Cloning of a CD59-like gene in rainbow trout. Expression and phylogenetic analysis of two isoforms

CD59, the major inhibitor of the complement membrane attack complex, is an 18-20 kDa glycoprotein, linked to the membrane via a glycosylphosphatidylinositol (GPI)-anchor. It restricts binding of C9 to the C5b-8 complex, preventing the formation of the complement membrane attack complex C5b-9. In this study we report the cloning of a second CD59-like gene in the rainbow trout, Oncorhynchus mykiss (referred to as CD59-2 and the previously deposited trout CD59-like gene as CD59-1). Trout CD59-2 is 56% identical to CD59-1 at the amino acid level. Both of trout CD59s show the highest identity score (54%) with putative CD59-like molecules from other teleost, and the overall identity with their mammalian orthologs is less than 30%. Trout CD59s are expressed in brain, heart, intestine, kidney, liver and spleen. Particularly, CD59-2 is abundant in trout brain, while CD59-1 seems to be absent in the trout spleen. Moreover, both of trout CD59 genes seems to be present as a single copy in trout genome.     

cDNA cloning and phylogenetic analysis of the sixth complement component in rainbow trout

The sixth complement protein (C6) is an essential component of the membrane attack complex (MAC); the end product of the lytic pathway of complement activation. The MAC complex constitutes a supramolecular assembly containing the five precursor proteins C5b, C6, C7, C8, and C9. Once assembled on the target surface it forms transmembrane channels that cause membrane damage and cytolysis of complement-opsonized pathogens. Besides mediating direct pathogen elimination, exposure of cells to sublytic doses of MAC can trigger diverse cellular responses such as, cell activation, induction of apoptosis, cell cycle re-entry and proliferation in various biological settings. The terminal complement components (C6-C9) are structurally related proteins, differing in size and complexity. In order to study their evolution, we report here the cloning and molecular characterization of C6 component in rainbow trout. The deduced amino acid sequence of trout C6 exhibits 55 and 44% identity with zebra fish and human orthologs, respectively. The 'domain' architecture of trout C6 resembles that of mammalian counterparts, and the cysteine backbone is also conserved. Finally, trout C6 gene appears to exist as a single copy in the trout genome, and is expressed in a wide range of trout tissues.     

Cloning and phylogenetic analysis of the alpha subunit of the eighth complement component (C8) in rainbow trout

The alpha subunit of the eighth complement component (C8) is a single-chain plasma glycoprotein which functions in the cytolytic process mediated by the complement system through a sequence of polymerization reactions with other terminal components. We have previously isolated and characterized the C8beta and C8gamma subunits of the eighth complement component in rainbow trout (Oncorhynchus mykiss). Here, we report the primary sequence, the tissue expression profile, the domain architecture and the phylogenetic analysis of the trout C8alpha gene. The deduced amino acid sequence of the trout C8alpha gene exhibits 44 and 43% identity with human and frog orthologs, respectively. The domain architecture of the trout C8alpha resembles that of mammalian orthologs, and the cysteine backbone shows a high degree of conservation. The trout C8alpha shows a similar expression profile with that of trout C8beta and C8gamma, pointing to the liver as the main source of the C8 genes expression. Although the presence of a fully developed lytic pathway of complement system is expected in teleost, this is the first report of the C8alpha gene in an organism other than mammalian.     

Clusterin induces matrix metalloproteinase-9 expression via ERK1/2 and PI3K/Akt/NF-κB pathways in monocytes/macrophages

Most solid tumor tissues possess a significant population of macrophages, which are known to be closely linked with tumor progression and metastasis. Clusterin has been reported to be overexpressed in various tumors and to have a tumor-promoting role. As clusterin induction and macrophage infiltration occur concurrently at the tumor site, it raises a possibility that clusterin may regulate the function of macrophages via facilitating ECM remodeling. Here, we demonstrate for the first time the expression of MMP-9 by clusterin in human primary monocytes as well as human and murine macrophage cell lines, THP-1, and Raw264.7. MMP-9 expression was accompanied by increased enzymatic activity, as revealed by gelatin zymography. The MMP-9 activity promoted by clusterin was found to be dependent on the activation of ERK1/2 and PI3K/Akt but not p38 or JNK pathways. Inhibition of PI3K activity did not affect the activation of ERK1/2 and vice versa, indicating that the two pathways were independently operated to stimulate MMP-9 activity. Moreover, clusterin facilitated nuclear translocation of NF-κB p65 along with IκB-α degradation and phosphorylation, which was critical for MMP-9 expression. As NF-κB is a central regulator of inflammation, clusterin may provide a molecular link between inflammation and cancer via up-regulating NF-κB and MMP-9. Collectively, these data highlight a novel role of clusterin as a stimulator for MMP-9 expression in macrophages, which may contribute to the tissue reorganization by serving as a modulator for ECM degradation.     

