No abstract

Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults.

A unique mutation in LRP5 is associated with high bone mass in man. Transgenic mice expressing this LRP5 mutation have a similar phenotype with high bone mass and enhanced strength. These results underscore the importance of LRP5 in skeletal regulation and suggest targets for therapies for bone disease.A mutation (G171V) in the low-density lipoprotein receptor related protein 5 (LRP5) has been associated with high bone mass (HBM) in two independent human kindreds. To validate the role of the mutation, several lines of transgenic mice were created expressing either the human LRP5 G171V substitution or the wildtype LRP5 gene in bone. Volumetric bone mineral density (vBMD) analysis by pQCT showed dramatic increases in both total vBMD (30 -55%) and trabecular vBMD (103-250%) of the distal femoral metaphysis and increased cortical size of the femoral diaphysis in mutant G171V transgenics at 5, 9, 17, 26, and 52 weeks of age (p < 0.01 for all). In addition, high-resolution microcomputed tomography (microCT) analysis of the distal femorae and lumbar vertebrae revealed an increase (110 -232%) in trabecular bone volume fraction caused by both increased trabecular number (41-74%) and increased trabecular thickness (34 -46%; p < 0.01 for all) in the mutant G171V mice. The increased bone mass was associated with significant increases in vertebral compressive strength (80 -140%) and the increased cortical size with significant increases in femoral bending strength (50 -130%). There were no differences in osteoclast number at 17 weeks of age. However, compared with littermate controls, the mutant G171V transgenic mice showed an increase in actively mineralizing bone surface, enhanced alkaline phosphatase staining in osteoblasts, and a significant reduction in the number of TUNEL-positive osteoblasts and osteocytes. These results suggest that the increased bone mineral density in mutant G171V mice was caused by increased numbers of active osteoblasts, which could in part be because of their increased functional lifespan. While slight bone anabolic activity was observed from overexpression of the wildtype LRP5 gene, it is clear that the G171V mutation, rather than overexpression of the receptor itself, is primarily responsible for the dramatic HBM bone effects. Together, these findings establish the importance of this novel and unexpected role of a lipoprotein receptor in regulating bone mass and afford a new model to explore LRP5 and its recent association with Wnt signaling in bone biology. (J Bone Miner Res 2003;18:960 -974)

IntroductionDegenerative joint diseases including osteoarthritis (OA) are common, particularly in the elderly. Early signs of OA include progressive loss from articular cartilage of the proteoglycan aggrecan, reflected by a loss of safranin O staining, excessive damage to type II collagen, and general degeneration and fibrillation of the cartilage surface, resulting ultimately in a loss of articular cartilage (1).One of the primary targets of this disease is type II collagen, the major structural collagen found in articular cartilage in healthy individuals. There is ordinarily a strict balance between the production of type II collagen and degradation of this protein by catabolic enzymes during normal remodeling of cartilage (1). Pathological conditions such as OA are characterized by a loss of this balance with increased proteolysis (1-5) and upregulation of the synthesis of type II procollagen (5) and aggrecan (6).Matrix metalloproteinases (MMPs) comprise a family of zinc-dependent enzymes that degrade extracellular matrix components. MMPs are synthesized in articulating joints by synovial cells and chondrocytes. In mature articular cartilage, chondrocytes maintain the cartilage-specific matrix phenotype. Elevated expression of MMPs is associated with cartilage degradation (1). MMP-13, also known as human collagenase-3, is thought to play an important role in type II collagen degradation in articular cartilage and especially in OA (4, 7-9). Type II collagen is the preferred substrate for MMP-13 (4, 7, 10). Expression and contents of MMP-1 (collagenase-1) and 11,12), expression of MMP-8 (collagenase-2), and collagenase activity (4,8) are upregulated in human OA cartilage.Spontaneous development of focal sites degeneration has been described in aging guinea pigs (13). Sublines of the inbred STR/ORT strain of mice also develop spontaneous OA with aging (14). Mice exhibit upregulated expression of MMP-13 and collagenase activity is upregulated in focal lesions (15). In guinea pigs, MMP-1 and MMP-13 are also upregulated in OA lesions associated with increased collagenase activity (16). It has been suggested that increased collagenase-3 (MMP-13) activity plays a pivotal role in the pathogenesis of osteoarthritis (OA). We have used tetracycline-regulated transcription in conjunction with a cartilage-specific promoter to target a constitutively active human MMP-13 to the hyaline cartilages and joints of transgenic mice. Postnatal expression of this transgene resulted in pathological changes in articular cartilage of the mouse joints similar to those observed in human OA. These included characteristic erosion of the articular cartilage associated with loss of proteoglycan and excessive cleavage of type II collagen by collagenase, as well as synovial hyperplasia. These results demonstrate that excessive MMP-13 activity can result in articular cartilage degradation and joint pathology of the kind observed in OA, suggesting that excessive activity of this proteinase can lead to this disease.

Parathyroid hormone (PTH) induces hypercalcemia in part through increasing bone resorption mediated by osteoclasts. Receptors for PTH are present not on osteoclasts, however, but on mesenchymal cells of the osteoblast lineage and stromal cells in the bone marrow (1). PTH must therefore act directly on these mesenchymal cells, which then -through direct cell-cell contact mediated by cellbound ligands such as osteoclast differentiation factor (ODF) and/or production of soluble ligands -modulates the activity of existing osteoclasts and the differentiation of osteoclasts from precursor cells (2, 3).Shortly after the discovery of the animal collagenases by Gross and Lapière (4), Walker et al. (5) found that bones removed from mice injected with doses of PTH sufficient to elevate serum calcium levels by ∼4 mg/dl produce collagenase activity at levels much higher than in bones removed from uninjected mice. The time course of the effects on inducing hypercalcemia and collagenase production was different in that increased collagenase production was not detected until several hours after serum calcium levels reached their peak (6). It had also been shown that the sustained hypercalcemia induced by PTH was obviated by inhibitors of mRNA synthesis (actinomycin D) (7,8) or protein synthesis (puromycin) (9), suggesting that, at least in part, PTH-induced hypercalcemia is dependent upon the synthesis of a protein in bone; collagenase was a candidate. In subsequent studies from several laboratories it was shown that in organ cultures of bone fragments, collagenase was released into the ambient medium and collagenase production could be stimulated by PTH (10-14). Later, it was possible to clone a collagenase gene from a cDNA library prepared from a rat osteogenic sarcoma cell line stimulated with PTH (15). A mouse collagenase cDNA was subsequently cloned (16) using the rat cDNA probe (15), and it was determined that the rodent enzymes had only ∼50% amino acid sequence similarity to human collagenase-1 (matrix metalloproteinase-1 [MMP-1]) but had high similarity to what was later identified as collagenase-3 (MMP-13) in humans (17). Osteoclasts, in addition to producing cysteine proteinases, also produce MMPs such as the 92-kDa gelatinase (gelatinase B) (18) and one of the membrane-bound (MT) MMPs, . In all but a few reported studies (e.g., ref. 20), however, it has not been possible to identify the expression of specific collagenases in osteoclasts using cDNA or cRNA probes. In contrast, several investigators have been readily able to measure collagenase produced by osteoblasts or stromal fibroblasts from different species, as described above, either constitutively or induced by several ligands, including PTH. When osteoblasts are exposed to PTH, they start producing collagenase and stop synthesizing collagen (21).During normal embryonic development, collagenase is expressed in cells (osteoblasts, stromal cells) in the bone shaft and in hypertrophic chondrocytes of the distal growth plate (22-25). More proximal hypertrophic cho...