Interaction Between Nitric Oxide Synthase and Cyclooxygenase Pathways in Osteoblastic MC3T3‐E1 Cells

Interleukin 1 (IL‐1) and tumor necrosis factor α (TNF‐α) have been implicated in the pathogenesis of osteoporosis. These proinflammatory cytokines induce both cyclooxygenase (COX) and nitric oxide synthase (NOS) with the release of prostaglandin (PG) and NO, respectively. The present study was undertaken to examine the interaction between COX and NOS pathways and their role in the regulation of osteoblastic function in MC3T3‐E1 cells. Addition of IL‐1α and TNF‐α induced a marked increase in the production of both NO and PGE2. Reverse transcription–polymerase chain reaction analysis showed that the increase in NO production was preceded by the expression of inducible NOS mRNA. The temporal profile of PGE2 production revealed a biphasic pattern: the first small peak at 3 h was caused by de novo synthesis of PGE2 through inducible COX (COX‐2) mRNA, while the subsequent progressive accumulation of PGE2 was mediated through the activation of COX pathway by NO since (1) aminoguanidine (AG), a selective inhibitor of inducible NOS, significantly suppressed the PGE2 production by IL‐1α and TNF‐α, (2) NOC‐18, an NO donor, reversed this suppression, and (3) NOC‐18 increased PGE2 production by itself. The increase in NO production in response to IL‐1α and TNF‐α was further stimulated by aspirin and inhibited by exogenous addition of PGE2, suggesting that PGE2 produced by the cytokines, in turn, negatively modulates NO production. IL‐1α and TNF‐α inhibited alkaline phosphatase (ALP) activity, which was significantly reversed by AG. NOC‐18 not only suppressed ALP activity by itself but also blocked the effect of AG, suggesting the role of NO in the inhibition of ALP activity. PGE2 decreased ALP activity, and the inhibitory effect of NOC‐18 was attenuated in the presence of aspirin, suggesting the involvement of PGE2 in the negative modulation of ALP activity by NO. These results suggest that NO produced in response to proinflammatory cytokines participates in the modulation of ALP activity via the activation of COX pathway. The interaction between NO and the COX pathways may play an important role in the regulation of osteoblastic functions under physiologic as well as pathologic conditions.

[1]  N. Kurihara,et al.  Inductive effects of prostaglandins on alkaline phosphatase in osteoblastic cells, clone MC3T3-E1. , 1985, Journal of biochemistry.

[2]  S. Yamamoto,et al.  Stimulation of prostaglandin E2 synthesis in cloned osteoblastic cells of mouse (MC3T3-E1) by epidermal growth factor. , 1986, The Journal of biological chemistry.

[3]  M. Currie,et al.  Regulation of prostaglandin production by nitric oxide; an in vivo analysis , 1995, British journal of pharmacology.

[4]  A. Ianaro,et al.  Modulation by nitric oxide of prostaglandin biosynthesis in the rat , 1995, British journal of pharmacology.

[5]  T. Evans,et al.  Aminoguanidine selectively inhibits inducible nitric oxide synthase , 1993, British journal of pharmacology.

[6]  L. Marnett,et al.  Prostaglandin endoperoxide synthase: structure and catalysis. , 1991, Biochimica et biophysica acta.

[7]  K. Ohya,et al.  Expression of prostaglandin E receptor subtypes in bone: expression of EP2 in bone development. , 1995, Bone.

[8]  A. Fujimori,et al.  Cyclooxygenase inhibitors enhance cell growth in an osteoblastic cell line, MC3T3‐E1 , 1989, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[9]  Shigehisa Yamamoto,et al.  Establishment of a clonal osteogenic cell line from newborn mouse calvaria , 1981 .

[10]  Roberto Pacifici,et al.  Estrogen, cytokines, and pathogenesis of postmenopausal osteoporosis , 1996, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[11]  S. Moncada,et al.  Nitric oxide: physiology, pathophysiology, and pharmacology. , 1991, Pharmacological reviews.

[12]  H. Kawaguchi,et al.  Differential regulation of inducible and constitutive prostaglandin endoperoxide synthase in osteoblastic MC3T3-E1 cells. , 1993, The Journal of biological chemistry.

[13]  M. Hirafuji,et al.  Role of endogenous PGE2 in osteoblastic functions of a clonal osteoblast-like cell, MC3T3-E1. , 1994, Prostaglandins, leukotrienes, and essential fatty acids.

[14]  C. Yallampalli,et al.  Nitric oxide donor alleviates ovariectomy-induced bone loss. , 1996, Bone.

[15]  Y. Vodovotz,et al.  Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta , 1993, The Journal of experimental medicine.

