Seventeen day chicken embryonic osteoblasts treated over a 30-day period with 1,25(OH)2 D3 showed a 2-10-fold decrease in collagen, osteopontin and osteocalcin protein accumulation, alkaline phosphatase enzyme activity, and mineral deposition. Comparable inhibition in the steady state mRNA levels for alpha 1(I) and alpha 2(I) collagen, osteocalcin, and osteopontin were observed, and the inhibitory action of the hormone was shown to be specific for only the late release populations of cells from sequential enzyme digestions of the chick calvaria. In order to determine whether the continuous hormone treatment blocked osteoblast differentiation, the cells were acutely treated for 24 h with 1,25(OH)2 D3 at culture periods when the cells proliferate (day 5), a culture period when the cells cease further cell division and are increasing in the expression of their differentiated functions (day 17), and a culture period when the cells are encapsulated within a mineralized extracellular matrix (day 30). Inhibition of the expression of collagen, osteocalcin, and osteopontin were observed at days 17 and 30, while no effect could be detected for the 5-day cultures. To further define whether the inhibitory effect was specific for cells expressing their differentiated phenotype, 1,25(OH)2 D3 treatment was initiated at day 17 and continued to day 30 after the cells have established their collagenous matrix. In these experiments further collagenous matrix deposition, mineral deposition, alkaline phosphatase activity, and osteocalcin synthesis were also inhibited after the hormone treatment was initiated. These results, in summary, show that 1,25(OH)2 D3 in primary avian osteoblast cultures derived from 17-day embryonic calvaria inhibits the expression of several genes associated with differentiated osteoblast function and inhibit extracellular matrix mineral deposition.

Osteocalcin is one of the major noncollagenous proteins specific to mineralized connective tissues of vertebrates. A cDNA clone encoding the chicken osteocalcin gene was isolated, and the complete coding sequence for the 97-amino-acid pre-pro-osteocalcin was deduced. The 48-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Lys-Arg sequence preceding the NH2-terminal His of the mature 49-amino-acid chicken osteocalcin, which is believed to be necessary for pro-peptide cleavage. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, including Phe at -16, which targets vitamin K-dependent gamma-carboxylation of the three specific Glu residues at positions 17, 21, and 24 in the mature protein. Northern blots of total RNA were prepared from embryonic and adult chicken tissues (bone, brain, heart, intestine, kidney, muscle) and probed with chicken osteocalcin cDNA. The appearance of a single 0.5 kb mRNA species confirms that bone is the major site of osteocalcin expression in vivo. In primary osteoblasts isolated from 17-day embryonic chicken calvaria, an osteocalcin mRNA of similar size is expressed concurrently with culture mineralization in vitro. Hypertrophic chondrocytes from 12-day ventral vertebrae and from the cephalic half of 17-day caudal sternae also express osteocalcin mRNA, but nonhypertrophic chondrocytes from the caudal half of 17-day sternae do not express osteocalcin mRNA.

A variable response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] hormone treatment was observed for osteoblast cell populations isolated from 12- and 17-day-old embryonic chick calvariae. The younger embryonic cell population showed 2- and 5-fold inductions of osteocalcin and osteopontin gene expression, respectively, and a 25% inhibition of collagen gene expression when treated with 1,25-(OH)2D3. In contrast, these same genes all displayed approximately 80% inhibition of their expression when the older embryonic cell populations were treated with hormone. The hormone response was related to the appearance of the vitamin D3 receptor (VDR) and the developmental state of teh two cell populations by assessing the numbers of cells that were immunologically labeled for two osteoblast lineage, stage-specific surface makers (alkaline phosphatase and SB-5, an osteocyte marker) and the VDR. Using the sequence of marker presentation, with VDR appearing first, followed by alkaline phosphatase and then SB-5, models were tested using logistic regression analysis to validate this order of marker presentation and establish that the two embryonic ages of the cell populations represent discrete stages of their lineage. This analysis indicated that 1,25-(OH)2D3 treatment progressed the 12-day-old embryo cell populations along their lineage and that the hormone promoted the appearance of its own receptor (P < 0.001) However, the appearance of the VDR does not appear to be a determinant in the variable responses of the different embryonic aged cell populations to the hormone. These data quantitatively establish the unique nature of osteoblast cell populations within their lineage progression for cells isolated from embryos of different ages, such that cell populations isolated from younger embryos are comprised of primarily presumptive or immature osteoblasts, whereas cells isolated from older embryos are comprised of mature osteoblasts. These data also demonstrate that the genomic effects of 1,25-(OH)2D3 are dependent on the developmental stage of the osteoblast lineage, and the stimulatory actions of the hormone are targeted to immature osteoblasts, whereas the effect of the hormone on mature osteoblasts is inhibitory.