The complete nuclear estrogen receptor family in the rainbow trout: discovery of the novel ERalpha2 and both ERbeta isoforms

Estrogen hormones interact with cellular ERs to exert their biological effects in vertebrate animals. Similar to other animals, fishes have two distinct ER subtypes, ERalpha (NR3A1) and ERbeta (NR3A2). The ERbeta subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ERalpha have not been demonstrated in any fish species. In the rainbow trout (Oncorhynchus mykiss), the only ER described is an isoform of the ERalpha subtype (i.e. ERalpha1, NR3A1a). The purpose of this study was to determine whether the gene for the other ERalpha isoform, ERalpha2 (i.e., NR3A1b), exists in the rainbow trout. A RT-PCR and cloning strategy, followed by screening a rainbow trout BAC library yielded a unique DNA sequence coding for 558 amino acids. The deduced amino acid sequence had a 75.4% overall similarity to ERalpha1. Both the rainbow trout ERbeta subtypes, ERbeta1 [NR3A2a] and ERbeta2, [NR3A2b] which were previously unknown in this species, were also sequenced as part of this study, and the amino acid sequences were found to be very different from the ERalphas (approximately 40% similarity). ERbeta1 and ERbeta2 had 594 and 604 amino acids, respectively, and had 57.6% sequence similarity when compared to one another. This information provides what we expect to be the first complete nuclear ER gene family in a fish. A comprehensive phylogenetic analysis with all other known fish ER gene sequences was undertaken to understand the evolution of fish ERs. The results show a single ERalpha subtype clade, with the closest relative to rainbow trout ERalpha2 being rainbow trout ERalpha1, suggesting a recent, unique duplication event to create these two isoforms. For the ERbeta subtype there are two distinct subclades, one represented by the ERbeta1 isoform and the other by the ERbeta2 isoform. The rainbow trout ERbeta1 and ERbeta2 are not closely associated with each other, but instead fall into their respective ERbeta subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms (ERalpha1, ERalpha2, ERbeta1, and ERbeta2) in stomach, spleen, heart, brain, pituitary, muscle, anterior kidney, posterior kidney, liver, gill, testis and ovary samples from rainbow trout. The mRNAs for each of the four ERs were detected in every tissue examined. The liver tended to have the highest ER mRNA levels along with the testes, while the lowest levels were generally found in the stomach or heart. The nuclear ERs have a significant and ubiquitous distribution in the rainbow trout providing the potential for complex interactions that involve the functioning of many organ systems.     

Linkage arrangement of Na,K-ATPase genes in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss)

As part of our efforts to characterize Na,K-ATPase isoforms in salmonid fish, we investigated the linkage arrangement of genes coding for the alpha and beta-subunits of the enzyme complex in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss). Genetic markers were developed from four of five previously characterized alpha-subunit isoforms (alpha1b, alpha1c, alpha2 and alpha3) and four expressed sequence tags derived from yet undescribed beta-subunit isoforms (beta1a, beta1b, beta3a and beta3b). Sex-specific linkage analysis of polymorphic loci in a reference meiotic panel revealed that Na,K-ATPase genes are generally dispersed throughout the rainbow trout genome. A notable exception was the colocalization of two alpha-subunit genes and one beta-subunit gene on linkage group RT-12, which may thus share a conserved orthologous segment with linkage group 1 in zebrafish (Danio rerio). Consistent with previously reported homeologous relationships among the chromosomes of the rainbow trout, primers designed from the alpha3-isoform detected a pair of duplicated genes on linkage groups RT-27 and RT-31. Similarly, the evolutionary conservation of homeologous regions on linkage groups RT-12 and RT-16 was further supported by the map localization of gene duplicates for the beta1b isoform. The detection of homeologs within each gene family also raises the possibility that novel isoforms may be discovered as functional duplicates.     

