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- Isolation and characterization of piscine osteonectin and downregulation of Its expression by PTH-related proteinPublication . Redruello, BegoƱa; EstĆŖvĆ£o, M. Dulce; Rotllant, J.; Guerreiro, P. M.; Anjos, Liliana; Canario, Adelino V. M.; Power, DeborahThe skeleton is the main source of osteonectin mRNA in adults of the seawater teleost sea bream Sparus auratus. It is expressed by cells forming the basement membrane of calcifying tissue indicating that, as in mammals, it may play a role in osteoblast differentiation. PTHrP induced downregulation of osteonectin mRNA in vitro in scales, a mineralizing tissue with bone-like metabolism. This indicates a means to redirect calcium to activities such as vitellogenesis when this ion is in high demand.
- Ligand binding and signalling pathways of PTH receptors in sea bream (Sparus auratus) enterocytesPublication . Rotllant, J.; Guerreiro, P. M.; Redruello, BegoƱa; Fernandes, H.; Apolonia, L.; Anjos, Liliana; Canario, Adelino V. M.; Power, DeborahWhole animal studies have indicated that Ca2+ uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using aminoterminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of 125I-(1ā35tyr) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (KD=2.59 nM; Bmax=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1ā34) PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The aminoterminal peptides (2ā34)PTHrP, (3ā34)PTHrP and (7ā34) PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1ā34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca2+ uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.
- Ontogeny of osteonectin expression in embryos and larvae of sea bream (Sparus auratus)Publication . EstĆŖvĆ£o, M. Dulce; Redruello, BegoƱa; Canario, Adelino V. M.; Power, DeborahOsteonectin (OSN) is a glycoprotein which is implicated in development, bone formation and mineralisation, tumorigenesis, angiogenesis, and wound healing. Regulation of its expression by hormones may be one of the mechanisms by which the endocrine system affects bone metabolism. As a first step to understanding OSN function in fish, the gene expression of the recently cloned cDNA for sea bream, Sparus auratus, osteonectin (sbOSN) was characterised during embryonic and larval development. sbOSN mRNA was first detected by semi-quantitative reverse transcription-polymerase chain reaction in embryos at early gastrula and its expression increased continuously until hatch, after which it decreased until 15 days post-hatch (dph), increased transiently until 24 dph and decreased thereafter. In situ hybridisation showed it had a differential tissue distribution which was age dependent. In general, sbOSN mRNA was identified in cartilaginous and calcified structures of both dermal and endochondral origin but its expression was not restricted to the skeleton. sbOSN transcripts were also detected in the skin, perichordal sheath, nerve cord, and kidney tubules.
- Expression of pituitary prolactin, growth hormone and somatolactin is modified in response to different stressors (salinity, crowding and food-deprivation) in gilthead sea bream Sparus auratusPublication . Laiz-CarriĆ³n, R.; Fuentes, J.; Redruello, BegoƱa; GuzmĆ”n, JosĆ© M.; RĆo, MarĆa P. MartĆn del; Power, Deborah; Mancera, J. M.Prolactin (PRL), growth hormone (GH) and somatolactin (SL) expression was studied in gilthead sea bream (Sparus auratus) in response to several different stressors (salinity, food deprivation or stocking density). In the first experiment, specimens were acclimated during 100 days at three different environmental salinities: low salinity water (LSW, 6 ppt), brackish water (BW, 12 ppt) and seawater (SW, 38 ppt). Osmoregulatory parameters corresponded to those previously reported for this species under similar osmotic conditions. Pituitary PRL expression increased with decreasing environmental salinity, and was significantly different between SW- and LSW-acclimated fish. Pituitary GH expression was similar between SW- and BW-acclimated fish but decreased in LSW-acclimated specimens. Pituitary SL expression had a āāU-shapedā relationship to environmental salinity with the lowest expression in BW-acclimated fish. In a second experiment SW-acclimated specimens were randomly assigned to one of four treatments and maintained for 14 days: (1) fed fish under low density (LD, 4 kg m!3); (2) fed fish under high density (HD, 70 kg m!3); (3) food deprived fish under LD; and (4) food deprived fish under HD. Plasma glucose and cortisol levels corresponded to those previously reported in S. auratus under similar experimental conditions. Pituitary PRL and SL expression increased in fish maintained under HD and decreased in food deprived fish. In conclusion, an effect of environmental salinity on pituitary PRL and GH expression has been demonstrated. In addition, crowding stress seems to interact with food deprivation in S. auratus and this is reflected by changes in pituitary PRL, GH and SL expression levels.
