Structure of the rat osteocalcin gene and regulation of vitamin D-dependent expression.

Abstract

The osteocalcin gene encodes a 6-kDa polypeptide, which represents one of the most abundant noncollagenous bone proteins, and the present studies establish that osteocalcin mRNA is detected only in bone tissue. An osteocalcin gene was isolated from a rat genomic DNA library, and sequence analysis indicated that the mRNA is represented in a 953-nucleotide segment of DNA consisting of four exons and three introns. A modular organization of the 5' flanking sequences of the gene is reflected by the presence of at least three classes of regulatory elements, which include the following: (i) RNA polymerase H canonical sequences; (U) a series of consensus sequences for hormone receptor binding sites and cyclic nucleotide responsive elements consistent with physiologic expression ofthe osteocalcin gene; and (Uii) a 24-nucleotide sequence in the proximal promoter region with a CAAT motif as a central element. We have designated this highly conserved sequence as an "osteocalcin box" since only 2 nucleotide substitutions are found in the rat and human osteocalcin genes. We have demonstrated two factors regulating osteocalcin gene expression. First, a 200-fold increase occurs in normal fetal calvaria osteoblasts producing a mineralizing matrix, compared to confluent osteoblasts in a nonmineralizing matrix. Second, contained within the 600 nucleotides immediately upstream from the transcription start site are sequences that support a 10-fold stimulated transcription of the gene by 1,25-dihydroxyvitamin D.There has been much interest in the vitamin K-dependent protein of bone, osteocalcin (bone Gla protein), since its discovery over a decade ago (1). A distinguishing feature of this 5.7-kDa protein (46-50 amino acids, depending on the species), and of functional significance, are 3 residues of the calcium binding amino acid, y-carboxyglutamic acid (Gla). Gla residues are posttranslationally synthesized from selected glutamic acid residues by a vitamin K-and C02-requiring enzyme complex (2). They are located at positions 17, 21, and 24 in all species from swordfish to mammals (1). This highly conserved sequence region from residues 20-34 in the central portion of the molecule, which also includes a disulfide loop (Cys-23-Cys-29), accounts for a structural conformation of the protein in the presence of calcium that promotes a tight binding of the protein to hydroxyapatite (1). The appearance of osteocalcin in embryonic bone coincident with mineral deposition (1), its association with the hydroxyapatite component of the matrix (3), its chemoattractant property for cells capable of bone resorption (4), and its modulated synthesis by the calcitrophic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (5-7) suggest a role for the protein in bone turnover. Although many properties of the protein have been identified, the precise function of osteocalcin is still unknown.Osteocalcin is synthesized de novo by osteoblasts as a 10,000-kDa precursor (8). While the majority ofthe processed osteocalcin peptide (5.7 kDa) is deposited in b...

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

“…Since the discovery of osteocalcin in the late 1970s (13,37,38), it has been used as a specific marker of osteoblast activity both in vivo (17,39,40) and in vitro (1,41) because of the restricted expression in the mature cells of osteoblastic lineage (15,42,43). Osteocalcin content in adult bone has been reported to range from 0.28 (human) to 2-2.5 (cow) mg/g of dry bone, and it represents up to 20% of noncollagenous bone matrix proteins (8,44).…”

CCAAT/Enhancer-binding protein homologous protein (CHOP) decreases bone formation and causes osteopenia

“…For this purpose, 1-10-μg of RNA were reversetranscribed using SuperScript III Platinum Two-Step qRT-PCR kit (Invitrogen, Carlsbad, CA) according to manufacturer's instructions and amplified in the presence of 5′-GACGCTTCACTACTCTTGACCCTGCG[FAM]C-3′ and 5′-GGATGTGCGTG TGACCTCTGT-3′ primers for CHOP; 5′-CACTTACGGCGCTACCTTGGGTAAGT [FAM] G-3′ and 5′-CCCAGCACAACTCCTCCCTA-3′ primers for osteocalcin; and 5′-CACGCTCTGGA AAGCTGTGGCG[FAM]G-3′ and 5′-AGCTTCCCGTTCAGCTCTGG-3′ primers for GAPDH and Platinum Quantitative PCR SuperMix-UDG (Invitrogen) at 54°-60°C for 45 cycles. Gene copy number was estimated by comparison with a standard curve constructed using chop or osteocalcin (J. Lian, University of Massachusetts, Worcester, MA) DNAs and corrected for gapdh (R. Wu, Cornell University, Ithaca, NY) copy number [24,36,37]. Reactions were conducted in a 96-well spectrofluorometric thermal iCycler (Bio-Rad), and fluorescence was monitored during every PCR cycle at the annealing step.…”

Functional Interference Between AP‐1 and the Vitamin D Receptor on Osteocalcin Gene Expression in Human Osteosarcoma Cells

“…Normal osteoblastic bone cells express the osteocalcin gene only after the proliferative stage (Owen et al, 1990) and its expression is regulated by calcitriol at the level of transcription, mRNA accumulation, and protein synthesis (Lian et al, 1989;Owen et al, 1991). We have previously shown by analysing mRNA from the human osteosarcoma cell lines MG-63, U2-0s, and SaOs-2 that osteocalcin gene expression is induced by calcitriol only in MG-63 cells (Mahonen et al, 1990).…”