38/RXR heterodimer is a potent sensor of a distinct class of physiologically relevant ecdysteroids (Baker, 2003).
An unusual characteristic of the Hr38/RXR heterodimer is that it required transactivation of both receptors to elicit an ecdysteroid response. In particular, the Hr38/RXR heterodimer fails to respond to ecdysteroid in the absence of ligand-activated RXR. Interestingly, Hr38 also fails to respond to ecdysteroid when Usp, the physiologic partner of Hr38, is used instead of RXR. These results raise the intriguing possibility that Usp, like RXR, must also be transactivated (e.g., by ligand) in order to enable the ecdysteroid response. Attempts were made to address this question by using VP16-Usp, a constitutively active form of Usp that circumvents the requirement for Usp ligand by fusing the strong transcriptional activation domain of the herpes simplex viral protein-16 (VP16) to Usp. As expected, in the absence of agonist, the GAL4-Hr38/VP16-Usp heterodimer shows a high constitutive level of basal activity that effectively mimics that of ligand-activated USP. Importantly, the addition of muristerone A to the GAL4-Hr38/VP16-Usp heterodimer elicits a significant increase in reporter gene activity, analogous to the effect seen with LG268-activated GAL4-Hr38/RXR. Similar to the results obtained above with ligand-activated RXR, the GAL4-Hr38/VP16-Usp heterodimer responds to a wide variety of ecdysteroids at comparably low concentrations. These results support the idea that Hr38 mediates a distinct heterodimer-dependent ecdysteroid signaling pathway (Baker, 2003).
To test the prediction that Hr38 is activated by ecdysteroids in Drosophila, transgenic flies were created that carry a heat-inducible hs-GAL4-Hr38 transgene in combination with a GAL4-dependent UAS-nlacZ reporter gene. Since target genes for Hr38 in the fly are unknown, this model permits the assaying of Hr38 transactivation directly in fly tissues. This transgenic fly model has been used to follow the ecdysteroid-dependent activation patterns of the EcR and Usp in Drosophila and has provided data consistent with the known biochemical and genetic activities of the full-length receptors in vivo (Kozlova, 2002). This strategy has also been employed to track ligand-dependent activation of the RAR and RXR ligand binding domains in the mouse central nervous system. To determine if GAL4-Hr38 is activated by ecdysteroids, third instar larval organs from this transgenic line were dissected at ~8 hr before puparium formation and cultured in the presence of either α-ecdysone or 3-epi-20E, two ecdysteroids that activate Hr38, but not EcR in SL2 cells. In the presence of 1 μM α-ecdysone, significant activation above background is seen for GAL4-Hr38. In contrast, these ecdysteroids have no effect on GAL4-EcR, although this same transgenic line shows robust activation by 20E (Kozlova, 2002). GAL4-Hr38 is also activated by 3-epi-20E in both the epidermis and fat body, consistent with the ability of this agonist to selectively activate Hr38 in SL2 cells. These organs contain significant amounts of endogenous Usp, consistent with the interpretation that GAL4-Hr38 ecdysteroid activation is dependent on heterodimerization with a Usp partner. Although similar results were seen in several independent experiments, not all hs-GAL4-Hr38; UAS-nlacZ animals display robust activation, indicating that a specific stage might be competent to respond to the hormone. In agreement with this idea, a complex and dynamic pattern of GAL4-Hr38 activation can be seen in untreated animals. This observation is consistent with the notion that the endogenous Hr38 response may be spatially and temporally regulated by the presence of a number of factors, including ecdysteroids, Hr38/Usp-specific coactivators, and potentially a Usp ligand (Baker, 2003).