- Stimulation of cortisol release by the N terminus of teleost parathyroid hormone-related protein in interrenal cells in vitroPublication . Rotllant, J.; Guerreiro, P. M.; Anjos, Liliana; Redruello, BegoƱa; Canario, Adelino V. M.; Power, DeborahThe mode of action of PTHrP in the regulation of sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system. Piscine (1ā34)PTHrP (10ā6ā10ā11 m) stimulated cortisol production in a dose-dependent manner. The ED50 of (1ā34)PTHrP was 2.8 times higher than that of (1ā39)ACTH, and maximum increase in cortisol production in response to 10ā8 m of (1ā34)PTHrP was approximately 7-fold lower than for 10ā8 m of (1ā39)ACTH. In contrast to (1ā34)PTHrP, piscine (10ā20)PTHrP, (79ā93)PTHrP, and (100ā125)PTHrP (10ā9ā10ā7 m) did not stimulate cortisol production. The effect of piscine (1ā34)PTHrP on cortisol production was abolished by N-terminal peptides in which the first amino acid (Ser) was absent and by simultaneous addition of inhibitors of the adenylyl cyclase-protein kinase A and phospholipase C-protein kinase C intracellular pathways but not by each separately. The PTHrP-induced signal transduction was further investigated by measurements of cAMP production and [H3]myo-inositol incorporation in an interrenal cell suspension. Piscine (1ā34)PTHrP increased cAMP and total inositol phosphate accumulation, which is indicative that the mechanism of action of PTHrP in interrenal tissue involves the activation of both the adenylyl cyclase-cAMP and phospholipase C-inositol phosphate signaling pathways. These results, together with the expression of mRNA for PTHrP and for PTH receptor (PTHR) type 1 and PTHR type 3 receptors in sea bream interrenal tissue, suggest a specific paracrine or autocrine steroidogenic action of PTHrP mediated by the PTHRs.
- Immunohistochemical detection of estrogen receptors in fish scalesPublication . Pinto, Patricia IS; EstĆŖvĆ£o, M. Dulce; Redruello, BegoƱa; Socorro, S.; Canario, Adelino V. M.; Power, DeborahCalcium mobilization from internal stores, such as scales, induced by 17b-estradiol during sexual maturation in salmonids is well documented. This calcium mobilization from scales is proposed to be mediated by the estrogen receptor (ER). However, the ER subtypes involved and signaling mechanisms responsible for this effect remain to be fully characterized. In the present study, we have localized ERa, ERba and ERbb proteins in juvenile and adult sea bream (Sparus auratus) and Mozambique tilapia (Oreochromis mossambicus) scales by immunohistochemistry with sea bream ER subtype specific antibodies. The three ERs were detected in isolated or small groups of round cells, in the basal layer of the scales of both juvenile and adult fish and the localization and signal intensity varied with the species and age of the animals. The ERs may be co-localized in cells of the scale posterior region that expressed tartrateresistant acid phosphatase (TRAP), a marker for osteoclasts. These results suggest that the calcium mobilizing action of 17b-estradiol on fish scales is via its direct action on ERs localized in osteoclasts.
- CRTAC1 homolog proteins are conserved from cyanobacteria to man and secreted by the teleost fish pituitary glandPublication . Redruello, BegoƱa; Louro, Bruno; Anjos, Liliana; Silva, NĆ”dia; Greenwell, Roger S.; Canario, Adelino V. M.; Power, DeborahCartilage acidic protein 1 (CRTAC1) gene expression is used as a marker for chondrocyte differentiation instem cell-based tissue engineering. It is also transcribed outside the skeleton where at least two different transcripts are expressed in lung and brain. In the pituitary gland of the teleost fish sea bream Sparus auratus, we have found a transcript with a high degree of sequence identity to CRTAC1 family members but lacking the EGF-like calcium-binding domain encoding sequence of CRTAC1 and designated it as CRTAC2. Database searches revealed many previously unidentified members of the CRTAC1 and CRTAC2 in phylogenetically distant organisms, such as cyanobacteria, bryophyta, lancelets, and diverse representatives of vertebrates. Phylogenetic analyses showed that the genes encoding CRTAC1 and CRTAC2 proteins coexist in teleost fish genomes. Structural prediction analysis identified the N-terminal region of the CRTAC1/CRTAC2 family members as a potential seven-bladed Ī² -propeller structure, closely related to those of integrin Ī± chains and glycosylphosphatidylinositol-specific phospholipase D1 protein families. This relationship is con fi rmed by phylogenetic analysis with the N-terminal domain of sea bream CRTAC2 as the most divergent sequence. Because teleost fi shes are the only phylogenetic group where both CRTAC1 and CRTAC2 genes are present, they occupy a pivotal position in studies of the mechanisms governing the speci fi c expression patterns of each gene/protein subfamily. This will be essential to elucidate their respective biological roles.
