To assess the effects of the orphan nuclear Estrogen receptor-related receptor gamma (ERR?) deficiency on skeletal development and bone turnover, we utilized an ERR? global knockout mouse line. While we observed no gross morphological anomalies or difference in skeletal length in newborn mice, by 8 weeks of age ERR? +/- males but not females exhibited increased trabecular bone, which was further increased by 14 weeks. The increase in trabecular bone was due to an increase in active osteoblasts on the bone surface, without detectable alterations in osteoclast number or activity. Consistent with the histomorphometric results, we observed an increase in gene expression of the bone formation markers alkaline phosphatase (Alp) and bone sialoprotein (Bsp) in bone and increase in serum ALP, but no change in the osteoclast regulators receptor activator of NF-?B ligand (RANKL) and osteoprotegerin (OPG) or the resorption marker carboxy-terminal collagen crosslinks (CTX). More colony forming units-alkaline phosphatase and -osteoblast (CFU-ALP, CFU-O respectively) but not CFU-fibroblast (CFU-F) formed in ERR? +/- versus ERR? +/+ stromal cell cultures, suggesting that ERR? negatively regulates osteoblast differentiation and matrix mineralization but not mesenchymal precursor number. By co-immunoprecipitation experiments, we found that ERR? and RUNX2 interact in an ERR? DNA binding domain (DBD)-dependent manner. Treatment of post-confluent differentiating bone marrow stromal cell cultures with Runx2 antisense oligonucleotides resulted in a reduction of CFU-ALP/CFU-O in ERR? +/- but not ERR? +/+ mice compared to their corresponding sense controls. Our data indicate that ERR? is not required for skeletal development but is a sex-dependent negative regulator of postnatal bone formation, acting in a RUNX2- and apparently differentiation stage-dependent manner.
Adult Ibsp-knockout mice (BSP-/-) display shorter stature, lower bone turnover and higher trabecular bone mass than wild type, the latter resulting from impaired bone resorption. Unexpectedly, BSP knockout also affects reproductive behavior, as female mice do not construct a proper "nest" for their offsprings. Multiple crossing experiments nonetheless indicated that the shorter stature and lower weight of BSP-/- mice, since birth and throughout life, as well as their shorter femur and tibia bones are independent of the genotype of the mothers, and thus reflect genetic inheritance. In BSP-/- newborns, µCT analysis revealed a delay in membranous primary ossification, with wider cranial sutures, as well as thinner femoral cortical bone and lower tissue mineral density, reflected in lower expression of bone formation markers. However, trabecular bone volume and osteoclast parameters of long bones do not differ between genotypes. Three weeks after birth, osteoclast number and surface drop in the mutants, concomitant with trabecular bone accumulation. The growth plates present a thinner hypertrophic zone in newborns with lower whole bone expression of IGF-1 and higher IHH in 6 days old BSP-/- mice. At 3 weeks the proliferating zone is thinner and the hypertrophic zone thicker in BSP-/- than in BSP+/+ mice of either sex, maybe reflecting a combination of lower chondrocyte proliferation and impaired cartilage resorption. Six days old BSP-/- mice display lower osteoblast marker expression but higher MEPE and higher osteopontin(Opn)/Runx2 ratio. Serum Opn is higher in mutants at day 6 and in adults. Thus, lack of BSP alters long bone growth and membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth. Compensatory high Opn may allow normal endochondral development in BSP-/- mice, while impairing primary mineralization.
While the role of estrogen receptor-related receptor alpha (ERR?) in chondrogenesis has been investigated, the involvement of ERR gamma (ERR?) has not been determined. To assess the effect of increased ERR? activity on cartilage development in vivo, we generated two transgenic (Tg) lines overexpressing ERR?2 via a chondrocyte-specific promoter; the two lines exhibited ?3 and ?5 fold increased ERR?2 protein expression respectively in E14.5 Tg versus wild type (WT) limbs. On postnatal day seven (P7), we observed a 4-10% reduction in the size of the craniofacial, axial and appendicular skeletons in Tg versus WT mice. The reduction in bone length was already present at birth and did not appear to involve bones that are derived via intramembranous bone formation as the bones of the calvaria, clavicle, and the mandible developed normally. Histological analysis of P7 growth plates revealed a reduction in the length of the Tg versus WT growth plate, the majority of which was attributable to a reduced proliferative zone. The reduced proliferative zone paralleled a decrease in the number of Ki67-positive proliferating cells, with no significant change in apoptosis, and was accompanied by large cell-free swaths of cartilage matrix, which extended through multiple zones of the growth plate. Using a bioinformatics approach, we identified known chondrogenesis-associated genes with at least one predicted ERR binding site in their proximal promoters, as well as cell cycle regulators known to be regulated by ERR?. Of the genes identified, Col2al, Agg, Pth1r, and Cdkn1b (p27) were significantly upregulated, suggesting that ERR?2 negatively regulates chondrocyte proliferation and positively regulates matrix synthesis to coordinate growth plate height and organization.
We reported previously that the orphan nuclear receptor, estrogen receptor-related receptor ? (ERR?), is expressed in articular chondrocytes and is dysregulated in a mouse model of inflammatory arthritis. The aim of this study, therefore, was to determine whether ERR? is also dysregulated in patients with osteoarthritis (OA).
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