Characterization of a Sea Urchin IQ Motif Containing Protein D as a Coactivator of Nuclear Receptors.

Nuclear receptor (NR) interacting proteins, such as coactivators and corepressors, play a crucial role in specifying the transcriptional activity of the receptor. However, little is known about the functional features of the NR coregulators in marine invertebrates. Using the yeast two-hybrid screening method, a sea urchin oocyte cDNA library was screened for proteins that interact with the ligand-binding domain of human RXRα (hRXRα) as the bait protein in the presence of 9-cis retinoic acid. Here, we describe IQ motif containing protein D (IQCD) as an RXR-interacting coactivator. The open reading frame of Strongylocentrotus nudus IQCD (SnIQCD) cDNA contains 1464 bp encoding a protein of 487 amino acids. SnIQCD and the vertebrate IQCDs contain well-conserved C-terminal IQ motifs and coiled-coil domains. The interactions between RXRα and IQCD were confirmed by an immunoprecipitation assay and a mammal two-hybrid assay. RXRα preferentially interacted with the C-terminal half including IQ motif than the N-terminal half of SnIQCD. The coactivator interacting LXXLL motif in SnIQCD is not directly involved in the interaction with RXRα. SnIQCD overexpression increased the basal RXR transactivation of a RXR-responsive reporter gene. Furthermore, SnIQCD enhanced the transcriptional activity of RXR heterodimeric partners such as RAR, PPAR, and the steroid hormone receptor family members from mammals, teleost fish, and sea urchin. Taken together, we suggest that IQCD orthologs are able to function as transcriptional coactivators cooperating with NRs.

Transcriptional activity and expression of liver X receptor in the ascidian Halocynthia roretzi.

Liver X receptors, LXRs, are ligand-activated transcription factors that belong to the group H nuclear receptor (NR) superfamily. In this study, an LXR (HrLXR) cDNA was cloned from the ascidian Halocynthia roretzi hepatopancreas and characterized to examine the functional conservation of ancestral LXRs in chordates. A phylogenetic analysis of HrLXR showed that it belongs to the tunicate (urochordate) LXR subgroup, which is distinct from vertebrate LXRs. Quantitative real-time PCR analysis revealed that HrLXR mRNA was expressed predominantly in the gills, and highly expressed in unfertilized eggs followed by decrease at later embryonic and larval stages. Unexpectedly, HrLXR was not activated by GW3965, whereas a synthetic ligand for a farnesoid X receptor, GW4064, activated HrLXR. This activation was abolished by the deletion of 51 amino acids from the N-terminus. In a mammalian two-hybrid system, HrLXR interacted with HrRXR in the presence of GW4064 or 9-cis retinoic acid. The injection of GW3965 and GW4064 in vivo increased the ATPbinding cassette sub-family G member 4 and HrLXR mRNA levels in the hepatopancreas and gills. These results suggest that the mRNA expression and transcriptional properties of HrLXR are different from those of vertebrate LXRs, although HrLXR is likely responsive to the related NR ligand, GW4064.

Molecular and expression analysis of the farnesoid X receptor in the urochordate Halocynthia roretzi.