A rapid PCR based cloning and screening strategy was used to identify new members of the nuclear hormone receptor superfamily that are expressed during the onset of Drosophila metamorphosis. Using this approach, three Drosophila genes, designated Hr38, DHR78, and DHR96, have been isolated. All three genes are expressed throughout third-instar larval and prepupal development. Hr38 is the Drosophila homolog of NGFI-B and binds specifically to an NGFI-B response element. The two adenosines upstream from the AGGTCA half-site are critical for Hr38 binding, as has been shown for NGF1-B. Furthermore, the ability of Hr38 to bind to a single AGGTCA half-site suggests that, like NGF1-B, this protein can bind DNA as a monomer (Fisk, 1995)
In Drosophila the response to the hormone ecdysone is mediated in part by Ultraspiracle (Usp) and ecdysone receptor (EcR), both of which are members of the nuclear receptor superfamily. Heterodimers of these proteins bind to ecdysone response elements (EcREs) and ecdysone to modulate transcription. Drosophila hormone receptor 38 (Hr38) and Bombyx hormone receptor 38 (BHR38) are two insect homologs of rat nerve growth factor-induced protein B (NGFI-B). Although members of the NGFI-B family are thought to function exclusively as monomers, Hr38 and BHR38 have been shown, in fact, to interact strongly with Usp and this interaction is evolutionarily conserved. Hr38 can compete in vitro against EcR for dimerization with Usp and consequently disrupt EcR-Usp binding to an EcRE. Moreover, transfection experiments in Schneider cells show that Hr38 can affect ecdysone-dependent transcription. This suggests that Hr38 plays a role in the ecdysone response and that more generally NGFI-B type receptors may be able to function as heterodimers with retinoid X receptor type receptors in regulating transcription (Sutherland, 1995).
Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, a `ligand sensor' system was used to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue- and stage-specific patterns of activation. These responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. Dynamic changes in activation were seen that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control (Palanker, 2006).
Nine GAL4-LBD ligand sensor lines described in this study show tissue-specific patterns of activity during development: EcR, USP, ERR, FTZ-F1, HNF4, E78, DHR3, DHR38 and DHR96. These transgenic lines will serve as valuable tools for the genetic and molecular dissection of the receptors they represent, the pathways they regulate and the upstream factors and co-factors that modulate their activity. Specifically, the data reported here show that these lines can be used to: (1) indicate tissues and stages in which the corresponding NRs are likely to function; (2) indicate where endogenous ligands and co-factors are likely to be found; (3) suggest NR biological functions; (4) suggest possible NR-NR interactions, cascades and target genes; (5) evaluate putative co-factors and ligands; (6) screen chemical compound libraries for new agonists and antagonists; and (7) screen genetically for new pathway components. The results of these studies will also provide important insights into the ligands, co-factors and functions of their vertebrate NR homologues (Palanker, 2006).
Examination of the nine active ligand sensor lines provided a number of insights into possible relationships between their corresponding NRs. For example, although each of these ligand sensors displays unique temporal and spatial patterns of activity, activation in specific tissues and stages is common to many. These common sites of LBD activity may indicate shared functions, hierarchical or physical interactions, or related ligands. Examples of tissues that represent hotspots for GAL4-LBD activation include the amnioserosa, yolk, midgut and fat body (Palanker, 2006).
Each of these tissues, and the stages at which they score positively, correlates well with the presence of putative ligands. The yolk, for example, is believed to act as a storage site for maternally provided ecdysteroids during embryogenesis. Work with other insects has shown that these ecdysteroids are conjugated in an inactive form to vitellin proteins via phosphate bridges. Around mid-embryogenesis, these yolk proteins and phosphate bonds are cleaved, thereby releasing what are presumed to be the earliest biologically active ecdysteroids in the embryo. Interestingly though, GAL4-EcR activation in the amnioserosa depends on the disembodied (dib) gene, which encodes a cytochrome P450 enzyme required in the penultimate step of Ecdysone (E) biosynthesis, suggesting that the final steps in the linear E biosynthetic pathway are required for EcR function in this tissue and contradicting the prediction that this activity would be dependent on maternal ecdysteroids and independent of the zygotic biosynthetic machinery. The mechanisms by which dib exerts this essential role in providing an EcR ligand, however, remain to be determined (Palanker, 2006).
