Department of Biochemical Sciences, Sapienza University of Roma, P,le Aldo Moro, 5, 00185 Roma, Italy.
Arthritis Res Ther. 2010;12(1):R18. doi: 10.1186/ar2920. Epub 2010 Jan 29.
Nuclear factor-kappaB (NF-kappaB) transcription factor regulates several cell signaling pathways, such as differentiation and inflammation, which are both altered in osteoarthritis. Inhibitor kappaB kinase (IKK)alpha and IKKbeta are kinases involved in the activation of the NF-kappaB transcription factor. The aim of the present study was to determine the effects of glucosamine (GlcN), which is administered in the treatment of osteoarthritis, and of its 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-beta-D-glucose (NAPA) derivative on IKK kinases and, consequently, on NF-kappaB activation in human chondrocytes.
The human chondrosarcoma cell line HTB-94 and human primary chondrocytes were stimulated with tumor necrosis factor (TNF)alpha after pre-treatment with GlcN or NAPA. Gene mRNA expression level was evaluated by real-time PCR. Inhibitor kappaB protein (IkappaB)alpha phosphorylation and p65 nuclear re-localization were analyzed by Western blotting; IKKalpha nuclear re-localization was also investigated by immunocytochemistry and Western blotting. IKK kinase activity was studied by in vitro kinase assay.
After TNFalpha stimulation, the mRNA expression level of some of the genes under NF-kappaB control, such as interleukin (IL)-6 and IL-8, increased, while treatment with GlcN and NAPA reverted the effect. We investigated the possibility that GlcN and NAPA inhibit IKK kinase activity and found that NAPA inhibits the IKKalpha kinase activity, whereas GlcN does not. Interestingly, both GlcN and NAPA inhibit IKKalpha nuclear re-localization.
Our results demonstrate that glucosamine and its peptidyl derivative can interfere with NF-kappaB signaling pathway by inhibiting IKKalpha activity in human chondrocytes. However, the mechanism of action of the two molecules is not completely overlapping. While NAPA can both specifically inhibit the IKKalpha kinase activity and IKKalpha nuclear re-localization, GlcN only acts on IKKalpha nuclear re-localization.
核因子-κB（NF-κB）转录因子调节多种细胞信号通路，如分化和炎症，这些通路在骨关节炎中都发生了改变。IKKα和 IKKβ激酶是参与 NF-κB 转录因子激活的激酶。本研究旨在确定氨基葡萄糖（GlcN）在骨关节炎治疗中的作用及其 2-（N-乙酰基）-L-苯丙氨酰基-2-脱氧-β-D-葡萄糖（NAPA）衍生物对人软骨细胞中 IKK 激酶的影响，进而对 NF-κB 的激活作用。
用人软骨肉瘤细胞系 HTB-94 和人原代软骨细胞，用肿瘤坏死因子（TNF）α刺激后，用 GlcN 或 NAPA 预处理。通过实时 PCR 评估基因 mRNA 表达水平。通过 Western blot 分析 IkappaB 蛋白（IkappaB）α磷酸化和 p65 核重新定位；免疫细胞化学和 Western blot 也研究了 IKKα核重新定位。通过体外激酶测定研究 IKK 激酶活性。
在 TNFα刺激后，一些受 NF-κB 控制的基因，如白细胞介素（IL）-6 和 IL-8 的 mRNA 表达水平增加，而 GlcN 和 NAPA 的治疗则逆转了这种效应。我们研究了 GlcN 和 NAPA 抑制 IKK 激酶活性的可能性，发现 NAPA 抑制 IKKα激酶活性，而 GlcN 则没有。有趣的是，GlcN 和 NAPA 均抑制 IKKα核重新定位。
我们的结果表明，氨基葡萄糖及其肽衍生物可通过抑制人软骨细胞中的 IKKα活性来干扰 NF-κB 信号通路。然而，这两种分子的作用机制并不完全重叠。虽然 NAPA 既能特异性抑制 IKKα激酶活性，又能抑制 IKKα核重新定位，但 GlcN 仅作用于 IKKα核重新定位。
