Deletion of the membrane complement inhibitor CD59a drives age and gender-dependent alterations to bone phenotype in mice

Degenerative joint diseases such as osteoarthritis are characterised by aberrant region-specificboneformationand abnormal bone mineral content. A recent study suggested a role for the complement membrane attack com-plex in experimental models of osteoarthritis. Since CD59a is the principal regulator of the membrane attackcomplex in mice, we evaluated the impact of CD59a gene deletion upon maintenance of bone architecture.In vivobone morphology analysis revealed that male CD59a-deficient mice have increased femur length and cor-tical bone volume, albeit with reduced bone mineral density. However, this phenomenon was not observed infemale mice. Histomorphometric analysis of the trabecular bone showed increased rates of bone homeostasis,with both increased bone resorption and mineral apposition rate in CD59a-deficient male mice. When bonecells were studied in isolation,in vitroosteoclastogenesis was significantly increased in male CD59a-deficientmice, although osteoblast formation was not altered.Our data reveal, for thefirst time, that CD59a is a regulator of bone growth and homeostasis. CD59a ablation inmale mice results in longer and wider bones, but with less density, which is likely a major contributing factorfor their susceptibility to osteoarthritis. Thesefindings increase our understanding of the role of complementregulation in degenerative arthritis

Acute Multiple Organ Failure in Adult Mice Deleted for the Developmental Regulator Wt1

There is much interest in the mechanisms that regulate adult tissue homeostasis and their relationship to processes governing foetal development. Mice deleted for the Wilms' tumour gene, Wt1, lack kidneys, gonads, and spleen and die at mid-gestation due to defective coronary vasculature. Wt1 is vital for maintaining the mesenchymal–epithelial balance in these tissues and is required for the epithelial-to-mesenchyme transition (EMT) that generates coronary vascular progenitors. Although Wt1 is only expressed in rare cell populations in adults including glomerular podocytes, 1% of bone marrow cells, and mesothelium, we hypothesised that this might be important for homeostasis of adult tissues; hence, we deleted the gene ubiquitously in young and adult mice. Within just a few days, the mice suffered glomerulosclerosis, atrophy of the exocrine pancreas and spleen, severe reduction in bone and fat, and failure of erythropoiesis. FACS and culture experiments showed that Wt1 has an intrinsic role in both haematopoietic and mesenchymal stem cell lineages and suggest that defects within these contribute to the phenotypes we observe. We propose that glomerulosclerosis arises in part through down regulation of nephrin, a known Wt1 target gene. Protein profiling in mutant serum showed that there was no systemic inflammatory or nutritional response in the mutant mice. However, there was a dramatic reduction in circulating IGF-1 levels, which is likely to contribute to the bone and fat phenotypes. The reduction of IGF-1 did not result from a decrease in circulating GH, and there is no apparent pathology of the pituitary and adrenal glands. These findings 1) suggest that Wt1 is a major regulator of the homeostasis of some adult tissues, through both local and systemic actions; 2) highlight the differences between foetal and adult tissue regulation; 3) point to the importance of adult mesenchyme in tissue turnover

The essential requirement for Runx1 in the development of the sternum

AbstractRunx1 is highly expressed in chondroprogenitor and osteoprogenitor cells and in vitro experiments suggest that Runx1 is important in the early stages of osteoblast and chondrocyte differentiation. However, because Runx1 knockout mice are early embryonic lethal due to failure of hematopoiesis, the role of Runx1 in skeletogenesis remains unclear. We studied the role of Runx1 in skeletal development using a Runx1 reversible knockout mouse model. By crossing with Tie2-Cre deletor mice, Runx1 expression was selectively rescued in the endothelial and hematopoietic systems but not in the skeleton. Although Runx1Re/Re embryos survived until birth and had a generally normal skeleton, the development of mineralization in the sternum and some skull elements was significantly disrupted. In contrast to wild-type embryos, the sternum of E17.5 Runx1Re/Re embryos showed high levels of Sox-9 and collagen type II expression and lack of development of hypertrophic chondrocytes. In situ hybridization analysis demonstrated that, in contrast to the vertebrae and long bones, the sternum of wild-type embryos expresses high levels of Runx1, but not Runx2, the master regulator of skeletogenesis. Thus, although Runx1 is not essential for major skeletal development, it does play an essential role in the development of the sternum and some skull elements

Endothelial nitric oxide synthase is not essential for nitric oxide production by osteoblasts subjected to fluid shear stress in vitro