Clusterin associates with altered elastic fibers in human photoaged skin and prevents elastin from ultraviolet-induced aggregation in vitro

Clusterin is a secreted glycoprotein with stress-induced expression in various diseased and aged tissues. It shares basic features with small heat shock proteins because it may stabilize proteins in a folding-competent state. Besides its presence in all human body fluids, clusterin associates with altered extracellular matrix proteins, such as beta-amyloid in Alzheimer senile plaques in the brain. Because dermal connective tissue alterations occur because of aging and UV radiation, we explored the occurrence of clusterin in young, aged, and sun-exposed human skin. Immunohistochemical analysis showed that clusterin is constantly associated with altered elastic fibers in aged human skin. Elastotic material of sun-damaged skin (solar elastosis), in particular, revealed a strong staining for clusterin. Because of the striking co-localization of clusterin with abnormal elastic material, we investigated the interaction of clusterin with elastin in vitro. A chaperone assay was established in which elastin was denatured by UV irradiation in the absence or presence of clusterin. This assay demonstrated that clusterin exerted a chaperone-like activity and effectively inhibited UV-induced aggregation of elastin. The interaction of both proteins was further analyzed by electron microscopy, size exclusion chromatography, and mass spectrometry, in which clusterin was found in a stable complex with elastin after UV exposure.     

Differential B cell expression of mouse Fc receptor homologs

Five Fc receptor homologs (FcRH1-5) possessing inhibitory and/or activating signaling motifs are differentially expressed during B cell differentiation in humans. In this analysis we describe their three mouse orthologs, moFcRH1, moFcRH2 and moFcRH3. The moFcRH genes are located in a chromosome 3 region that is syntenic with the FcRH locus on human chromosome 1. They encode proteins with 2-5 Ig-like domains that share 20-61% extracellular identity with their human counterparts. One moFcRH1 isoform lacks a transmembrane domain as do both moFcRH2 isoforms. The other moFcRH1 isoform and two moFcRH3 isoforms have transmembrane domains and cytoplasmic ITIM and ITAM-like consensus sequences implying their inhibitory or activating signaling potential. Whereas the moFcRH1 and moFcRH3 orthologs are preferentially expressed at different stages in B cell differentiation, the structurally novel moFcRH2 gene is expressed in non-lymphoid tissues. The highly restricted pattern of moFcRH3 expression suggests this member of the phylogenetically conserved FcRH family may have an important immunoregulatory role in marginal zone B cells.     

Clusterin expression in follicular dendritic cells associated with prion protein accumulation

Peripheral accumulation of abnormal prion protein (PrP) in variant Creutzfeldt-Jakob disease and some animal models of transmissible spongiform encephalopathies (TSEs) may occur in the lymphoreticular system. Within the lymphoid tissues, abnormal PrP accumulation occurs on follicular dendritic cells (FDCs). Clusterin (apolipoprotein J) has been recognized as one of the molecules associated with PrP in TSEs, and clusterin expression is increased in the central nervous system where abnormal PrP deposition has occurred. We therefore examined peripheral clusterin expression in the context of PrP accumulation on FDCs in a range of human and experimental TSEs. PrP was detected immunohistochemically on tissue sections using a novel highly sensitive method involving detergent autoclaving pretreatment. A dendritic network pattern of clusterin immunoreactivity in lymphoid follicles was observed in association with the abnormal PrP on FDCs. The increased clusterin immunoreactivity appeared to correlate with the extent of PrP deposition, irrespective of the pathogen strains, host mouse strains or various immune modifications. The observed co-localization and correlative expression of these proteins suggested that clusterin might be directly associated with abnormal PrP. Indeed, clusterin immunoreactivity in association with PrP was retained after FDC depletion. Together these data suggest that clusterin may act as a chaperone-like molecule for PrP and play an important role in TSE pathogenesis.     