[16]  P. Nibbering,et al.  Inducible production of nitric oxide in osteoblast-like cells and in fetal mouse bone explants is associated with suppression of osteoclastic bone resorption. , 1994, The Journal of clinical investigation.

[17]  S. Narumiya,et al.  Prostaglandin E receptor subtypes in mouse osteoblastic cell line. , 1996, Endocrinology.

[18]  M. Mcdaniel,et al.  IL-1 beta induces the coexpression of both nitric oxide synthase and cyclooxygenase by islets of Langerhans: activation of cyclooxygenase by nitric oxide. , 1993, Biochemistry.

[19]  M. Currie,et al.  Nitric oxide activates cyclooxygenase enzymes. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R. Jilka,et al.  Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. , 1995, The New England journal of medicine.

[21]  J. Polak,et al.  Cytokine-stimulated expression of inducible nitric oxide synthase by mouse, rat, and human osteoblast-like cells and its functional role in osteoblast metabolic activity. , 1995, Endocrinology.

[22]  P. Hauschka,et al.  Cytokines induce nitric oxide production in mouse osteoblasts. , 1994, Biochemical and biophysical research communications.

[23]  H. Kawaguchi,et al.  The role of prostaglandins in the regulation of bone metabolism. , 1995, Clinical orthopaedics and related research.

[24]  P. López-Jaramillo,et al.  The L-arginine: nitric oxide pathway. , 1993, Current opinion in nephrology and hypertension.

[25]  H. Datta,et al.  Osteoclastic inhibition: an action of nitric oxide not mediated by cyclic GMP. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. Cunningham,et al.  Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line. , 1992, The Journal of biological chemistry.

[27]  Y. Amagai,et al.  In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria , 1983, The Journal of cell biology.

[28]  S. Ralston,et al.  Mechanisms of cytokine induced bone resorption: role of nitric oxide, cyclic guanosine monophosphate, and prostaglandins. , 1996, Bone.

[29]  Y. Xia,et al.  Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression. , 1993, Archives of biochemistry and biophysics.

[30]  J. Vane,et al.  A better understanding of anti-inflammatory drugs based on isoforms of cyclooxygenase (COX-1 and COX-2). , 1995, Advances in prostaglandin, thromboxane, and leukotriene research.

[31]  S. Ralston,et al.  Human osteoblast-like cells produce nitric oxide and express inducible nitric oxide synthase. , 1994, Endocrinology.

[32]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[33]  Y. Xia,et al.  Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide. , 1995, The Journal of clinical investigation.

[34]  S. Srivastava,et al.  Cross-talk between cyclooxygenase and nitric oxide pathways: prostaglandin E2 negatively modulates induction of nitric oxide synthase by interleukin 1. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[35]  P. Collin‐Osdoby,et al.  Bone cell function, regulation, and communication: A role for nitric oxide , 1995, Journal of cellular biochemistry.

[36]  J. Riancho,et al.  Expression and functional role of nitric oxide synthase in osteoblast‐like cells , 1995, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[37]  D. Hwang,et al.  Involvement of Reactive Oxygen Intermediates in Cyclooxygenase-2 Expression Induced by Interleukin-1 , Tumor Necrosis Factor-a , and Lipopolysaccharide , 2022 .

[38]  R. Russell,et al.  An interleukin 1 like factor stimulates bone resorption in vitro , 1983, Nature.

[39]  S. Tannenbaum,et al.  Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. , 1982, Analytical biochemistry.

[40]  J. Morrow,et al.  Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[41]  M. Prystowsky,et al.  Glyceraldehyde-3-phosphate dehydrogenase mRNA is a major interleukin 2-induced transcript in a cloned T-helper lymphocyte. , 1990, Gene.

[42]  P. W. Johnston,et al.  Nitric oxide: A cytokine‐induced regulator of bone resorption , 1995, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[43]  S. Narumiya,et al.  International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. , 1994, Pharmacological reviews.

[44]  P. Damoulis,et al.  Nitric Oxide Acts in Conjunction with Proinflammatory Cytokines to Promote Cell Death in Osteoblasts , 1997, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[45]  J. Roberts,et al.  Nitric oxide produced by endothelial cells increases production of eicosanoids through activation of prostaglandin H synthase. , 1995, Circulation research.

[46]  S. Moncada,et al.  The L-arginine-nitric oxide pathway. , 1993, The New England journal of medicine.

[47]  K. M. Davies,et al.  "NONOates" (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors: convenient nitric oxide dosage forms. , 1996, Methods in enzymology.

[48]  T. Bringman,et al.  Stimulation of bone resorption and inhibition of bone formation in vitro by human tumour necrosis factors , 1986, Nature.

[49]  T. Kasten,et al.  Potentiation of osteoclast bone-resorption activity by inhibition of nitric oxide synthase. , 1994, Proceedings of the National Academy of Sciences of the United States of America.