CYTOKINE-STIMULATED EXPRESSION OF INDUCIBLE NITRIC-OXIDE SYNTHASE BY MOUSE, RAT, AND HUMAN OSTEOBLAST-LIKE CELLS AND ITS FUNCTIONAL-ROLE INOSTEOBLAST METABOLIC-ACTIVITY

Recent evidence suggests that the production of nitric oxide (NO) may have important roles in the regulation of osteoblast and osteoclast me tabolism. The present study was performed to investigate the effects o f interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alp ha), and interferon-gamma (IFN-gamma) on the expression of inducible N O-synthase (iNOS) and to measure high-output production of NO by prima ry rat osteoblasts and osteoblastic cell lines ROS 17/2.8, MC3T3-E1 an d MG-63. In addition, we have investigated if NO may mediate some of t he effects of these cytokines on osteoblast metabolism. Northern blots and immunocytochemistry revealed time-dependent iNOS messenger RNA an d protein expression in primary rat osteoblasts in response to cytokin e treatment. Reverse transcription polymerase chain reaction amplified an 807-base pair (bp) product from ROS 17/2.8 cells, which had a size and restriction enzyme-cut pattern identical to that predicted for au thentic rat iNOS. Nitrite accumulation in culture medium was induced b y IFN-gamma in a time- and dose-dependent manner and inhibited by cotr eatment with inhibitors of NOS activity and by dexamethasone. IL-1 bet a, TNF-alpha, and bacterial lipopolysaccharide were found to have weak stimulatory effects on nitrite production on their own. However, IL-1 beta and TNF-alpha showed strong synergy with IFN-gamma, but, surpris ingly, lipopolysaccharide was found to exert potent inhibitory effects on IFN-gamma-induced nitrite synthesis. Basal production of nitrite a nd induction of its synthesis was similarly observed with primary rat osteoblasts as well as ROS 17/2.8, MC3T3-E1, and MG-63 cell lines. Cyt okine-induced NO production significantly reduced osteoblast activity, as was evidenced by inhibition of DNA synthesis, cell proliferation, alkaline phosphatase activity, and osteocalcin production. The results provide evidence for a basal expression of iNOS activity and show tha t the iNOS messenger RNA, protein, and enzyme activity are all induced by cytokines across the species. The data further suggest that osteob last-derived NO may have an important role in mediation of localized b one destruction associated with inflammatory bone diseases such as rhe umatoid arthritis.