The farnesoid X receptors (FXRs) are the major transcriptional regulators of bile salt synthesis in vertebrates. However, the structural conservation of invertebrate FXRs has only been studied for the major model organisms and studies on additional invertebrate FXRs are clearly required to obtain better resolution of FXR phylogeny and comparative developmental insights in chordates. In the present study, the cDNA encoding the farnesoid X receptor, HrFXR, was cloned from a marine invertebrate Halocynthia roretzi. The open reading frame of HrFXR encoded 688 amino acids including a longer N-terminal region and showed overall sequence identities of 28-41% to vertebrate and Ciona intestinalis FXRs. The N-terminal activation function 1 (AF-1) and hinge domains of HrFXR displayed relatively low identities (<20%), whereas the DNA-binding and ligand-binding domains showed relatively high (>73%) and intermediate (21-50%) identities, respectively. Based on a phylogenetic analysis, HrFXR belonged to a urochordate group, which was placed differently from vertebrate FXRα and FXRß subgroups. Real-time quantitative PCR analysis revealed that the HrFXR mRNA originated maternally and was highly expressed in adult gonads. Additionally, HrFXR mRNA levels in the gills and hepatopancreas showed significantly higher values in animals with soft tunic syndrome compared to those of normal individuals. Furthermore, direct injection of cholic acid significantly increased HrFXR transcript levels in vivo, although an expression vector containing HrFXR cDNA did not show a significant transactivation function in response to a well-known ligand for vertebrate FXR, GW4064, in HepG2 cells. These results suggest that the tunicate FXR has different structural and expressional characteristics compared to those of vertebrate FXRs.

Bi-functional induction of the quinone reductase and cytochrome P450 1A1 by youngiasides via Nrf2-ARE and AhR-XRE pathways.

Many phytochemicals are known to exert cancer chemopreventive activity by eliminating chemical carcinogens or toxic xenobiotics through the action of detoxification enzymes. In this study, we investigated the cancer chemopreventive effects of youngiasides isolated from Crepidiastrum denticulatum. These youngiasides significantly induced quinone reductase (QR) activity in mouse hepatoma Hepa-1c1c7 cells, and showed a relatively high chemoprevention index (CI; divided IC(50) value with CD value). The youngiasides also significantly induced transcriptional activation of QR in Hepa-QR-secreted alkaline phosphatase (SEAP) cells, which is a stable cell line containing the intact promoter region of QR. In order to determine if upregulation of QR by the youngiasides was mediated through a mono-functional or bi-functional mechanism, we examined the nuclear factor-E2 p45-related factor 2(Nrf2)-antioxidant response element (ARE) and aryl hydrocarbon receptor (AhR)-xenobiotic response element (XRE) pathways, which are two major pathways, involved in regulation of Phase I and/or Phase II detoxification enzymes. The youngiasides increased the cytochrome P450 1A1 (CYP1A1) mRNA and protein levels in human colorectal cancer Caco-2 cells and also increased the QR mRNA and protein levels in Caco-2 cells through ARE and XRE activation which resulted from translocation of Nrf2 and AhR into the nucleus. These results suggest that regulation of QR by the youngiasides was due to bi-functional induction through the Nrf2-ARE and AhR-XRE pathways. Thus, these youngiasides as bi-functional inducers of QR have potential as cancer chemopreventive agents.

Production of recombinant Japanese eel gonadotropins by baculovirus in silkworm larvae.

Recombinant follicle-stimulating hormone (reFSH) and luteinizing hormone (reLH) of the Japanese eel Anguilla japonica were produced by baculovirus in silkworm Bombyx mori larvae. cDNAs encoding Japanese eel gonadotropin subunits (i.e., FSH beta, LH beta, and common alpha) were introduced into the baculovirus, which was infected into silkworm larvae after propagation of the recombinant virus in B. mori culture cells. A 100ml solution of pooled hemolymph from silkworm larvae containing reFSH or reLH were obtained from approximately 250 infected larvae. Ten milliliters of hemolymph were applied to Ni-affinity choromatography, and 5.6 and 3.5mg of partially purified reFSH and reLH were obtained, respectively. Using Western blot analysis concentrations of reFSH and reLH in the original hemolymph was estimated to be 2.2 and 1.1mg/ml, respectively. Biological activities of reFSH and reLH were assessed in vitro and in vivo. Purified reFSH and reLH induced eel oocyte maturation in vitro, and administration of hemolymph containing reFSH or reLH induced spermatogenesis in vivo in sexually immature Japanese eel. The present study indicates that a baculovirus-silkworm system could produce large amounts of biologically active recombinant fish gonadotropins for use in investigations in reproductive endocrinology and/or aquaculture of fish.

