Cloning of the cDNA for sea bream (Sparus aurata) parathyroid hormone-related protein.
ABSTRACT This paper reports cloning of the cDNA for sea bream (Sparus aurata) parathyroid hormone-related protein (PTHrP). The gene codes for a 125-amino acid mature protein with a 35-residue prepeptide. The total gene sequence is 1.8 kb with approximately 75% noncoding. The N-terminus of the protein resembles mammalian and chicken PTHrP peptides with 12 of the first 21 amino acids identical and for which there is homology with mammalian parathyroid hormone. Toward the C-terminus, the nuclear transporter region between residues 79 and 93 in sea bream is 73% homologous to tetrapod PTHrP, and the RNA binding domain, 96-117, is 50% homologous, moreover starting with the conserved lysine and terminating with the lysine/arginine sequence. Sea bream PTHrP differs significantly from mammalian and chicken PTHrP, having a novel 16-amino acid segment between residues 38 and 54 and completely lacking the terminal domain associated in mammals with inhibition of bone matrix lysis. RT-PCR and in situ hybridization of sea bream tissues show that the gene is expressed widely and the results confirm observations of a PTHrP-like factor in sea bream detected with antisera to human PTHrP.
Article: Isolation and characterization of piscine osteonectin and downregulation of its expression by PTH-related protein.[show abstract] [hide abstract]
ABSTRACT: The 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. Osteonectin is a unique matricellular calcium-binding glycoprotein and a major noncollagenous constituent of higher eukaryote bone. In terrestrial vertebrates, it has been associated with development, remodeling, cell turnover, and tissue repair, all processes involving substantial changes in extracellular matrix (ECM) structure. In skeleton biology, osteonectin has been described as a positive factor in the mineralization process as well as in osteoblastic cell lineage differentiation and is downregulated by the hypercalcemic hormone PTH. In this study, we report the cloning and characterization of bream S. auratus osteonectin cDNA and its tissue and cellular distribution. Its high expression by fish scales provides a unique in vitro bioassay with which to study regulation of osteonectin gene expression by the recently isolated piscine PTH-related peptide (PTHrP). An intervertebral tissue cDNA library from S. auratus was the source of the full-length cDNA clone for osteonectin. Expression studies were performed by semiquantitative RT-PCR, Northern blot, and in situ hybridization analysis. Moreover, an in vitro bioassay with S. auratus scales was specifically developed for measuring the effect of PTHrP on osteonectin expression. Phylogenetic analysis showed that S. auratus osteonectin is highly homologous with previously reported osteonectins, supporting the idea of a conserved function for this protein in the ECM. Its expression pattern in adult tissues from S. auratus was markedly biased toward skeletal structures of both dermal or endochondral origin. More specifically, the localization of the osteonectin mRNA in the basement membrane that separates the epithelia from the underlying mineralized connective tissue supports a role for this protein in calcified matrix turnover. Furthermore, the recently identified piscine hypercalcemic factor PTHrP downregulates osteonectin expression in scales, suggesting a catabolic action for this hormone on these structures.Journal of Bone and Mineral Research 05/2005; 20(4):682-92. · 6.37 Impact Factor