Rosen, Clifford J.

Abstract Introduction: The metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A) functions to enhance local insulin-like growth factor (IGF)-I bioavailability through cleavage of inhibitory IGF binding proteins. Because IGF-I is an important regulator of skeletal growth and remodeling and PAPP-A is highly expressed by osteoblastic cells, we hypothesized that, in the absence of PAPP-A, bone physiology would be compromised because of a blunting of local IGF-I action even in the presence of normal circulating IGF-I levels. Materials and Methods: pQCT, μCT, histomorphometry, and mechanical strength testing were performed on bones from PAPP-A knockout (KO) mice and wildtype (WT) littermates at 2–12 mo of age. IGF-I levels and bone formation and resorption markers were determined in sera from these animals. Results: Volumetric BMD in PAPP-A KO mice measured by pQCT at the femoral midshaft, which is primarily cortical bone, was 10% less than WT at 2 mo. This difference was maintained at 4, 6, and 12 mo. Cortical thickness at this site was similarly decreased. On the other hand, trabecular bone at the distal femur (pQCT) and in the tibia (μCT) showed age-progressive decreases in bone volume fraction in PAPP-A KO compared with WT mice. Tibial μCT indicated a 46% relative decrease in trabecular bone volume/total volume (BV/TV) and a 28% relative decrease in trabecular thickness in PAPP-A KO compared with WT mice at 6 mo. These trabecular deficiencies in PAPP-A KO mice corresponded to a weakening of the bone. Serum markers and bone histomorphometry indicated that the primary impact of PAPP-A is on skeletal remodeling resulting in a state of low-turnover osteopenia in adult PAPP-A KO mice. Circulating IGF-I levels were not altered in PAPP-A KO mice. Conclusions: PAPP-A is a bone growth regulatory factor in vivo and, in its absence, mice show skeletal insufficiency in mass, density, architecture, and strength. The data suggest a primary role for PAPP-A in modulating local IGF bioavailability for trabecular bone remodeling.

Abstract ERα acts either through classical (ERE-mediated) or nonclassical (non-ERE) pathways. The generation of mice carrying a mutation that eliminates classical ERα signaling presents a unique opportunity to study the relative roles of these pathways in bone. This study defines the skeletal phenotype and responses to ovariectomy and estrogen replacement in these mice. Introduction: Estrogen receptor α (ERα) can act either through classical estrogen response elements (EREs) or through non-ERE (nonclassical) pathways. To unravel these in bone, we crossed mice heterozygous for a knock-in mutation abolishing ERE binding (nonclassical ERα knock-in [NERKI]) with heterozygote ERα knockout mice and studied the resulting female ERα+/+, ERα+/NERKI, and ERα−/NERKI mice. The only ERα present in ERα−/NERKI mice is incapable of activating EREs but can signal through nonclassical pathways, whereas ERα+/NERKI mice may have a less drastic alteration in the balance between classical and nonclassical estrogen signaling pathways. Materials and Methods: BMD was measured using DXA and pQCT at 3 months of age (n = 46–48/genotype). The mice were randomly assigned to sham surgery, ovariectomy, ovariectomy + estradiol (0.25 μg/day), or ovariectomy + estradiol (1.0 μg/day; n = 10–12/group) and restudied 60 days later. Results and Conclusions: At 3 months of age, both the ERα+/NERKI and ERα−/NERKI mice had deficits in cortical, but not in trabecular, bone. Remarkably, changes in cortical bone after ovariectomy and estrogen replacement in ERα−/NERKI mice were the opposite of those in ERα+/+ mice. Relative to sham mice, ovariectomized ERα−/NERKI mice gained more bone (not less, as in ERα+/+ mice), and estrogen suppressed this increase (whereas augmenting it in ERα+/+ mice). Estrogen also had opposite effects on bone formation and resorption parameters on endocortical surfaces in ERα−/NERKI versus ERα+/+ mice. Collectively, these data show that alteration of the balance between classical and nonclassical ERα signaling pathways leads to deficits in cortical bone and also represent the first demonstration, in any tissue, that complete loss of classical ERE signaling can lead to paradoxical responses to estrogen. Our findings strongly support the hypothesis that there exists a balance between classical and nonclassical ERα signaling pathways, which, when altered, can result in a markedly aberrant response to estrogen.

Abstract IGF-I and their binding proteins are important in bone health. Examination of BMD, osteoblast proliferation, and markers of bone resorption in transgenic mice that constitutively overexpress IGFBP-3 indicates that overexpression of IGFBP-3 increases osteoclast number and bone resorption, impairs osteoblast proliferation, and has a significant negative effect on bone formation. Introduction: Low serum insulin-like growth factor I (IGF-I) levels correlate with an increased risk of osteoporotic fractures. Serum IGF-I is largely bound to IGF-binding protein-3 (IGFBP-3), which can inhibit IGF-I action and enhance delivery of IGF-I to tissues. Its role in bone biology is unclear. Methods: Bone mineral density (BMD), osteoblast proliferation, and markers of bone resorption were examined in transgenic (Tg) mice that constitutively overexpressed human IGFBP-3 cDNA driven by either the cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter. Results: Cultured calvarial osteoblasts from Tg mice expressed the transgene and grew more slowly than cells from wild-type (Wt) mice, and the mitogenic response to IGF-I was attenuated in osteoblasts from Tg mice. Total volumetric BMD and cortical BMD, measured in the femur using peripheral quantitative computed tomography (pQCT) were significantly reduced in both Tg mouse strains compared with Wt mice. PGKBP-3 Tg mice showed the most marked reduction in bone density. Osteocalcin levels were similar in Wt and CMVBP-3 Tg mice but were significantly reduced in PGKBP-3 Tg mice. Urinary deoxypyridinoline and osteoclast perimeter, markers of bone resorption, were significantly increased in both Tg mouse strains compared with Wt mice. Using double labeling with tetracycline, we demonstrated that pericortical and endocortical mineral apposition rate was significantly reduced in PGKBP-3 Tg mice compared with Wt mice. Conclusions: These data show that overexpression of IGFBP-3 increases osteoclast number and bone resorption, impairs osteoblast proliferation, and has a significant negative effect on bone formation.