Nitric Oxide Boosts TLR-4 Mediated Lipocalin 2 Expression in Chondrocytes Rodolfo Go ´ mez, 1 Morena Scotece, 1 Javier Conde, 1 Veronica Lopez, 1 Jesus Pino, 2 Francisca Lago, 3 Juan J. Go ´ mez-Reino, 1 Oreste Gualillo 1 1 SERGAS, Santiago University Clinical Hospital Research Laboratory 9 (NEIRID LAB: Neuroendocrine Interactions in Rheumatology and Inflammatory Diseases), Institute of Medical Research (IDIS), Santiago de Compostela, 15706, Spain, 2 Division of Orthopaedic Surgery, SERGAS, Santiago University Clinical Hospital, Santiago de Compostela, 15706, Spain, 3 SERGAS, Santiago University Clinical Hospital, Research Laboratory 7 (Molecular and Cellular Cardiology), Institute of Medical Research (IDIS), Santiago de Compostela, 15706, Spain Received 29 October 2012; accepted 5 February 2013 Published online 11 March 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22331 ABSTRACT: Lipocalin 2 (LCN2) has recently emerged as a novel adipokine involved in different processes including arthritis and chondrocyte inflammatory response. However, little is known about its activity on chondrocyte homeostasis and its regulation by nitric oxide (NO) Hence, we performed a set of experiments aimed to achieve a better understanding of this relationship. Cell vitality was tested in the ATDC5 cell line by the MTT colorimetric assay. Protein expression and gene expression was evaluated by Western blot and real time RT-PCR, respectively. NO production (determined as nitrite accumulation) was assayed by the Griess reaction. First, we demonstrated that LCN2 decreased murine chondrocytes vitality. Next, LCN2 co-stimulation with LPS enhanced NOS2 protein expression by murine chondrocytes. In addition, inhibition of LPS-induced nitric oxide production by aminoguanidine, a selective NOS2 inhibitor, significantly reduced LPS-mediated LCN2 expression. In contrast, treatment of murine chondrocytes with sodium nitroprussiate (SNP), a classic NO donor, scarcely induced LCN2 expression. Intriguingly, SNP addition to LPS-challenged chondrocytes, treated with aminoguanidine, provoked a strong induction of LCN2 expression. Finally, murine ATDC5 cells, co-cultured with LPS pre-challenged macrophages, had higher LCN2 expression in comparison with murine chondrocytes co-cultured with non pre-challenged macrophages. In this work we have described for the first time that NO is able to exert a control on LCN2 expression, suggesting the existence of a feedback loop regulating its expression. # 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1046–1052, 2013. Keywords: chondrocyte; inflammation; adipokines; nitric oxide; TLR4; LCN2 Chondrocytes are the unique cells in mature cartilage that synthesize and maintain the integrity of carti- lage-specific extracellular matrix. In rheumatoid ar- thritis (RA) and osteoarthritis (OA) the phenotype of chondrocytes changes, and apoptosis and extracellular matrix degradation occur. These severe perturbations in cartilage homeostasis are mediated in part by the activation of toll-like receptors (TLRs) in chondro- cytes. 1,2 These receptors are members of a highly conserved family of pattern recognition receptors, which recognize pathogen-associated molecular pat- terns (PAMPs). However, TLRs also recognize dam- age-associated molecular patterns (DAMPs), that are host-derived molecules released upon tissue injury, as a stress response. 3 Among TLRs, TLR4 has been related with several diseases since it is able to recognize numerous DAMPs such as: Hsp60, Hsp70, fibronectin, hyaluronic acid, heparan sulphate, fibrino- gen, and tenascin-C. 3 Specifically, TLR4 receptor has been involved in RA and OA. 4,5 TLR4 activation involves the production of nitric oxide (NO), 6 several pro-inflammatory cytokines 7 and adipokines 8 that may work together boosting cartilage degradation. 9,10 NO, a highly reactive gas, is involved in the pathogenesis of arthritis. In fact, it is known that NO production is greatly increased in RA or OA chondro- cytes in comparison to healthy chondrocytes. 11,12 In addition, it has been reported that stimulated chon- drocytes are the cells with the highest NO production, even more than macrophages. 13 Increased NO produc- tion is supported by an increased nitric oxide synthase (NOS2) expression, that in turn, could be induced by mechanical stimulus or by biochemical stimuli such as IL-1, TNF, LPS, and several pro-inflammatory adipo- kines. 9,10,14–16 Also, higher NO amounts are accompa- nied by chondrocytes apoptosis, 17 lower proteoglycan and collagen synthesis, 18 metalloproteinases activa- tion, 19 and cartilage inflammation. 6 During the last decade, adipokines have been involved in several rheumatic diseases including RA and OA. 20,21 In these pathologies, leptin and adiponec- tin have been associated with pro-inflammatory and catabolic processes. However, little is known about the role of novel adipokines in the progression of these pathologies. 20,21 Among these, adipokines is LCN2. This novel adipokine could be found as a 25 kDa monomer, as a 46 kDa homodimer, and in a covalent The authors declare that they have no conflicts of interest. Authors’ contributions: All authors revised critically the manu- script for important intellectual content, and all authors ap- proved the final version to be submitted for publication. Study conception and design: O.G., F.L., R.G. Acquisition of data: R. G., M.S., J.C., V.L. Analysis and interpretation of data: O.G., R. G., M.S., J.C., V.L., F.L., J.J.G.-R. Manuscript preparation: R.G., O.G. Grant sponsor: Instituto de Salud Carlos III; Grant numbers: PI11/00497; PI11/01073; RIER RD08/0075; Grant sponsor: Xunta de Galicia; Grant number: 10CSA918029PR; Grant sponsor: REDINSCOR; Grant number: RD06/0003/0016; Grant sponsor: Spanish Ministry of Education; Grant sponsor: IDIS; Grant sponsor: Spanish National Institute of Health “Carlos III”; Grant sponsor: Xunta de Galicia. Correspondence to: Oreste Gualillo (T: 34-981950905; F: 34- 981950905; E-mail: oreste.gualillo@sergas.es) # 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. 1046 JOURNAL OF ORTHOPAEDIC RESEARCH JULY 2013