Original ArticlesNitric Oxide Synthase Expression in Bone Cells
Introduction
Nitric oxide (NO) plays an important role as a mediator of diverse cellular functions in a variety of cell types, and is an effector of cytotoxicity in macrophage killing.19., 20. Its production is mediated by nitric oxide synthase, which is broadly divided into constitutive (cNOS) and inducible (iNOS) isoforms. The three most widely studied nitric oxide synthases are those present in the endothelium, brain, and macrophages.20 A role for nitric oxide in bone has recently been suggested. Nitric oxide has been shown to modulate osteoclast recruitment and activity,3., 6., 11., 16., 23. and nitric oxide is also thought to play a role in osteoblast function.
In vitro studies suggest that high levels of nitric oxide, as typically generated by iNOS, suppress osteoblast proliferation and differentiation, but that lower levels may stimulate osteoblast function.24., 25. Recently, osteoblastic cells have been shown to respond to mechanical strain and shear stress, resulting in a rapid increase in nitric oxide production.8., 22., 27. Furthermore, in a rat-tail model of mechanical stimulation, the induction of new bone formation by mechanical loading was suppressed by administration of a nitric oxide synthase inhibitor, l-NMMA, before, but not after, a single episode of loading.7 This suggests that the osteogenic response of bone to mechanical loading is dependent upon early production of nitric oxide.
We have also found, using an experimental model of inflammatory bowel disease, that cancellous bone formation is markedly suppressed in the presence of active colonic inflammation.15 iNOS is known to be induced in osteoblasts by cytokines, such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and interferon (INF).5., 10., 24. Thus it is likely that these inflammatory mediators may induce iNOS expression in osteoblasts, and that the NO may be responsible for the suppression of bone formation.
Despite the apparent importance of nitric oxide in bone physiology, data on the localization of expression of nitric oxide synthase in bone cells in vivo are sparse.9 In this communication, we have used immunohistochemistry to localize the expression of the three main nitric oxide synthases in the bone cells in normal rats and humans. We also investigated the expression of iNOS in osteoblasts in tibiae of rats with experimental colitis.
Section snippets
NOS expression in normal bones
Four 13-week-old female Wistar rats were used. The rats were perfusion fixed with 4% paraformaldehyde in phosphate buffered saline (PBS). Tibiae and caudal vertebrae were then postfixed in the same fixative for 3 h and decalcified for 10 days in 10% ethylenediamine tetraacetic acid (EDTA)/PBS at 4°C. Three-day-old neonatal rats were also studied. These were fixed in 4% paraformaldehyde in PBS for 3 h and then decalcified for 24 h in 10% EDTA/PBS at 4°C. The rat tissues were processed through
Results
Specificity of staining was confirmed for the three antibodies used. bNOS positivity was observed in the molecular and granular layers of the cerebellum, and in ganglion cells and nonadrenergic, noncholinergic fibers of the colon (Figure 1). These cells did not stain with eNOS or iNOS. eNOS, but not bNOS or iNOS, positivity was observed in the vessels in the hilum of the ovary (Figure 1). eNOS positivity was consistently noted in the endothelium and media of blood vessels in the periosteal
NOS expression in tibiae of rats with experimental colitis
Plump cuboidal osteoblasts in the primary spongiosa and on the corticoendosteal surface of tibiae of animals with experimental colitis exhibited strong positivity with iNOS (Figure 5). Chondrocytes and young osteocytes were also positive for iNOS. A few osteoclasts also stained weakly positively, as in the normal animals. As in normal animals, cuboidal osteoblasts were consistently negative in the control animals treated with intrarectal vehicle.
Osteoclastoma
A high proportion of mononuclear stromal cells in the osteoclastomas exhibited strong cytoplasmic positivity for eNOS but not bNOS. The osteoclasts were either negative or showed weak positivity for eNOS (Figure 5). Unlike eNOS, mononuclear stromal cells in the osteoclastomas were negative for iNOS, but the numerous osteoclasts that are characteristic of the tumor showed strong positivity (Figure 5).
Discussion
We present the first detailed immunohistochemical study of nitric oxide synthases in bone cells in rats and humans in vivo. Previous investigations of the expression of nitric oxide synthases in bone have centered on cells in tissue culture.3., 24., 25. The main enzyme that has been detected in bone cells using these techniques is the inducible isoform, together with smaller quantities of bNOS.24., 25. This is not surprising, because iNOS is upregulated by numerous cytokines present in tissue
Acknowledgements
The authors wish to thank V. Emmons for assistance with preparation of the manuscript. This work was supported by the Arthritis and Rheumatism Council and the Wellcome Trust.
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