Role of the nitric oxide synthase pathway in bone cell function and bone disease
Nitric oxide acts as a signalling molecule in several tissues and organ systems and recent work has shown that it is also produced in bone. The aim of this thesis was to investigate the role of the nitric oxide synthase pathway in bone cell function and bone disease. Studies with primary human osteoblasts indicated that they had the ability to produce nitric oxide under conditions of cytokine stimulation using the iNOS pathway and in response to fluid flow using the ecNOS pathway. Cytokine induced nitric oxide had a differentiation-inducing effect as reflected by a decrease in cell proliferation and an increase in the expression of alkaline phosphatase. Further investigations showed that p27Kjpl was induced in response to cytokine stimulation in primary human osteoblasts suggesting that this may have been responsible for cytokine induced inhibition of cell growth. An investigation of the expression of the different isoforms of nitric oxide synthase in human metabolic bone diseases revealed that ecNOS was the predominantly expressed isoform in bone. The primary human osteoblasts also showed widespread expression of ecNOS protein. In view of this, a genetic association study was carried out to investigate the relationship between a polymorphism in the ecNOS gene, bone mineral density and fracture incidence. A significant relationship was found between bone mineral density and presence of the ecNOS polymorphism, but the polymorphism was not found to be associated with osteoporotic fracture. In summary, nitric oxide produced via the inducible nitric oxide synthase pathway in human osteoblast-like cells inhibits cell proliferation and increases cell differentiation. This inhibition of cell proliferation was associated with increased expression of the cell cycle inhibitory protein p27Kipl. ecNOS was shown to be the predominant isoform of nitric oxide synthase expressed under basal conditions and in response in fluid flow. A polymorphism in this gene was shown to be associated with decreased bone mass.