- Cellular morphology and markers of cartilage and bone in the marine teleost Sparus auratusPublication . EstĆŖvĆ£o, M. Dulce; Silva, Nadia; Redruello, BegoƱa; Costa, Rita; Gregorio, Silvia; Canario, Adelino V. M.; Power, DeborahModifications have been characterised in terms of cellular organisation and the extracellular matrix (ECM) during bone ontogeny in the sea bream (Sparus auratus). During endochondral development, the agglomeration of matrix-secreting cells gives rise to chondrones; these chondrones frequently contain proliferating-cell-nuclearantigen-positive cells, which subsequently become large collagen-II-positive cells with the characteristics of chondrocytes. Moreover, the matrix:cell ratio within the perichondrium increases, accompanied by a modification in ECM composition. Mineralisation of cartilage ECM is marked by a rapid fall in cell number, the switching off of collagen II transcription and the switching on of collagen X transcription, followed by collagen I transcription and bone mineralisation. The formation of dermal structures initiated upon the condensation of mesenchyme cells defines the future location of the dermal bone. Subsequent cellular differentiation gives rise to cells on the bone surface; these cells are positive for collagen I and osteonectin transcripts. The fish skeleton, with the exception of vertebrae, tends to comprise flattened bones that are covered by a monolayer of cells, the periosteum. A third type of tissue, present in gills, consists of chondrocyte-like cells embedded in a mineralised matrix resembling chondroid bone in mammals. The results suggest that the cellular organisation and ontogeny of endochondral and dermal bone in the sea bream are similar to those described in other vertebrates.
- Cloning of the glucocorticoid receptor (GR) in gilthead seabream (Sparus aurata)Publication . Acerete, L.; Balasch, J. C.; Castellana, B.; Redruello, BegoƱa; Roher, N.; Canario, Adelino V. M.; Planas, J. V.; MacKenzie, S.; Tort, L.In order to determine the cortisol response after an immune challenge in the gilthead seabream (Sparus aurata), a cortisol receptor (GR) was cloned, sequenced and its expression determined after lipopolysaccharide (LPS) treatment. To clone the gilthead seabream GR (sbGR), consecutive PCR amplifications and screening of a pituitary cDNA library were performed. We obtained a clone of 4586 bp encoding a 784aa protein. Northern blot analysis from head kidney, heart and intestine revealed that the full length sbGR mRNA was approximately 6.5 Kb. A LPS treatment, used as an acute stress model, was employed to characterise the expression of sbGR and some selected genes involved in the immune response (IL-1Ī², TNF-Ī±, Mx protein, cathepsin D and PPAR-Ī³). All genes were expressed in all tissues examined and responses were tissue and time dependent revealing differential gene expression profiles after LPS administration. Furthermore, analysis of plasma cortisol levels after LPS injection, showed an acute response to inflammatory stress with a significant increase two and six h after injection, recovering to basal levels 12 h post-stress in all LPS concentrations tested.
- Calcium mobilization from fish scales is mediated by parathyroid hormone related protein via the parathyroid hormone type 1 receptorPublication . Rotllant, J.; Redruello, BegoƱa; Guerreiro, P. M.; Fernandes, H.; Canario, Adelino V. M.; Power, DeborahThe scales of bony fish represent a significant reservoir of calcium but little is known about their contribution, as well as of bone, to calcium balance and how calcium deposition and mobilization are regulated in calcified tissues. In the present study we report the action of parathyroid hormone-related protein (PTHrP) on calcium mobilization from sea bream (Sparus auratus) scales in an in vitro bioassay. Ligand binding studies of piscine 125I-(1 ā 35tyr)PTHrP to the membrane fraction of isolated sea bream scales revealed the existence of a single PTH receptor (PTHR) type. RT-PCR of fish scale cDNA using specific primers for two receptor types found in teleosts, PTH1R, and PTH3R, showed expression only of PTH1R. The signalling mechanisms mediating binding of the N-terminal amino acid region of PTHrP were investigated. A synthetic peptide (10 8 M) based on the N-terminal 1 ā 34 amino acid residues of Fugu rubripes PTHrP strongly stimulated cAMP synthesis and [3 H]myo-inositol incorporation in sea bream scales. However, peptides (10 8 M) with N-terminal deletions, such as (2 ā 34), (3 ā 34) and (7 ā 34)PTHrP, were defective in stimulating cAMP production but stimulated [3 H]myo-inositol incorporation. (1 ā 34)PTHrP induced significant osteoclastic activity in scale tissue as indicated by its stimulation of tartrate-resistant acid phosphatase. In contrast, (7 ā 34)PTHrP failed to stimulate the activity of this enzyme. This activity could also be abolished by the adenylyl cyclase inhibitor SQ-22536, but not by the phospholipase C inhibitor U-73122. The results of the study indicate that one mechanism through which N-terminal (1 ā 34)PTHrP stimulates osteoclastic activity of sea bream scales, is through PTH1R and via the cAMP/AC intracellular signalling pathway. It appears, therefore, that fish scales can act as calcium stores and that (1 ā 34)PTHrP regulates calcium mobilization from them; it remains to be established if this mechanism contributes to calcium homeostasis in vivo.