Endothelial nitric oxide synthase (eNOS) has long been held responsible for NO production by mechanically stimulated osteoblasts, but this has recently been disputed. We investigated whether one of the three known NOS isoforms is essential for NO production by mechanically stimulated osteoblasts in vitro and revisited the bone phenotype of the eNOS-/- mouse. Osteoblasts, obtained as outgrowths from mouse calvaria or long bones of wild-type (WT), eNOS-/-, inducible NOS-/- (iNOS-/-), or neuronal NOS-/- (nNOS-/-) mice, were subjected to mechanical stimulation by means of pulsating fluid flow (PFF); and NO production was determined. Tibiae and femora from 8-week-old mice were subjected to μCT and three-point bending tests. Deletion of single NOS isoforms did not lead to significant upregulation of alternate isoforms in cultured osteoblasts from WT, eNOS-/-, iNOS-/-, or nNOS-/- mice. Expression of eNOS mRNA in osteoblasts was below our detection limit, and no differences in growth between WT and eNOS-/- osteoblasts were found. PFF increased NO production by approximately fourfold in WT and eNOS-/- osteoblasts and significantly stimulated NO production in iNOS-/- and nNOS-/- osteoblasts. Tibiae and femora from WT and eNOS-/- mice showed no difference in bone volume and architecture or in mechanical parameters. Our data suggest that mechanical stimuli can enhance NO production by cultured osteoblasts singly deficient for each known NOS isoform and that lack of eNOS does not significantly affect bone mass and strength at 8 weeks of age. Our data challenge the notion that eNOS is a key effector of mechanically induced bone maintenance.</p

Endothelial nitric oxide synthase is not essential for nitric oxide production by osteoblasts subjected to fluid shear stress in vitro

Endothelial nitric oxide synthase (eNOS) has long been held responsible for NO production by mechanically stimulated osteoblasts, but this has recently been disputed. We investigated whether one of the three known NOS isoforms is essential for NO production by mechanically stimulated osteoblasts in vitro and revisited the bone phenotype of the eNOS-/- mouse. Osteoblasts, obtained as outgrowths from mouse calvaria or long bones of wild-type (WT), eNOS-/-, inducible NOS-/- (iNOS-/-), or neuronal NOS-/- (nNOS-/-) mice, were subjected to mechanical stimulation by means of pulsating fluid flow (PFF); and NO production was determined. Tibiae and femora from 8-week-old mice were subjected to μCT and three-point bending tests. Deletion of single NOS isoforms did not lead to significant upregulation of alternate isoforms in cultured osteoblasts from WT, eNOS-/-, iNOS-/-, or nNOS-/- mice. Expression of eNOS mRNA in osteoblasts was below our detection limit, and no differences in growth between WT and eNOS-/- osteoblasts were found. PFF increased NO production by approximately fourfold in WT and eNOS-/- osteoblasts and significantly stimulated NO production in iNOS-/- and nNOS-/- osteoblasts. Tibiae and femora from WT and eNOS-/- mice showed no difference in bone volume and architecture or in mechanical parameters. Our data suggest that mechanical stimuli can enhance NO production by cultured osteoblasts singly deficient for each known NOS isoform and that lack of eNOS does not significantly affect bone mass and strength at 8 weeks of age. Our data challenge the notion that eNOS is a key effector of mechanically induced bone maintenance.</p

Endothelial nitric oxide synthase is not essential for nitric oxide production by osteoblasts subjected to fluid shear stress in vitro

Endothelial nitric oxide synthase (eNOS) has long been held responsible for NO production by mechanically stimulated osteoblasts, but this has recently been disputed. We investigated whether one of the three known NOS isoforms is essential for NO production by mechanically stimulated osteoblasts in vitro and revisited the bone phenotype of the eNOS-/- mouse. Osteoblasts, obtained as outgrowths from mouse calvaria or long bones of wild-type (WT), eNOS-/-, inducible NOS-/- (iNOS-/-), or neuronal NOS-/- (nNOS-/-) mice, were subjected to mechanical stimulation by means of pulsating fluid flow (PFF); and NO production was determined. Tibiae and femora from 8-week-old mice were subjected to μCT and three-point bending tests. Deletion of single NOS isoforms did not lead to significant upregulation of alternate isoforms in cultured osteoblasts from WT, eNOS-/-, iNOS-/-, or nNOS-/- mice. Expression of eNOS mRNA in osteoblasts was below our detection limit, and no differences in growth between WT and eNOS-/- osteoblasts were found. PFF increased NO production by approximately fourfold in WT and eNOS-/- osteoblasts and significantly stimulated NO production in iNOS-/- and nNOS-/- osteoblasts. Tibiae and femora from WT and eNOS-/- mice showed no difference in bone volume and architecture or in mechanical parameters. Our data suggest that mechanical stimuli can enhance NO production by cultured osteoblasts singly deficient for each known NOS isoform and that lack of eNOS does not significantly affect bone mass and strength at 8 weeks of age. Our data challenge the notion that eNOS is a key effector of mechanically induced bone maintenance.</p