Computational Study of Evolutionary Selection Pressure on Rainbow Trout Estrogen Receptors

Molecular dynamics simulations were used to determine the binding affinities between the hormone 17-estradiol (E2) and different estrogen receptor (ER) isoforms in the rainbow trout, Oncorhynchus mykiss. Previous phylogenetic analysis indicates that a whole genome duplication prior to the divergence of ray-finned fish led to two distinct ER isoforms, ER and ER, and the recent whole genome duplication in the ancestral salmonid created two ER isoforms, ER and ER. The objective of our computational studies is to provide insight into the underlying evolutionary pressures on these isoforms. For the ER subtype our results show that E2 binds preferentially to ER over ER. Tests of lineage specific N/S ratios indicate that the ligand binding domain of the ER gene is evolving under relaxed selection relative to all other ER genes. Comparison with the highly conserved DNA binding domain suggests that ER may be undergoing neofunctionalization possibly by binding to another ligand. By contrast, both ER and ER bind similarly to E2 and the best fitting model of selection indicates that the ligand binding domain of all ER genes are evolving under the same level of purifying selection, comparable to ER.     

ORP3 splice variants and their expression in human tissues and hematopoietic cells

ORP3 is a member of the newly described family of oxysterol-binding protein (OSBP)-related proteins (ORPs). We previously demonstrated that this gene is highly expressed in CD34(+) hematopoietic progenitor cells, and deduced that the "full-length" ORP3 gene comprises 23 exons and encodes a predicted protein of 887 amino acids with a C-terminal OSBP domain and an N-terminal pleckstrin homology domain. To further characterize the gene, we cloned ORP3 cDNA from PCR products and identified multiple splice variants. A total of eight isoforms were demonstrated with alternative splicing of exons 9, 12, and 15. Isoforms with an extension to exon 15 truncate the OSBP domain of the predicted protein sequence. In human tissues there was specific isoform distribution, with most tissues expressing varied levels of isoforms with the complete OSBP domain; while only whole brain, kidney, spleen, thymus, and thyroid expressed high levels of the isoforms associated with the truncated OSBP domain. Interestingly, the expression in cerebellum, heart, and liver of most isoforms was negligible. These data suggest that differential mRNA splicing may have resulted in functionally distinct forms of the ORP3 gene.     

A putative beta2-adrenoceptor from the rainbow trout (Oncorhynuchus mykiss). Molecular characterization and pharmacology.

Extensive molecular characterization of mammalian beta-adrenoceptors has revealed complex modes of regulation and interaction. Relatively little attention, however, has focused on adrenoceptors from early branching vertebrates such as fish. Using an RT-PCR approach we have cloned a rainbow trout beta2-adrenoceptor gene that codes for a 409-amino-acid protein with the same seven transmembrane domain structure as its mammalian counterparts. This rainbow trout beta2-adrenoceptor shares a high degree of amino-acid sequence conservation with other vertebrate beta2-adrenoceptors. The conclusion that this sequence is a rainbow trout beta2-adrenoceptor is further supported by phylogenetic analysis of vertebrate beta-adrenoceptor sequences and competitive pharmacological binding data. RNase protection assays demonstrate that the rainbow trout beta2-adrenoceptor gene is highly expressed in the liver and red and white muscle, with lower levels of expression in the gills, heart, kidney and spleen of the rainbow trout. The lack of regulatory phosphorylation sites within the G-protein-binding domain of the rainbow trout beta2-adrenoceptor sequence suggests that the in vivo control of trout beta2-adrenoceptor signaling differs substantially from that of mammals.     

Expression and immunochemical analysis of rat and human fibroblast growth factor receptor (flg) isoforms.