The response of the EcR and USP ligand sensors in the adjacent amnioserosa tissue shows that active ecdysteroids are not present until the hormone reaches the amnioserosa. A recent study of yolk-amnioserosa interactions has revealed dynamic transient projections that emanate from one tissue and contact the other, suggesting that there may be functional interactions between these two cell types. It is possible that these projections mediate the transfer of lipophilic ligand precursors from the yolk to the amnioserosa. This transfer, in turn, could determine the proper timing of EcR activation in the amnioserosa, thus triggering the major morphogenetic movements that establish the body plan of the first instar larva (Palanker, 2006).
Studies of the DHR38 receptor have demonstrated that it can be activated by a distinct set of ecdysteroids from those that activate EcR, through a novel mechanism that does not involve direct ligand binding. The activation of GAL4-DHR38 that was observed in the embryonic amnioserosa is consistent with this model of DHR38 regulation. First, exogenous 20E can only weakly activate GAL4-DHR38, relative to the strong ectopic activation seen with 20E on the EcR ligand sensor. This correlates with the weak ability of 20E to activate DHR38 in cell culture transfection assays relative to the strong 20E activation of EcR. Second, the DHR38 ligand sensor is activated in the amnioserosa earlier than the EcR construct, suggesting that it is responding to a different signal. It is possible that this signal is an ecdysteroid precursor that can act on DHR38 but not EcR - paralleling the ability of DHR38 to be activated by E, the precursor to 20E, which activates EcR. This putative ecdysteroid must be produced in a manner independent of the conventional ecdysteroid biosynthetic pathway, however, since a zygotic dib mutation has no effect on GAL4-DHR38 activation in the amnioserosa. Rather, this early activation may be due to maternal ecdysteroids that are conjugated and inactive in the yolk and transferred to the amnioserosa. These studies highlight the value of combining mutations in hormone biosynthesis with ligand sensor activation as a powerful means of dissecting hormone signaling pathways. Further studies of DHR38 function and regulation in embryos could help clarify the potential significance of this distinct activation response (Palanker, 2006).
DHR3, DHR38 and HNF4 ligand sensors appear to respond to metabolic signals Interestingly, the midgut continues to be a hotspot for ligand sensor activity long after it has engulfed the yolk during embryogenesis. This seems logical, as the midgut is responsible for most lipid absorption and release, and many vertebrate NRs are involved in fatty acid, cholesterol and sterol metabolism and homeostasis. The observed restriction of ligand sensor activity to a narrow group of cells located at the base of the gastric caeca is of particular interest. This is the site where nutrients in a feeding larva are absorbed into the circulatory system. The activation of DHR3, DHR38 and HNF4 ligand sensors in this region of the gastric caeca suggests that these receptors are activated by one or more small nutrient ligands. Moreover, this suggests that the corresponding receptors may exert crucial metabolic functions by acting as nutrient sensors (Palanker, 2006).
Further evidence of metabolic functions for DHR3, DHR38 and HNF4 arises from their ligand sensor activation patterns in the embryonic yolk and larval fat body. The yolk is the main nutrient source for the developing embryo and represents an abundant source of lipids, correlating with specific activation of DHR3, DHR38 and HNF4 ligand sensors in this cell type during embryogenesis. Upon hatching into a larva, the fat body acts as the main metabolic organ of the animal, functionally equivalent to the mammalian liver. Upon absorption by the gastric caeca, nutrients travel through the circulatory system and are absorbed by the fat body, where they are broken down and stored as triglycerides, glycogen and trehalose. Once again, the efficient activation of the DHR3, DHR38 and HNF4 ligand sensors in the fat body of metabolically active third instar larvae, and lack of sensor activity in non-feeding prepupae, supports the model that the corresponding NRs operate as metabolic sensors. This proposed function is consistent with the roles of their vertebrate orthologs. Mammalian ROR, the ortholog of DHR3, binds cholesterol and plays a crucial role in lipid homeostasis. Similarly, mammalian HNF4 can bind C14-18 fatty acids, is required for proper hepatic lipid metabolic gene regulation and lipid homeostasis, and is associated with human Maturity-Onset Diabetes of the Young (MODY1). The studies described here suggest that DHR3 and HNF4 may perform similar metabolic functions in flies, defining a new genetic model system for characterizing these key NRs (Palanker, 2006).
