Release of Intact and Fragmented Osteocalcin Molecules from Bone Matrix during Bone Resorption in Vitro

Abstract

Osteocalcin detected from serum samples is considered a specific marker of osteoblast activity and bone formation rate. However, osteocalcin embedded in bone matrix must also be released during bone resorption. To understand the contribution of each type of bone cell in circulating osteocalcin levels, we used immunoassays detecting different molecular forms of osteocalcin to monitor bone resorption in vitro. Osteoclasts were obtained from rat long bones and cultured on bovine bone slices using osteocalcin-depleted fetal bovine serum. In addition, human osteoclasts differentiated from peripheral blood mononuclear cells were used. Both rat and human osteoclasts released osteocalcin from bovine bone into medium. The amount of osteocalcin increased in the presence of parathyroid hormone, a stimulator of resorption, and decreased in the presence of bafilomycin A1, an inhibitor of resorption. The amount of osteocalcin in the medium correlated with a well characterized marker of bone resorption, the C-terminal telopeptide of type I collagen (r > 0.9, p < 0.0001). The heterogeneity of released osteocalcin was determined using reverse phase high performance liquid chromatography, and several molecular forms of osteocalcin, including intact molecule, were identified in the culture medium. In conclusion, osteocalcin is released from the bone matrix during bone resorption as intact molecules and fragments. In addition to the conventional use as a marker of bone formation, osteocalcin can be used as a marker of bone resorption in vitro. Furthermore, bone matrix-derived osteocalcin may contribute to circulating osteocalcin levels, suggesting that serum osteocalcin should be considered as a marker of bone turnover rather than bone formation. Osteocalcin (OC)1 is a 6-kDa noncollagenous protein produced by osteoblasts (1), osteocytes (2), and odontoblasts (3).Osteocalcin messenger RNA has also been detected in tissues other than bone, but it appears to be processed properly only in the bone microenvironment (4, 5). The structure of osteocalcin is characterized by three glutamic acid residues, which undergo a vitamin K-dependent carboxylation. The ␥-carboxyglutamic acid residues (Gla) provide osteocalcin with the ability to bind bone hydroxyapatite with a high affinity (6, 7). Osteocalcin is the second most abundant protein in the bone matrix, and it is highly conserved among all vertebrate species (8). The biological function of osteocalcin is probably related to the regulation of bone turnover and/or mineralization (9, 10).The expression of osteocalcin is a marker of late osteoblast differentiation and is induced only after the expression of other osteoblastic markers such as alkaline phosphatase and type I collagen (11,12). Newly synthesized osteocalcin is mostly (60 -90%) adsorbed to the bone hydroxyapatite via the Gla residues, but a part of it leaks into the circulation where it can be detected (13,14). Although osteoblasts synthesize only intact osteocalcin (15), osteocalcin may further undergo intracellular processing or ...

Methionine synthase reductase polymorphisms are associated with serum osteocalcin levels in postmenopausal women

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Osteocalcin gene polymorphisms influence concentration of serum osteocalcin and enhance fracture identification

“…(5) OC also may be released from the bone matrix during bone resorption, and therefore, serum OC can be considered a marker of bone turnover instead of solely a marker of bone formation. (6) The genetic evidence suggesting osteocalcin as a candidate gene comes from several sources. In a genome-wide linkage analysis, chromosomal region 1q21-23 was identified with the maximum logarithm (base 10) of odds (LOD) score of 3.1 with bone mineral density (BMD) phenotype.…”

The role of osteocalcin in the endocrine cross-talk between bone remodelling and energy metabolism

“…The significance of these findings for osteocalcin biology derives from two observations originally made at least two decades ago. The first is that carboxylated osteocalcin bound to the mineralised bone matrix via its Gla residues can be released upon resorption of this matrix by osteoclasts [11][12][13][14]. The second observation is that Gla residues can be decarboxylated when exposed to acid pH [15], and that bone resorption precisely involves acidification of the bone matrix.…”