Young Chang Sohn

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.

Conserved properties of a urochordate estrogen receptor-related receptor (ERR) with mammalian ERRalpha

Estrogen receptor-related receptors (ERRs) were the first orphan nuclear receptors identified on the basis of their sequence similarity to the estrogen receptors. Although unique ERRs were found in some marine invertebrates, the molecular functions of these receptors are not well understood. In the present study, we identified three transcript variants of the tunicate Halocynthia roretzi ERR (Hr-ERR), varying in their 3 untranslated regions, and putatively encoding a unique receptor deriving from an ancestor protein common to vertebrate ERRalpha/beta/gamma. Maternal mRNA of Hr-ERR was detected throughout the entire egg cytoplasm and early embryos. Zygotic Hr-ERR was predominantly expressed in the heart, and at lower levels in muscle, stomach, gonad and digestive glands. Electrophoretic mobility shift assay demonstrated that Hr-ERR directly binds to the estrogen-response element (ERE) and ERR-response element (ERRE). Gene reporter assays also showed that Hr-ERR activates transcription through ERE and ERRE. Hr-ERR-mediated transactivation was modulated by various cofactors for mammalian ERRs, such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha and small heterodimer partner. In addition, the ERR antagonists 4-hydroxytamoxifen and diethylstilbestrol inhibited the Hr-ERR-mediated transactivation, whereas Hr-ERR activity on ERE was further induced by genistein, an ERRalpha agonist. Taken together, our results show that Hr-ERR is an unduplicated ERR that however, possesses functional properties common to ERRalpha and not to ERRbeta/gamma.

Effects of Shortened Photoperiod on Gonadotropin-Releasing Hormone, Gonadotropin, and Vitellogenin Gene Expression Associated with Ovarian Maturation in Rainbow Trout

Reproductive activities of salmonids are synchronized by changes in photoperiod, which control the endocrine system via the brain-pituitary-gonadal axis. Gonadotropin-releasing hormone (GnRH) in the brain regulates synthesis and release of the pituitary gonadotropins (GTHs; FSH and LH). FSH and LH in turn stimulate the production of sex steroids for oocyte growth and maturation-Inducing steroid hormones for oocyte maturation and ovulation, respectively, in female salmonids. To clarify effects of long-term photoperiod manipulations on the reproductive activity of salmonids from early recrudescence to ovulation, we Investigated the gene expression profiles of GnRH, GTHs, and vitellogenin (VTG), and plasma sex steroids in female rainbow trout (Oncorhynchus mykiss). In addition, the percentages of eyed embryos and hatched alevins were examined together with the number of ovulated eggs to evaluate the effects of photoperiod regimes on egg quality. During late summer, the mRNA levels of GnRHs, GTH&agr;, and LH&bgr;, and the plasma level of a maturational steroid (17&agr;,20&bgr;-dihydroxy-4-pregnen-3-one; 17,20&bgr;-P) were significantly elevated by a gradually shortened photoperiod under constant temperature, in accordance with accelerated sexual maturation. The percentages of eyed embryos and hatched alevins from fish ovulated in August were comparable to those of control fish observed in December. These results clearly indicate that syntheses of GnRHs, LH, VTG, and 17,20&bgr;-P are effectively accelerated by a programmed long-short photoperiod regime in early recrudescent female rainbow trout, without a marked deterioration in egg quality.

9-Cis-retinoic acid induces growth inhibition in retinoid-sensitive breast cancer and sea urchin embryonic cells via retinoid X receptor α and replication factor C3.

