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Comparative Study
. 2005;7(3):R581-91.
doi: 10.1186/ar1708. Epub 2005 Mar 4.

Signalling pathway involved in nitric oxide synthase type II activation in chondrocytes: synergistic effect of leptin with interleukin-1

Affiliations
Comparative Study

Signalling pathway involved in nitric oxide synthase type II activation in chondrocytes: synergistic effect of leptin with interleukin-1

Miguel Otero et al. Arthritis Res Ther. 2005.

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 micromol/l] and LY294002 [1, 2.5, 5 and 10 micromol/l]), mitogen-activated protein kinase kinase (MEK)1 (PD098059 [1, 5, 10, 20 and 30 micromol/l]) and p38 kinase (SB203580 [1, 5, 10, 20 and 30 micromol/l]) were added 1 hour before stimulation. Nitric oxide synthase type II mRNA expression in ATDC5 chondrocytes was investigated by real-time 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.

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Figures

Figure 1
Figure 1
Leptin synergizes with IL-1 in inducing nitric oxide synthase (NOS) type II. Synergistic effect of leptin (OB) on nitrite (NO2-) accumulation and NOS type II protein expression induced by IL-1. Stimulations were conducted in serum-free conditions (a,b) in ATDC5 chondrogenic cells and (c) in human primary chondrocytes. NO2- accumulation is selectively inhibited by aminoguanidine (AG) both in (d) ATDC5 cells and in (panel c) human primary chondrocytes. Values are expressed as mean ± standard error of the mean. WB, western blot.
Figure 2
Figure 2
Janus kinase (JAK)2 inhibition blocks leptin/IL-1-induced nitric oxide (NO) production and nitric oxide synthase (NOS) type II protein expression. Effect of tyrphostin AG490 and Tkip on NO production and NOS II protein expression. The effect of tyrphostin AG490 was evaluated in terms of (a) nitrite accumulation in ATDC5 cells stimulated with leptin and IL-1, and in terms of (d) NOS II protein expression. The effect of Tkip was evaluated by nitrite accumulation in (b) leptin/IL-1 ATDC5 co-stimulated cells and in (c) IL-1 stimulated cells (panel c). (e) Effect of Tkip on NOS type II protein expression in leptin/IL-1 co-stimulated cells.
Figure 3
Figure 3
Involvement of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase kinase (MEK)-1 and p38-kinase in leptin/IL-1-induced nitric oxide synthase (NOS). Dose-dependent effect of (a,a1) LY294002, (b,b1) PD098059 and (c,c1) SB203580 on nitrite (NO2-) production and NOS type II protein expression in stimulated and unstimulated ATDC5 cells. Stimulations were conducted in serum-free conditions. Each inhibitor was added 1 hour before cytokine co-stimulation. Values are expressed as mean ± standard error of the mean. OB, leptin; WB, western blot.
Figure 4
Figure 4
Leptin synergism does not depend upon chondrocyte differation state. Effect of different inhibitors on nitrite (NO2-) accumulation in 15-day differentiated ATDC5 cells stimulated or not with leptin, alone or in combination with IL-1, during (a) 24 and (b) 48 hours. The effect of inhibitors was also evaluated in 21-day differentiated ATDC5 cells, after (c) 24 or (d) 48 hours of stimulation with leptin and IL-1 (alone or in combination). Values are expressed as mean ± standard error of the mean. OB, leptin.
Figure 5
Figure 5
Leptin acts synergistically with IL-1 in human primary chondrocytes. Nitrite (NO2-) accumulation in leptin (OB)/IL-1 co-stimulated human primary chondrocytes. Stimulations were conducted in serum-free conditions in the presence or absence of tyrphostin AG490, wortmannin, LY294002, PD98059 and SB203580 inhibitors. Values are expressed as mean ± standard error of the mean.
Figure 6
Figure 6
Effect of leptin/IL-1 co-stimulation on nitric oxide synthase (NOS) type II mRNA expression. Real-time RT-PCR analysis of the expression of the inducible NOS type II mRNA in leptin (OB)/IL-1 co-stimulated ATDC5 cells. Stimulations (24 hours) were conducted in serum-free conditions. Specific inhibitors were added 1 hour before cytokine co-stimulation. Values are expressed as mean ± standard error of the mean.

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