Potentially 96 splice variants among four genes that code for the human heparin-binding fibroblast growth factor receptor family complicate study of structure, metabolism, and function of single isoforms in mammalian cells. As an alternative, we expressed structural subdomains and isoforms of the flg receptor gene in bacteria and baculoviral-infected insect cells. We developed and characterized a panel of 16 isoform and domain-specific polyclonal and monoclonal antibodies. The panel of antibodies was used to distinguish mature glycosylated ligand-binding and kinase-active and -inactive recombinant isoforms in baculoviral insect cells and transfected mammalian cells and natural isoforms in rat prostate and human liver cells. The results revealed a cell type-specific expression of the flg gene and isoforms that result from combinations of splice variations. Reactive epitopes of monoclonal antibodies against both the three (alpha) and two (beta) immunoglobulin-like disulfide loop extracellular domain isoforms were mapped by cross-reactivity with synthetic polypeptide sequences and deletion mutants expressed in bacteria. The native alpha and beta receptor isoforms differed in display of shared epitopes and suggested that the NH2-terminal Loop I and COOH-terminal Loops II and III of the alpha isoform are interactive. Although the common Loops II and III appear qualitatively sufficient for ligand binding, the results suggest that tertiary relationships among loops in the three and two loop isoforms are distinct and, therefore, the two isoforms may have distinct activities. Spatial models for arrangement of immunoglobulin-like loops in the extracellular domain of the two isoforms are presented.     

Estrogen regulation of human osteoblast function is determined by the stage of differentiation and the estrogen receptor isoform

Although osteoblasts have been shown to respond to estrogens and express both isoforms of the estrogen receptor (ER alpha and ER beta), the role each isoform plays in osteoblast cell function and differentiation is unknown. The two ER isoforms are known to differentially regulate estrogen-inducible promoter-reporter gene constructs, but their individual effects on endogenous gene expression in osteoblasts have not been reported. We compared the effects of 17 beta-estradiol (E) and tamoxifen (TAM) on gene expression and matrix formation during the differentiation of human osteoblast cell lines stably expressing either ER alpha (hFOB/ER alpha 9) or ER beta (hFOB/ER beta 6). Expression of the appropriate ER isoform in these cells was confirmed by northern and western blotting and the responses to E in the hFOB/ER beta 6 line were abolished by an ER beta-specific inhibitor. The data demonstrate that (1) in both the hFOB/ER cell lines, certain responses to E or TAM (including alkaline phosphatase, IL-6 and IL-11 production) are more pronounced at the late mineralization stage of differentiation compared to earlier stages, (2) E exerted a greater regulation of bone nodule formation and matrix protein/cytokine production in the ER alpha cells than in ER beta cells, and (3) the regulated expression of select genes differed between the ER alpha and ER beta cells. TAM had no effect on nodule formation in either cell line and was a less potent regulator of gene/protein expression than E. Thus, both the ER isoform and the stage of differentiation appear to influence the response of osteoblast cells to E and TAM.     

Two inducible, functional cyclooxygenase-2 genes are present in the rainbow trout genome

The cyclooxygenases (Cox) catalyze the initial reactions in prostanoid biosynthesis, and produce the common prostanoids precursor, PGH(2). Mammalian species have two Cox isoforms; constitutively expressed cyclooxygenase-1 (Cox-1) and inducible cyclooxygenase-2 (Cox-2). Database searches suggest three Cox genes are present in many fish species. In this study, we cloned and characterized a second Cox-2 cDNA, Cox-2b, from the rainbow trout. Rainbow trout Cox-2b protein contains all the functionally important conserved amino acids for Cox enzyme activity. Moreover, the Cox-2b message contains AU-rich elements (AREs) in the 3' untranslated region (3'UTR) characteristic of inducible Cox-2 mRNAs. We took advantage of the existence of a rainbow trout cell line to demonstrate that expression from both the originally reported Cox-2 (Cox-2a) and Cox-2b genes is inducible. However, differential induction responses to alternative inducers are observed for rainbow trout Cox-2a and Cox-2b. Both Cox-2a and Cox-2b proteins expressed in COS cells are enzymatically active. Thus the rainbow trout has two functional, inducible Cox-2 genes. The zebrafish also contains two Cox-2 genes. However, genome structure analysis suggests diversion of the Cox-2a gene between zebrafish and rainbow trout.     

