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Abstract

The objective of the present study was to investigate the effect of leptin, alone or in combination with IL-1, on nitric oxide synthase (NOS) type II activity in vitro in human primary chondrocytes, in the mouse chondrogenic ATDC5 cell line, and in mature and hypertrophic ATDC5 differentiated chondrocytes. For completeness, we also investigated the signalling pathway of the putative synergism between leptin and IL-1. For this purpose, nitric oxide production was evaluated using the Griess colorimetric reaction in culture medium of cells stimulated over 48 hours with leptin (800 nmol/l) and IL-1 (0.025 ng/ml), alone or combined. Specific pharmacological inhibitors of NOS type II (aminoguanidine [1 mmol/l]), janus kinase (JAK)2 (tyrphostin AG490 and Tkip), phosphatidylinositol 3-kinase (PI3K; wortmannin [1, 2.5, 5 and 10 µmol/l] and LY294002 [1, 2.5, 5 and 10 µmol/l]), mitogen-activated protein kinase kinase (MEK)1 (PD098059 [1, 5, 10, 20 and 30 µmol/l]) and p38 kinase (SB203580 [1, 5, 10, 20 and 30 µmol/l]) were added 1 hour before stimulation. Nitric oxide synthase type II mRNA expression in ATDC5 chondrocytes was investigated by realtime PCR and NOS II protein expression was analyzed by western blot. Our results indicate that stimulation of chondrocytes with IL-1 results in dose-dependent nitric oxide production. In contrast, leptin alone was unable to induce nitric oxide production or expression of NOS type II mRNA or its protein. However, co-stimulation with leptin and IL-1 resulted in a net increase in nitric oxide concentration over IL-1 challenge that was eliminated by pretreatment with the NOS II specific inhibitor aminoguanidine. Pretreatment with tyrphostin AG490 and Tkip (a SOCS-1 mimetic peptide that inhibits JAK2) blocked nitric oxide production induced by leptin/IL-1. Finally, wortmannin, LY294002, PD098059 and SB203580 significantly decreased nitric oxide production. These findings were confirmed in mature and hypertrophic ATDC5 chondrocytes, and in human primary chondrocytes. This study indicates that leptin plays a proinflammatory role, in synergy with IL-1, by inducing NOS type II through a signalling pathway that involves JAK2, PI3K, MEK-1 and p38 kinase.

Transcriptional and post-transcriptional regulation of iNOS expression in human chondrocytes

“…Similar data regarding NF-κB-dependent iNOS expression were published for human primary chondrocytes [45]. In the murine chondrogenic cell line ADTC5, critical dependence of iNOS induction from the JAK2 activity has been described [46]. Therefore, cytokine-induced iNOS expression in chondrocytes depends on the activation of the JAK2-STAT-1α and NF-κB pathways.…”

Leptin regulates chondrogenic differentiation in ATDC5 cell-line through JAK/STAT and MAPK pathways

“…Signal transduction by leptin has been studied extensively in numerous experimental systems, in vitro and in vivo (see reviews [11,23]), but only one study examined leptin-activated signal transduction pathways in chondrocytes [24]. The authors found that JAK2, PI3K, p38, and ERK were activated in the ATDC5 cell model during synergistic stimulation of nitric oxide type II by leptin and interleukin-1 (IL-1).…”

Exploratory secondary analyses of a cognitive-behavioral intervention for knee osteoarthritis demonstrate reduction in biomarkers of adipocyte inflammation

“…Although there are well documented benefits of weight loss in persons with knee OA, including reduction in knee joint compressive loads [44], improvement of knee OA symptoms [17, 25, 26], the extent to which reductions in circulating leptin contribute to beneficial clinical outcomes remains uncertain. The fact that leptin, in conjunction with IL-1β, can induce nitric oxide synthase activity [45] and alone, has the ability to stimulate proinflammatory cytokines from adipose tissue including IL-1β, PGE 2 , TNF-α, and IL-6 [46] may provide a means by which leptin exerts its effect in mediating the inflammation associated with OA.…”