There is widespread interest in defining factors and mechanisms that suppress the proliferation of cancer cells. Retinoic acid (RA) is a potent suppressor of mammary cancer and developmental embryonic cell proliferation. However, the molecular mechanisms by which 9-cis-RA signaling induces growth inhibition in RA-sensitive breast cancer and embryonic cells are not apparent. Here, we provide evidence that the inhibitory effect of 9-cis-RA on cell proliferation depends on 9-cis-RA-dependent interaction of retinoid X receptor α (RXRα) with replication factor C3 (RFC3), which is a subunit of the RFC heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp on DNA. An RFC3 ortholog in a sea urchin cDNA library was isolated by using the ligand-binding domain of RXRα as bait in a yeast two-hybrid screening. The interaction of RFC3 with RXRα depends on 9-cis-RA and bexarotene, but not on all-trans-RA or an RA receptor (RAR)-selective ligand. Truncation and mutagenesis experiments demonstrated that the C-terminal LXXLL motifs in both human and sea urchin RFC3 are critical for the interaction with RXRα. The transient interaction between 9-cis-RA-activated RXRα and RFC3 resulted in reconfiguration of the PCNA-RFC complex. Furthermore, we found that knockdown of RXRα or overexpression of RFC3 impairs the ability of 9-cis-RA to inhibit proliferation of MCF-7 breast cancer cells and sea urchin embryogenesis. Our results indicate that 9-cis-RA-activated RXRα suppresses the growth of RA-sensitive breast cancer and embryonic cells through RFC3.

Characterization of steroid receptor coactivator in sea urchin, Strongylocentrotus nudus, and its involvement in embryonic development.

Ligand-bound nuclear receptors (NRs) recruit coactivators such as members of the p160 steroid receptor coactivator (SRC) family and cyclic AMP responsive element binding protein (CREB)-binding protein (CBP) to specific enhancer elements and activate target gene transcription. In the present study, we isolated a novel SRC from the sea urchin Strongylocentrotus nudus (SnSRC) by using the ligand-binding domain of retinoid X receptor as a bait in a yeast two-hybrid screening. The SnSRC and vertebrate SRCs are different in size but share the overall characteristic domains, such as NR interacting domain (NID), CBP-binding and glutamine-rich regions. SnSRC mRNA showed highest expression levels at the 32-cell, 64-cell and pluteus larval stages. Full-length SnSRC (1992 amino acids) interacted with several NRs, including sea urchin estrogen receptor-related receptor (ERR), human and masu salmon estrogen receptors (ERα), mouse ERRγ, rat glucocorticoid receptor α, and rat thyroid receptor β. The SnSRC possesses two functional NIDs, both of which are dependent on their core LxxLL motifs. Furthermore, preferential interacting domains for ERα in the SnSRC are located in the central LxxLL motifs, revealed by the truncation and mutagenesis studies. Strikingly, the SnSRC has a single transcription activation domain, which interacts with CBP, a transcriptional integrator. In addition, transient knockdown of the SnSRC gene in the sea urchin embryo using morpholino antisense RNA induced abnormal phenotypes at gastrulation stage such as the lack of primary invagitation and exogastrulation. These results suggest that the SnSRC is a new member of the SRC family and plays an important role during early embryonic development.

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.

Changes in Prolactin and Growth Hormone Gene Expression of Rainbow Trout Oncorhynchus mykiss Adapted to Seawater

Prolactin (PRL) plays an important role in freshwater (FW) osmoregulation by preventing the loss of ions and the uptake of water in fish. Growth hormone (GH) promotes acclimation to seawater (SW) in several teleosts. We acclimated rainbow trout Oncorhynchus mykiss weighting 68.2±16.6, 138.3±24, and 287.5±42.1 g in separate experiments to SW under slow-acclimation (SSW) or acute-acclimation (ASW) conditions, and then examined the PRL and GH mRNA levels using the real-time quantitative polymerase chain reaction. The PRL mRNA levels in all three experimental groups decreased significantly with both the SSW and ASW treatments, as compared to a control group kept in FW for 30days. The GH mRNA levels increased with ASW in the largest fish, whereas the levels in the other groups did not change significantly. The mortality rate of the largest fish was lower than for the other groups, whereas the growth rate among the three experimental groups did not differ significantly. The growth rate of the ASW group was highest for the smallest fish. These results suggest that SW acclimation is associated with the gene expression levels of PRL and GH in relatively large rainbow trout. In addition, the fish mortality and growth rate on FW-SW transfer seem to be related to body weight, and the SW acclimation method may be applied to the hatcheries industry.