Comparative expression analysis of two paralogous Hsp70s in rainbow trout cells exposed to heat stress

Heat-shock protein 70 (Hsp70) is the major stress-inducible protein in vertebrates and highly conserved throughout evolution. To accurately investigate the mRNA expression profiles of multiple Hsp70s in rainbow trout Oncorhynchus mykiss, we isolated full-length cDNA clones encoding Hsp70 from the fish and investigated their mRNA expression profiles during heat stress. Consequently, two Hsp70s, Hsp70a and Hsp70b, were identified and found to have 98.1% identity in their deduced amino acid sequences. Southern blot analysis indicated that the two Hsp70s are encoded by distinct genes in the genome. Northern blot analysis showed that each of Hsp70a and Hsp70b expressed two mRNA species having different sizes by heat stress in rainbow trout RTG-2 cells. The induction levels of total Hsp70b mRNAs were consistently higher than Hsp70a counterparts during heat stress, although the expression profiles of the two genes were similar to each other in temperature shift and time course experiments. Interestingly, an mRNA species with a larger molecular size was expressed only under severe heat stress not less than 28 degrees C irrespective of Hsp70a and Hsp70b. These results suggest that the comprehensive identification of duplicated genes is a prerequisite to examining the gene expression profiles for tetraploid species such as rainbow trout.     

The fastest contracting muscles of nonmammalian vertebrates express only one isoform of the ryanodine receptor.

The skeletal muscles of chickens, frogs, and fish have been reported to express two isoforms (alpha and beta) of the sarcoplasmic reticulum calcium release channel (ryanodine receptor or RYR), while mammals express only one. We have studied patterns of RYR isoform expression in skeletal muscles from a variety of fish, reptiles, and birds with immunological techniques. Immunoblot analysis with a monoclonal antibody that recognizes both nonmammalian RYR isoforms and a polyclonal antibody specific to the alpha isoform show two key results: (a) two reptilian orders share with mammals the pattern of expressing only the alpha (skeletal) RYR isoform in skeletal muscle; and (b) certain functionally specialized muscles of fish and birds express only the alpha RYR isoforms. While both isoforms are expressed in the body musculature of fish and birds, the alpha isoform is expressed alone in extraocular muscles and swimbladder muscles. The appearance of the alpha RYR isoform alone in the extraocular muscles and a fast-contracting sonic muscle in fish (toadfish swimbladder muscle) provides evidence that this isoform is selectively expressed when rapid contraction is required. The functional and phylogenetic implications of expression of the alpha isoform alone are discussed in the context of the mechanism and evolution of excitation-contraction coupling.     

STAT4 isoforms differentially regulate Th1 cytokine production and the severity of inflammatory bowel disease

STAT4, a critical regulator of inflammation in vivo, can be expressed as two alternative splice forms, a full-length STAT4alpha, and a STAT4beta isoform lacking a C-terminal transactivation domain. Each isoform is sufficient to program Th1 development through both common and distinct subsets of target genes. However, the ability of these isoforms to mediate inflammation in vivo has not been examined. Using a model of colitis that develops following transfer of CD4(+) CD45RB(high) T cells expressing either the STAT4alpha or STAT4beta isoform into SCID mice, we determined that although both isoforms mediate inflammation and weight loss, STAT4beta promotes greater colonic inflammation and tissue destruction. This correlates with STAT4 isoform-dependent expression of TNF-alpha and GM-CSF in vitro and in vivo, but not Th1 expression of IFN-gamma or Th17 expression of IL-17, which were similar in STAT4alpha- and STAT4beta-expressing T cells. Thus, higher expression of a subset of inflammatory cytokines from STAT4beta-expressing T cells correlates with the ability of STAT4beta-expressing T cells to mediate more severe inflammatory disease.