Martin G, Andriamanalijaona R, Grässel S, Dreier R, Mathy-Hartert M, Bogdanowicz P, Boumédiene K, Henrotin Y, Bruckner P, Pujol J-P
Laboratory of Connective Tissue Biochemistry, Faculty of Medicine, 14032 Caen Cedex, France.
Arthritis Rheum. 2004 Nov;50(11):3549-60. doi: 10.1002/art.20596.
To determine the effects of hypoxia and reoxygenation on the metabolism of chondrocytes and their response to interleukin-1beta (IL-1beta). The study included activation of hypoxia-inducible factor 1 (HIF-1), NF-kappaB, and activator protein 1 (AP-1) transcription factors, expression of matrix components and metalloproteases and transforming growth factor beta (TGFbeta) and TGFbeta receptors, and production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)).
Bovine articular chondrocytes (BACs) were cultured to confluency in either 5% O(2) (hypoxia) or 21% O(2) (normoxia) in media supplemented with 10% fetal calf serum (FCS). BACs were preincubated for 18 hours in media with 1% FCS only and then incubated for 24 hours in the presence of IL-1beta. For reoxygenation experiments, cells were treated in the same way in 5% O(2), except that cultures were transferred to normal atmospheric conditions and used after 4 hours for RNA extraction or after 30 minutes for cytoplasmic or nuclear protein extraction.
In hypoxic and reoxygenated chondrocytes, we observed strong DNA binding of HIF-1. IL-1beta-induced DNA binding of NF-kappaB and AP-1 was significantly higher in hypoxic and reoxygenated cultures than in normoxia. Greater activation of the MAPKs was also observed with IL-1beta treatment in hypoxia compared with normoxia. Steady-state levels of type II collagen and aggrecan core protein messenger RNA (mRNA) were decreased by IL-1beta in all instances. Matrix metalloprotease 1 (MMP-1) and MMP-3 mRNA were increased by IL-1beta in normoxia and hypoxia, whereas only MMP-3 mRNA was enhanced in reoxygenated cultures. The MMP-2 mRNA level was not significantly affected by IL-1beta in normoxia or hypoxia, whereas it was enhanced in reoxygenated cultures. MMP-9 mRNA was dramatically decreased by IL-1beta only in low oxygen tension. Tissue inhibitor of metalloproteinases 1 (TIMP-1) message was significantly enhanced by the cytokine in most instances, whereas TIMP-2 message was markedly decreased by IL-1beta in reoxygenated cultures. Stimulation of TGFbeta1 expression by IL-1beta was observed only in normal atmospheric conditions. One of the more striking findings of the study was the greater stimulating effect of IL-1beta on NO production observed in hypoxia, which was much higher than in normoxia, whereas the reverse was observed for IL-1beta-induced PGE(2) production.
Oxygen level and reoxygenation stress significantly modulate gene expression and the response of articular chondrocytes to cytokines such as IL-1beta. In hypoxic conditions, which mimic the in vivo condition of cartilage, the effects of IL-1beta on both synthesis and degradative processes are significantly different from those in normoxia, conditions that are unlikely encountered by chondrocytes in a normal state. In low oxygen tension, high IL-1beta-induced NO production is associated with a significant decrease in PGE(2) synthesis. These data should influence our concept of the role of oxygen in the pathophysiology of joint disease and may help define the best conditions in which to develop bioartificial cartilage.
确定缺氧和复氧对软骨细胞代谢及其对白介素-1β（IL-1β）反应的影响。研究内容包括缺氧诱导因子1（HIF-1）、核因子κB（NF-κB）和活化蛋白1（AP-1）转录因子的激活，基质成分、金属蛋白酶、转化生长因子β（TGFβ）及其受体的表达，以及一氧化氮（NO）和前列腺素E2（PGE2）的产生。
将牛关节软骨细胞（BACs）在补充有10%胎牛血清（FCS）的培养基中，于5%氧气（缺氧）或21%氧气（常氧）条件下培养至汇合。BACs先在仅含1% FCS的培养基中预孵育18小时，然后在IL-1β存在下孵育24小时。对于复氧实验，细胞在5%氧气条件下以相同方式处理，只是将培养物转移至正常大气条件下，4小时后用于RNA提取，或30分钟后用于细胞质或细胞核蛋白提取。
在缺氧和复氧的软骨细胞中，我们观察到HIF-1有强烈的DNA结合。IL-1β诱导的NF-κB和AP-1的DNA结合在缺氧和复氧培养物中显著高于常氧培养物。与常氧相比，缺氧条件下IL-1β处理也观察到丝裂原活化蛋白激酶（MAPKs）有更大程度的激活。在所有情况下，IL-1β均降低了Ⅱ型胶原蛋白和聚集蛋白聚糖核心蛋白信使核糖核酸（mRNA）的稳态水平。在常氧和缺氧条件下，IL-1β使基质金属蛋白酶1（MMP-1）和MMP-3 mRNA增加，而在复氧培养物中只有MMP-3 mRNA增强。在常氧或缺氧条件下，IL-1β对MMP-2 mRNA水平无显著影响，而在复氧培养物中其水平增强。仅在低氧张力下，IL-1β使MMP-9 mRNA显著降低。在大多数情况下，细胞因子显著增强了金属蛋白酶组织抑制剂1（TIMP-1）的信使核糖核酸水平，而在复氧培养物中，IL-1β使TIMP-2信使核糖核酸水平显著降低。仅在正常大气条件下观察到IL-1β对TGFβ1表达的刺激作用。该研究更显著的发现之一是，IL-1β在缺氧条件下对NO产生的刺激作用更大，远高于常氧条件，而对于IL-1β诱导的PGE2产生则观察到相反情况。
氧水平和复氧应激显著调节基因表达以及关节软骨细胞对细胞因子如IL-1β的反应。在模拟软骨体内状况的缺氧条件下，IL-1β对合成和降解过程的影响与常氧条件下显著不同，而常氧条件是软骨细胞在正常状态下不太可能遇到的。在低氧张力下，高IL-1β诱导的NO产生与PGE2合成的显著减少相关。这些数据应会影响我们对氧在关节疾病病理生理学中作用的概念，并可能有助于确定开发生物人工软骨的最佳条件。