Characterization of FMN2 gene at human chromosome 1q43

Mouse Formin (Fmn1) is an actin regulator interacting with Profilin, SRC, EMS1, FNBP1, FNBP2, FNBP3, FNBP4, WBP4 and alpha-catenin. FMN1, FHOD1, FHOD3, GRID2IP and FHDC1 are non-FDD-type Formin homology proteins, while FMNL1, FMNL2, FMNL3, DIAPH1, DIAPH2, DIAPH3, DAAM1 and DAAM2 are FDD-type Formin homology proteins. Here, we characterized human FMN2 gene by using bioinformatics. Complete coding sequence of human FMN2 cDNA was determined by assembling AL359918, AL513342, AL590490, AL646016 genome sequences, AF218941 partial cDNA, and AF218942 partial cDNA. FMN2 mRNA was expressed in fetal brain, adult whole brain, hypothalamus, retina, pancreatic islet and germinal-center B cells. Among various human tumors, FMN2 mRNA was expressed in parathyloid tumor, glioblastoma, retinoblastoma and chondrosarcoma. Human FMN2 (1722 aa) showed 74.7% total-amino-acid identity with mouse Fmn2, and 31.9% total-amino-acid identity with human FMN1. Although N-terminal half was divergent between FMN2 orthologs and FMN1 orthologs, FH1 and FH2 domains were conserved among FMN2 and FMN1 orthologs. Exon-intron structure was conserved between FMN2 and FMN1 genes. RYR2-FMN2-CKTSF1B2 (PRDC) locus at human chromosome 1q43 and RYR3-FMN1-CKTSF1B1 (Gremlin) locus at human chromosome 15q13-q14 were paralogous regions (paralogons) within the human genome. This is the first report on comprehensive characterization of the human FMN2 gene.     

Clusterin gene expression in the rat thymus is not modulated by dexamethasone treatment.

Clusterin, a multifunctional glycoprotein, characterized as a potent inhibitor of the membrane attack complex of complement, is also known to be the product of a gene that is highly up-regulated in certain tissues undergoing programmed cell death. We have studied the expression of this gene in the rat thymus after the induction of thymocyte programmed cell death (PCD) by in vivo dexamethasone administration. Northern blot analysis of clusterin mRNA 2, 4, 6 and 8 hr after dexamethasone administration in a total of 21 rats revealed no modification in the level of clusterin gene expression. In situ hybridization demonstrated that clusterin gene expression is macroscopically confined to the medullary region of the thymus, and that this distribution is not modified by dexamethasone administration. These results strongly suggest that in the rat, clusterin gene expression is not associated with the programmed cell death of thymocytes following in vivo dexamethasone administration. In situ hybridization of the clusterin cRNA to thymus cryostat sections confirmed the results obtained by Northern blot analysis. Indeed, no consistent increase in the amount of clusterin mRNA was detectable at any of the time-points studied. The macroscopic distribution of clusterin mRNA in the rat thymus was identical to that previously observed in the human thymus, clusterin message being essentially detected within the medullary regions. No modification in the macroscopic distribution of clusterin gene expression was detected after dexamethasone administration. These results suggest that, like the human thymus, medullary epithelial cells are the site of clusterin gene expression in the rat thymus. Moreover they indicate that an increase in the extent of ongoing thymocyte PCD does not significantly modify the rate or site of clusterin gene expression within the thymus.     

Divergence of Spatial Gene Expression Profiles Following Species-Specific Gene Duplications in Human and Mouse

To examine the process by which duplicated genes diverge in function, we studied how the gene expression profiles of orthologous gene sets in human and mouse are affected by the presence of additional recent species-specific paralogs. Gene expression profiles were compared across 16 homologous tissues in human and mouse using microarray data from the Gene Expression Atlas for 1575 sets of orthologs including 250 with species-specific paralogs. We find that orthologs that have undergone recent duplication are less likely to have strongly correlated expression profiles than those that remain in a one-to-one relationship between human and mouse. There is a general trend for paralogous genes to become more specialized in their expression patterns, with decreased breadth and increased specificity of expression as gene family size increases. Despite this trend, detailed examination of some particular gene families where species-specific duplications have occurred indicated several examples of apparent neofunctionalization of duplicated genes, but only one case of subfunctionalization. Often, the expression of both copies of a duplicated gene appears to have changed relative to the ancestral state. Our results suggest that gene expression profiles are surprisingly labile and that expression in a particular tissue may be gained or lost repeatedly during the evolution of even small gene families. We conclude that gene duplication is a major driving force behind the emergence of divergent gene expression patterns.     

