Otero Miguel, Lago Rocío, Gómez Rodolfo, Lago Francisca, Gomez-Reino Juan Jesús, Gualillo Oreste
NeuroEndocrine Interactions in Rheumatology and Inflammatory Diseases Laboratory, Santiago University Clinical Hospital, Research Laboratory 4, Santiago de Compostela, Spain.
Life Sci. 2007 Oct 27;81(19-20):1452-60. doi: 10.1016/j.lfs.2007.09.007. Epub 2007 Sep 26.
In a previous study, we established that leptin acts synergistically with interferon-gamma in inducing nitric oxide synthase type II in cultured chondrocytes via Janus kinase-2 activation. However, the exact molecular mechanism that accounts for this synergism is not completely understood. The aim of the present study was to further delineate the signalling pathway used by leptin/interferon-gamma in the nitric oxide synthase type II induction in chondrocytes. Consequently, the roles of PI-3 kinase, MEK1 and p38 kinase were investigated using specific pharmacological inhibitors (Wortmannin, LY 294002, PD 098,059 and SB 203580). For this purpose, the amount of stable nitrite, the end product of NO generation by activated chondrocytes, has been evaluated by Griess colorimetric reaction in culture medium of human primary chondrocytes and in the murine ATDC5 cell line stimulated with leptin (400 nM) and interferon-gamma (1 ng/ml), alone or in combination. Specific inhibitors for PI-3K, MEK1 and p38 were added 1 h before stimulation. Nitric oxide synthase type II mRNA was investigated by real-time RT-PCR and NOS type II protein expression has been evaluated by western blot analysis. Our results showed that, as expected, leptin synergizes with IFN-gamma in inducing NO accumulation in the supernatant of co-stimulated cells. Pre-treatment with Wortmannin, LY 294002, PD 098,059 and SB 203580 caused a significant decrease in nitrite production, NOS type II protein expression and NOS type II mRNA expression induced by leptin and interferon-gamma co-stimulation. These findings were confirmed in 15 and 21-day differentiated ATDC5 cells, and in normal human primary chondrocytes. This is the first report showing that NOS type II induction triggered by co-stimulation with leptin and interferon-gamma is mediated by a signaling pathway involving PI-3K, MEK1 and p38.
在之前的一项研究中，我们证实，瘦素通过激活Janus激酶-2，与干扰素-γ协同作用，诱导培养的软骨细胞中Ⅱ型一氧化氮合酶的产生。然而，导致这种协同作用的确切分子机制尚未完全明确。本研究的目的是进一步阐明瘦素/干扰素-γ在软骨细胞中诱导Ⅱ型一氧化氮合酶时所使用的信号通路。因此，我们使用特异性药理抑制剂（渥曼青霉素、LY 294002、PD 098059和SB 203580）研究了PI-3激酶、MEK1和p38激酶的作用。为此，通过格里斯比色反应，在人原代软骨细胞培养基以及用瘦素（400 nM）和干扰素-γ（1 ng/ml）单独或联合刺激的小鼠ATDC5细胞系中，评估了活化软骨细胞产生的NO的终产物——稳定亚硝酸盐的量。在刺激前1小时加入PI-3K、MEK1和p38的特异性抑制剂。通过实时RT-PCR研究Ⅱ型一氧化氮合酶mRNA，并通过蛋白质印迹分析评估Ⅱ型一氧化氮合酶蛋白表达。我们的结果表明，正如预期的那样，瘦素与干扰素-γ协同作用，诱导共刺激细胞上清液中NO的积累。用渥曼青霉素、LY 294002、PD 098059和SB 203580预处理后，瘦素和干扰素-γ共刺激诱导的亚硝酸盐产生、Ⅱ型一氧化氮合酶蛋白表达和Ⅱ型一氧化氮合酶mRNA表达显著降低。这些发现在分化15天和21天的ATDC5细胞以及正常人原代软骨细胞中得到了证实。这是第一份表明瘦素和干扰素-γ共同刺激引发的Ⅱ型一氧化氮合酶诱导是由涉及PI-3K、MEK1和p38的信号通路介导的报告。
