Identification of an osteopontin‐like protein in fish associated with mineral formation

Abstract

Fish has been recently recognized as a suitable vertebrate model and represents a promising alternative to mammals for studying mechanisms of tissue mineralization and unravelling specific questions related to vertebrate bone formation. The recently developed Sparus aurata (gilthead seabream) osteoblast‐like cell line VSa16 was used to construct a cDNA subtractive library aimed at the identification of genes associated with fish tissue mineralization. Suppression subtractive hybridization, combined with mirror orientation selection, identified 194 cDNA clones representing 20 different genes up‐regulated during the mineralization of the VSa16 extracellular matrix. One of these genes accounted for 69% of the total number of clones obtained and was later identified as theS. aurata osteopontin‐like gene. The 2138‐bp full‐length S. aurata osteopontin‐like cDNA was shown to encode a 374 amino‐acid protein containing domains and motifs characteristic of osteopontins, such as an integrin receptor‐binding RGD motif, a negatively charged domain and numerous post‐translational modifications (e.g. phosphorylations and glycosylations). The common origin of mammalian osteopontin and fish osteopontin‐like proteins was indicated through an in silico analysis of available sequences showing similar gene and protein structures and was further demonstrated by their specific expression in mineralized tissues and cell cultures. Accordingly, and given its proven association with mineral formation and its characteristic protein domains, we propose that the fish osteopontin‐like protein may play a role in hard tissue mineralization, in a manner similar to osteopontin in higher vertebrates.

Establishment of primary cell cultures from fish calcified tissues

“…osteocalcin, osteonectin, osteopontin, matrix Gla protein, bone morphogenetic protein 2, alkaline phosphatase, etc.) and their regulation during in vitro mineralization were also demonstrated, further confirming their suitability to identify molecular determinants of tissue calcification (Laizé et al 2005;Rafael et al 2006;Fonseca et al 2007). Finally, these cells are well adapted to grow in standard media (DMEM or L15) in the presence of mammalian serum (10% fetal bovine serum), seriously decreasing costs associated to cell maintenance (fish sera are available, but expensive) and allowing direct comparison with studies using mammalian bone-derived cells (Kellermann et al 1990;Fournier and Price 1991;Stanford et al 1995;Costa and Fernandes 2000).…”

Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A

“…However, the increase in bglap expression at older stages of development (52-60 dph) seemed to be due to the completion of the ossification of skeletal structures in the axial skeleton [9-11,18], similar to that found in European sea bass [88,89]. Considering that osteopontin and osteocalcin are involved in the modulation of hydroxyapatite crystallization [90], the advanced SPP1 expression might be related to the inhibition of osteoblast mineralization [91] occurring in vertebrae centra during their intramembranous ossification, thus allowing osteoblast to maintain their proliferative state; while bglap expression would be an indicator of the osteoblast mineralization in those structures [89]. …”

Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadate

“…A two-class SAM analysis identified mineralogenic genes, some of which had already been previously investigated during in vitro mineralization of VSa13 and VSa16 cells through a candidate gene approach, e.g. tissue non-specific alkaline phosphatase ( TNAP; unpublished data), BMP-2 [21], SPP1 [20], MGP and OC [19]. At first glance, a good correlation ( i.e.…”