Four flavonoids from Echinosophora koreensis and their effects on alcohol metabolizing enzymes.

A new prenylated dihydroflavonol, 3-hydroxy-kenusanone B 1, as well as three other known isoflavanones, sophoronol 2, sophoraisoflavanone A 3 and kenusanone H 4, were isolated from the rhizomes of Echinosophora koreensis. The structures of these compounds were elucidated using spectroscopic analyses that included extensive 2D NMR, optical rotation spectrometry and mass spectrometry. All four flavonoids enhanced the activities of alcohol metabolizing enzymes such as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) at micromolar concentrations. Sophoronol 2 showed a nine-fold increased activation of alcohol dehydrogenase and aldehyde dehydrogenase than a negative control group at concentrations of 100 microg/mL and 50 microg/mL, respectively. This study suggests that prenylated flavonoids have the potential to prevent 'hangovers' after alcohol intake.

Tectoridin, a poor ligand of estrogen receptor alpha, exerts its estrogenic effects via an ERK-dependent pathway.

Phytoestrogens are the natural compounds isolated from plants, which are structurally similar to animal estrogen, 17beta-estradiol. Tectoridin, a major isoflavone isolated from the rhizome of Belamcanda chinensis. Tectoridin is known as a phytoestrogen, however, the molecular mechanisms underlying its estrogenic effect are remained unclear. In this study we investigated the estrogenic signaling triggered by tectoridin as compared to a famous phytoestrogen, genistein in MCF-7 human breast cancer cells. Tectoridin scarcely binds to ER alpha as compared to 17beta-estradiol and genistein. Despite poor binding to ER alpha, tectoridin induced potent estrogenic effects, namely recovery of the population of cells in the S-phase after serum starvation, transactivation of the estrogen response element, and induction of MCF-7 cell proliferation. The tectoridin-induced estrogenic effect was severely abrogated by treatment with U0126, a specific MEK1/2 inhibitor. Tectoridin promoted phosphorylation of ERK1/2, but did not affect phosphorylation of ER alpha at Ser(118). It also increased cellular accumulation of cAMP, a hallmark of GPR30-mediated estrogen signaling. These data imply that tectoridin exerts its estrogenic effect mainly via the GPR30 and ERK-mediated rapid nongenomic estrogen signaling pathway. This property of tectoridin sets it aside from genistein where it exerts the estrogenic effects via both an ER-dependent genomic pathway and a GPR30-dependent nongenomic pathway.

cDNA cloning of Japanese flounder stanniocalcin 2 and its mRNA expression in a variety of tissues.

Stanniocalcin 1 (STC1) is a glycoprotein hormone important in the maintenance of calcium and phosphate homeostasis in both fishes and mammals. Although two related STC genes, STC1 and STC2, were found to be expressed in multiple tissues as paracrine regulators in mammals, spatial expression pattern of stc2 mRNA has not been elucidated in fishes in contrast to that of clearly described stc1. In the present study, we have cloned and characterized a full-length stc2 cDNA from Japanese flounder (Paralichyhus olivaceus) ovary and analyzed expression pattern of stc2 in both sexes. The flounder stc2 cDNA (1501 nucleotides) encoded a putative prehormone of 286 amino acids (aa) with a signal peptide of 20 aa and a mature protein of 266 aa. The deduced aa sequence of flounder stc2 showed high sequence identity with those of pufferfish, zebrafish, and human (57.7-89.0%), whereas it showed less identity with that of flounder stc1 (24.3%). RT-PCR analysis revealed that the flounder stc2 gene is expressed in all examined tissues including the pituitary, brain, heart, kidney, gills, stomach, spleen, skin, dorsal fin, skeletal muscle, liver, corpuscles of Stannius, intestine, ovary and testis. Our data indicate that fish stc2 gene, like stc1, is expressed in a wide variety of tissues.