We have previously described osteoblast/osteocyte factor 45 (OF45), a novel bone-specific extracellular matrix protein, and demonstrated that its expression is tightly linked to mineralization and bone formation. In this report, we have cloned and characterized the mouse OF45 cDNA and genomic region. Mouse OF45 (also called MEPE) was similar to its rat orthologue in that its expression was increased during mineralization in osteoblast cultures and the protein was highly expressed within the osteocytes that are imbedded within bone. To further determine the role of OF45 in bone metabolism, we generated a targeted mouse line deficient in this protein. Ablation of OF45 resulted in increased bone mass. In fact, disruption of only a single allele of OF45 caused significantly increased bone mass. In addition, knockout mice were resistant to aging-associated trabecular bone loss. Cancellous bone histomorphometry revealed that the increased bone mass was the result of increased osteoblast number and osteoblast activity with unaltered osteoclast number and osteoclast surface in knockout animals. Consistent with the bone histomorphometric results, we also determined that OF45 knockout osteoblasts produced significantly more mineralized nodules in ex vivo cell cultures than did wild type osteoblasts. Osteoclastogenesis and bone resorption in ex vivo cultures was unaffected by OF45 mutation. We conclude that OF45 plays an inhibitory role in bone formation in mouse.The constant modulation of the balance between skeletal strength and mineral availability in bone is effected by competing cell types in response to physiological needs. Osteoblasts produce, organize, and mineralize bone matrix in forming bone. Osteoclasts break down matrix by forming a lytic pocket in which bone is degraded and calcium is released. The generation and activity of these cell types is tightly regulated to provide equilibrium between formation and resorption and, thereby, an appropriate balance of strength and mineral release. Under certain conditions, such as aging, postmenopausal estrogen deficiency, or some pathophysiological states, there can exist an imbalance between bone resorption and bone formation. As a result, skeletal mass and strength are compromised and osteoporotic fractures can occur in the afflicted individuals.Bone is produced by the organization and mineralization of the extracellular matrix produced by osteoblasts. The major component of the extracellular matrix of these cells is Type I collagen, which functions as a scaffold for new bone. In addition, non-collagenous matrix proteins have been identified that influence the operations of bone turnover, formation, and repair. These proteins are generally acidic and highly posttranslationally modified by phosphorylation, glycosylation, or sulfation (1).Targeted deletion of extracellular matrix genes in mice has been a useful method to determine the in vivo functions of several matrix proteins. For example, osteocalcin is an abundant gamma carboxyl glutamic acid-containing bone matrix...

We describe the cloning and characterization of a novel bone-specific cDNA predicted to encode an extracellular matrix protein. This cDNA was identified by subtractive hybridization based upon its high expression in bone marrow-derived osteoblasts. By Northern blot analysis, we detected a single 2-kilobase mRNA transcript in bone, whereas no expression was detected in other tissues. Immunohistochemistry revealed that the protein was expressed highly in osteocytes within trabecular and cortical bone. RNA and protein expression analysis using in vivo marrow ablation as a model of bone remodeling demonstrated that this gene was expressed only in cells that were embedded within bone matrix in contrast to the earlier expression of known osteoblast markers. The cDNA was predicted to encode a serine/glycine-rich secreted peptide containing numerous potential phosphorylation sites and one RGD sequence motif. The interaction of RGD domain-containing peptides with integrins has been shown previously to regulate bone remodeling by promoting recruitment, attachment, and differentiation of osteoblasts and osteoclasts. Secretion of this RGD-containing protein from osteocytes has the potential to regulate cellular activities within the bone environment and thereby may impact bone homeostasis. We propose the name OF45 (osteoblast/osteocyte factor of 45 kDa) for this novel cDNA.Bone is a highly dynamic tissue that undergoes continual processes of remodeling and modeling (1). In the growing skeleton, the amount of mineralized bone formed exceeds the amount lost through resorption, whereas in the mature adult lost bone mineral is precisely balanced by an equivalent amount of formation, thereby preserving the integrity of the skeleton. Under certain conditions such as aging, postmenopausal estrogen deficiency, or prolonged steroid treatment, the amount of bone formed is not sufficient to compensate for the quantity lost by resorption. Over time, this imbalance results in reduced bone mass, compromises the structural integrity of the skeleton, and, ultimately, can lead to osteoporotic bone fractures. Osteoporosis and the resulting fractures are causes of significant morbidity and mortality within the aging population.Bone remodeling is a very complex process of tightly coordinated action by the bone resorbing osteoclasts and the bone forming osteoblasts. Osteoblasts are derived from a mesenchymal cell lineage and are responsible for the formation of new bone matrix in their differentiated state (2). In addition, osteoblasts produce factors that regulate the formation of osteoclasts and osteoclastic bone resorbing activity in response to endocrine signals such as parathyroid hormone and 1,25-dihydroxyvitamin D 3 . It has been postulated that bone loss associated with aging is caused by a defect in the osteoblast cell lineage (3-6). Either the mesenchymal precursor population is insufficient or has lost the capacity to proliferate and/or differentiate into sufficient numbers of functioning osteoblasts.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) gene superfamily of growth and differentiation factors. Members of the BMP family were originally cloned and characterized by their ability to induce ectopic bone formation. Of the various BMPs cloned, the bone inductive ability of BMP-7 (OP-1) and BMP-2 has been well characterized. Both BMP-7 and -2 have been shown to have clinical utility in the healing of non-union fractures. However, in spite of the various advances in BMP research, the physiological regulation of BMPs is not well understood. Here we studied the expression of BMP-7 by cloning a 4.6-kB fragment of the human BMP-7 promoter (hBMP-7p) and placing it upstream of a luciferase reporter. The promoter reporter construct was stably transfected into different cell backgrounds and its regulation by various factors was investigated. We show that retinoic acid (RA) treatment results in an upregulation of the hBMP-7p reporter activity. This regulation of the hBMP-7p was further confirmed by Northern blot, PCR, and Western blot analyses, which showed an increase in both BMP-7 mRNA and protein expression upon treatment with RA. We further show that RA specifically upregulates expression of osteocalcin via activation of BMP-7 mRNA and protein in vitro. Similarly, prostaglandin E(2) (PGE(2)) treatment increases BMP-7 mRNA and protein levels, but does not transcriptionally activate the hBMP-7p. Additionally, in vivo expression of BMP-7 in bone was increased upon PGE(2) treatment. In conclusion, RA and PGE(2) upregulate BMP-7 protein expression both in vitro and in vivo.