Expression and functional role of nitric oxide synthase isoforms in human osteoblast-like cells

Previous studies have shown evidence of constitutive and cytokine-inducible nitric oxide (NO) synthase activity in cultured osteoblast-like cells from various species. Although cytokine-induced NO production has been found to inhibit osteoblast growth, the role of constitutive NO production in regul ating osteoblast function is less clear and the isoforms of nitric oxide sy nthase (NOS) that are expressed by human osteoblasts have not been determin ed. Here, we investigated NOS expression in cultured human osteoblast-like cells and studied the effects of constitutive and cytokine-induced NO on os teoblast growth and differentiation. Low levels of NO were produced constit utively by osteoblast-like cells as reflected by analysis of medium nitrite concentrations, and evidence of ecNOS mRNA, protein, and bioactivity was f ound in primary osteoblasts (hOBs), TE85, and MG63 osteosarcoma cells. None of the osteoblast-like cells expressed nNOS, however, and iNOS was produce d only by hOB cells after stimulation with the cytokines IL-1 beta, TNF-alp ha, and IFN-gamma, The NOS inhibitor, L-NMMA, did not affect growth or alka line phosphatase activity in unstimulated osteoblasts, Incubation of hOB ce lls with cytokines inhibited growth and stimulated alkaline phosphatase act ivity and these effects were abrogated by L-NMMA, Cytokines also inhibited growth of TE85 cells and MG63 cells, but these effects appeared to be NO in dependent because they were not influenced by L-NMMA, Our experiments show that human osteoblasts constitutively produce NO through the ecNOS pathway, but demonstrate that this does not appear to exert an appreciable effect o n osteoblast growth or differentiation under basal conditions. In contrast, IL-1 beta, TNF-alpha, and IFN-gamma exerted growth-inhibiting and differen tiation-inducing effects on osteoblasts that were partly NO dependent, indi cating that NO may act predominantly as a modulator of cytokine-induced eff ects on osteoblast function, (Bone 24: 179-185; 1999) (C) 1999 by Elsevier Science Inc. All rights reserved.