The epigenetic effect of glucosamine and a nuclear factor-kappa B (NF-kB) inhibitor on primary human chondrocytes--implications for osteoarthritis.
The epigenetic effect of glucosamine and a nuclear factor-kappa B (NF-kB) inhibitor on primary human chondrocytes--implications for osteoarthritis.
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DOI：
10.1016/j.bbrc.2011.01.007
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发表时间：
2011-02-18
影响因子：
3.1
通讯作者：
Oreffo RO
中科院分区：
文献类型：
--
作者：
Imagawa K;de Andrés MC;Hashimoto K;Pitt D;Itoi E;Goldring MB;Roach HI;Oreffo RO
特发性骨关节炎是全球最常见的骨关节炎（OA）形式，并且仍然是日益老龄化的人口中残疾和相关社会经济负担的主要原因。传统上，OA被认为是一种退行性关节疾病，其特征在于关节软骨的进行性破坏和软骨下骨的变化，最终导致关节衰竭。然而，OA的病因是多因素的，涉及遗传、机械和环境因素。治疗方式包括止痛、类固醇或透明质酸联合注射、口服补充剂（包括葡萄糖胺和硫酸软骨素）以及物理疗法。因此，对疾病修饰剂的发现存在显著兴趣。一种这样的药剂，葡糖胺（GlcN）是常用的处方，即使治疗功效和作用机制仍然存在争议。炎性细胞因子（包括IL-1 β）和蛋白酶（如MMP-13）与其启动子中相关的CpG去甲基化一起参与OA的发病机制和进展。我们已经研究了GlcN调节NF-κ B活性的潜力，并在关节软骨细胞中研究了甘氨酸诱导的异常基因表达，关键是，这是否与表观遗传过程有关。在股骨颈骨折手术后，从经伦理许可获得的股骨头的关节软骨中分离人软骨细胞。软骨细胞分6组培养5周;（i）对照培养物，（ii）用2.5ng/ml IL-1 β和2.5ng/ml制瘤素M（OSM）的混合物培养，（iii）用2mM N-乙酰基GlcN（Sigma-Aldrich），（iv）用2.5 ng/ml IL-1 β、2.5 ng/ml OSM和2 mM GlcN的混合物培养，（v）用1.0 μ M BAY 11 - 7082（BAY; NF-kB抑制剂：Calbiochem，达姆施塔特，德国）和，（vi）用2.5 ng/ml IL-1 β、2.5 ng/ml OSM和1.0 μ M BAY的混合物培养。采用qRT-PCR检测IL 1B和MMP 13 mRNA水平。通过焦磷酸测序定量IL 1 β和MMP 13近端启动子的CpG位点中的DNA甲基化百分比。在IL-1 β和OSM处理的关节软骨细胞中，IL-1 β表达增强超过580倍。GlcN显著地改善了精氨酸诱导的表达4倍。单独BAY使IL 1 β表达增加3倍。在BAY存在下，IL-1 β诱导的IL 1B mRNA水平降低6倍。在对照培养物中观察到的IL-1 β启动子中− 256 CpG位点的平均甲基化百分比为65%，在IL-1 β/OSM存在下降低至36%。单独的GlcN和BAY对IL1B启动子的甲基化状态具有可忽略的影响。GlcN和BAY分别将细胞因子诱导的IL 1B启动子甲基化状态丧失改善至44%和53%。IL-1 β/OSM处理增加MMP 13 mRNA水平，与GlcN或BAY无关，并且未观察到MMP 13启动子甲基化状态的变化。我们首次证明GlcN和BAY可以阻止IL 1 β启动子中特定CpG位点的甘氨酸诱导的去甲基化，这与IL 1 β表达的降低有关。这些研究提供了一个潜在的作用机制，通过NF-κ B的OA疾病修饰剂，关键是，证明需要进一步的研究，以阐明NF-κ B可能发挥的作用，在人类软骨细胞中的DNA去甲基化。
Idiopathic osteoarthritis is the most common form of osteoarthritis (OA) world-wide and remains the leading cause of disability and the associated socio-economic burden in an increasing aging population. Traditionally, OA has been viewed as a degenerative joint disease characterized by progressive destruction of the articular cartilage and changes in the subchondral bone culminating in joint failure. However, the etiology of OA is multifactorial involving genetic, mechanical and environmental factors. Treatment modalities include analgesia, joint injection with steroids or hyaluronic acid, oral supplements including glucosamine and chondroitin sulfate, as well as physiotherapy. Thus, there is significant interest in the discovery of disease modifying agents. One such agent, glucosamine (GlcN) is commonly prescribed even though the therapeutic efficacy and mechanism of action remain controversial. Inflammatory cytokines, including IL-1β, and proteinases such as MMP-13 have been implicated in the pathogenesis and progression of OA together with an associated CpG demethylation in their promoters. We have investigated the potential of GlcN to modulate NF-kB activity and cytokine-induced abnormal gene expression in articular chondrocytes and, critically, whether this is associated with an epigenetic process. Human chondrocytes were isolated from the articular cartilage of femoral heads, obtained with ethical permission, following fractured neck of femur surgery. Chondrocytes were cultured for 5 weeks in six separate groups; (i) control culture, (ii) cultured with a mixture of 2.5 ng/ml IL-1β and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2 mM N-acetyl GlcN (Sigma–Aldrich), (iv) cultured with a mixture of 2.5 ng/ml IL-1β, 2.5 ng/ml OSM and 2 mM GlcN, (v) cultured with 1.0 μM BAY 11-7082 (BAY; NF-kB inhibitor: Calbiochem, Darmstadt, Germany) and, (vi) cultured with a mixture of 2.5 ng/ml IL-1β, 2.5 ng/ml OSM and 1.0 μM BAY. The levels of IL1B and MMP13 mRNA were examined using qRT-PCR. The percentage DNA methylation in the CpG sites of the IL1β and MMP13 proximal promoter were quantified by pyrosequencing. IL1β expression was enhanced over 580-fold in articular chondrocytes treated with IL-1β and OSM. GlcN dramatically ameliorated the cytokine-induced expression by 4-fold. BAY alone increased IL1β expression by 3-fold. In the presence of BAY, IL-1β induced IL1B mRNA levels were decreased by 6-fold. The observed average percentage methylation of the −256 CpG site in the IL1β promoter was 65% in control cultures and decreased to 36% in the presence of IL-1β/OSM. GlcN and BAY alone had a negligible effect on the methylation status of the IL1B promoter. The cytokine-induced loss of methylation status in the IL1B promoter was ameliorated by both GlcN and BAY to 44% and 53%, respectively. IL-1β/OSM treatment increased MMP13 mRNA levels independently of either GlcN or BAY and no change in the methylation status of the MMP13 promoter was observed. We demonstrate for the first time that GlcN and BAY can prevent cytokine-induced demethylation of a specific CpG site in the IL1β promoter and this was associated with decreased expression of IL1β. These studies provide a potential mechanism of action for OA disease modifying agents via NF-kB and, critically, demonstrate the need for further studies to elucidate the role that NF-kB may play in DNA demethylation in human chondrocytes.
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影响因子：
6
作者：
Fan, Zhiyong;Soeder, Stephan;Aigner, Thomas
通讯作者：
Aigner, Thomas
影响因子：
4.8
作者：
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影响因子：
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作者：
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通讯作者：
Roach, Helmtrud I.
DOI：
10.1152/ajpheart.01025.2008
发表时间：
2009-02-01
影响因子：
4.8
作者：
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通讯作者：
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影响因子：
3.7
作者：
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通讯作者：
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