Identification and characterization of rat Dact1 and Dact2 genes in silico

DACT1 (DAPPER1), Frizzled receptors, MUSK receptor, VANGL1, VANGL2, PRICKLE1, PRICKLE2, DAAM1, Casein kinases, MARK3 (PAR1), PP2C, AXIN1, AXIN2, NKD1, NKD2, FRAT1, FRAT2 and CXXC4 are WNT signaling molecules associating with Dishevelled family proteins. Human DACT1 is the ortholog of Xenopus Dapper and Frodo, and human DACT2 (DAPPER2) is the paralog of human DACT1. Here, we identified and characterized rat Dact1 (Dapper1) and Dact2 (Dapper2) genes by using bioinformatics. Rat Dact1 gene, consisting of four exons, was located within AC136677.3 genome sequence. Rat Dact2 gene, consisting of four exons, was located within AC139434.3 genome sequence. Dact1 was mapped to rat chromosome 6q24, and Dact2 gene to rat chromosome 1q12. Rat Dact1 (778 aa) showed 93.7, 82.9, 60.3, 58.7 and 48.6% total-amino-acid identity with mouse Dact1, human DACT1, Xenopus Dapper, Xenopus Frodo and zebrafish dact1, respectively. Rat Dact2 (768 aa) showed 86.6, 59.6 and 38.3% total-amino-acid identity with mouse Dact2, human DACT2 and zebrafish dact2, respectively. Dact1 orthologs were more evolutionarily conserved than Dact2 orthologs. Seven DAPH domains (DAPH1-DAPH7), originally identified as the regions conserved between human DACT1 and DACT2, were conserved among mammalian Dact1 orthologs and Dact2 orthologs. DAPH2 domain, corresponding to the Leucine zipper motif, was located within the coiled-coil region. DAPH3 domain was the Serine rich region. DAPH7 domain was the C-terminal PDZ binding region. This is the first report on the rat Dact1 and Dact2 genes.     

Tubular up-regulation of clusterin mRNA in murine lupus-like nephritis.

Clusterin, a widely distributed glycoprotein, is detected in most tissues and in numerous physiological fluids. In the kidney, this protein is constitutively expressed in tubular epithelial cells, and its expression is enhanced following tubular injuries. In addition, clusterin has been detected in glomerular immune deposits of glomerulonephritis. The present study was designed to define the sites of clusterin mRNA accumulation in murine lupus-like nephritis in comparison with murine tubulopathies. In lupus-like nephritis, a significant increase of clusterin mRNA abundance was demonstrated. This up-regulation was localized exclusively in tubular epithelial cells exhibiting tubulointerstitial alterations, whereas no clusterin mRNA was detectable in diseased glomeruli, excluding an active synthesis of clusterin by glomerular cells. A similar tubular increase of clusterin mRNA abundance was observed in myeloma-like cast nephropathy induced by IgG3 monoclonal cryoglobulins and even in the absence of any detectable histological alterations in a model of septic shock induced by the injection of bacterial lipopolysaccharides. Our results suggest that tubular epithelial cells are the only sites of clusterin mRNA accumulation during the course of lupus-like nephritis and that the tubular up-regulation of clusterin gene expression may reflect the cellular response to various types of tubular injuries.     

Rainbow trout suppressor of cytokine signalling (SOCS)-1, 2 and 3: molecular identification, expression and modulation

Suppressor of cytokine signalling (SOCS) proteins are inhibitors of cytokine signalling pathways. Three SOCS genes, SOCS-1, 2 and 3, have been identified and their sequences analyzed in an economically important fish, rainbow trout (Oncorhynchus mykiss, Walbaum). In general, these three SOCS molecules are well conserved especially in the SRC homology 2 and the SOCS domains, with sequence identities between trout and mammals ranging from 41 to 42, 50 to 51, and 58 to 61% for SOCS-1, 2 and 3, respectively. The identities within fish species are slightly higher, with sequence identities between trout and the other fish species at 44-46, 64-70, and 71-76% for SOCS-1, 2 and 3, respectively. All the SOCS-1, as well as all the SOCS-2 or 3 molecules from different species are grouped together in phylogenetic tree analysis with high bootstrap support, with the fish molecules in each type grouping closely together. The expression of the trout SOCS-1, 2 and 3 genes are detectable by real-time PCR in all the eight tissues studied; the gills, skin, muscle, liver, spleen, head kidney, intestine and brain. SOCS-1 is highly expressed in intestine, head kidney, spleen, gills and skin. SOCS-2 is highly expressed in brain, head kidney, muscle, spleen, gills, skin and intestine. The expression of SOCS-3 is the highest among the three SOCS genes in all the tissues except in intestine, brain and liver. The modulation of SOCS gene expression is shown to be cytokine and cell type dependent. While interferon-gamma up-regulates the expression of all the three SOCS genes in both the fibroid RTG-2 and the monocyte/macrophage RTS-11 cell lines, interleukin-1beta only up-regulates SOCS gene expression in the RTG-2 cell line, with little, if any, effect in the RTS-11 cell line.