Several vertebrate NRs play a central role in xenobiotic responses by directly binding toxic compounds and inducing the expression of key detoxification enzymes such as cytochrome P450s and glutathione transferases. Ligand sensor activation observed in the gut, epidermis, tracheae or fat body could represent xenobiotic responses insofar as toxic compounds could enter the organism through any of these tissues. Directed screens that test xenobiotic compounds for their ability to activate Drosophila NR ligand sensors will provide a means of identifying potential xenobiotic receptors. Understanding these response systems, in turn, could facilitate the production of insect resistant crops and the development of more effective pesticides (Palanker, 2006).
Like its vertebrate orthologs SXR/PXR and CAR, DHR96 has been recently shown to act in insect xenobiotic responses, providing resistance to the sedative effect of phenobarbital and lethality caused by chronic exposure to DDT (King-Jones et al., 2006). DHR96 is also required for the proper transcriptional response of a subset of phenobarbital-regulated genes. DHR96 can be activated by the CAR-selective agonist CITCO, suggesting that it may be regulated in a manner similar to that of the vertebrate xenobiotic receptors. It is also interesting to note that angelicin was found to activate the USP ligand sensor fusion. Angelicin is an angular furanocoumarin that has the furan ring attached at the 7,8 position of the benz-2-pyrone nucleus. Detailed studies have shown that insects have adapted to the presence of furanocoumarins in their host plants by expressing specific cytochrome P450 enzymes that detoxify these compounds. In the black swallowtail butterfly (Papilio polyxenes), furanocoumarins induce the transcription of P450 genes through an unknown regulatory pathway, thereby aiding in xenobiotic detoxification. The observation that angelicin, and not the linear furanocoumarins 8-methoxypsoralen (xanthotoxin) or 5-methoxypsoralen (bergapten), can activate GAL4-USP suggests that NRs may mediate this detoxification response and may be capable of distinguishing between the linear and angular chemical forms. It is possible that USP may mediate this effect on its own or, more likely, as a heterodimer partner with another NR. Similarly, the activation of GAL4-USP by fenoxycarb may represent a xenobiotic response. This activation, however, is weaker and more variable than the activation observed with angelicin. Identifying other factors that mediate xenobiotic responses in Drosophila would provide a new basis for dissecting the control of detoxification pathways in higher organisms (Palanker, 2006).
GAL4-ERR displays a remarkable switch in activity during mid-embryogenesis, from strong activation in the myoblasts to specific and strong activation in the CNS. The ERR ligand sensor also shows widespread transient activation in the mid-third instar, a time when larval ERR gene expression begins, together with a global switch in gene expression that prepares the animal for entry into metamorphosis 1 day later. This so-called mid-third instar transition includes upregulation of EcR, providing sufficient receptor to transduce the high titer late larval 20E hormone pulse, upregulation of the Broad-Complex, which is required for entry into metamorphosis, and induction of the genes that encode a polypeptide glue used to immobilize the puparium for metamorphosis. The signal and receptor that mediate this global reprogramming of gene expression remain undefined. The widespread activation of GAL4-ERR at this stage raises the interesting possibility that it may play a role in this transition. Moreover, given that the only ligand sensors to display widespread transient activation are EcR and USP, in response to 20E, it is possible that this response reflects a systemic mid-third instar pulse of a ERR hormone. Vertebrate members of the ERR family can bind the synthetic estrogen diethylstilbestrol and the selective ER modulator tamoxifen, as well as its metabolite, 4-hydroxytamoxifen, suppressing their otherwise constitutive activity in cell culture. This is notably different from the highly restricted patterns of ERR ligand sensor activity that was detected in Drosophila, which suggests that it does not function as a constitutive activator in vivo. Rather, it is envisioned that the patterns of ERR activation are precisely modulated by protein co-factors and/or one or more ligands to direct the dynamic shifts in activation that are detect during embryogenesis and third instar larval development. Functional studies of the Drosophila homolog of the ERR receptor family may provide a basis for understanding these dynamic shifts in LBD activation, as well as revealing a natural ligand for this NR (Palanker, 2006).
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