Does protein kinase R mediate TNF-alpha- and ceramide-induced increases in expression and activation of matrix metalloproteinases in articular cartilage by a novel mechanism?
Does protein kinase R mediate TNF-alpha- and ceramide-induced increases in expression and activation of matrix metalloproteinases in articular cartilage by a novel mechanism?
复制标题
DOI：
10.1186/ar1024
复制
发表时间：
2004
影响因子：
4.9
通讯作者：
Mason DJ
中科院分区：
文献类型：
--
作者：
Gilbert SJ;Duance VC;Mason DJ
我们研究了促炎细胞因子TNF-α、第二信使C2-神经酰胺和蛋白激酶R（PKR）在牛关节软骨降解中的作用。用C2-神经酰胺或TNF-α刺激牛关节软骨外植体24小时。为了抑制PKR的活化，将2-氨基嘌呤加入到重复培养物中。通过明胶酶谱法分析培养基中的基质金属蛋白酶（MMP）表达和活化，通过二甲基亚甲基蓝测定法分析蛋白聚糖释放，并通过Cytotox 96®测定法分析细胞活力。C2-神经酰胺治疗软骨外植体导致在一个显着的释放到介质中的前和活性MMP-2。TNF-α治疗组也观察到小幅增加。在TNF-α或C2-神经酰胺处理前用2-氨基嘌呤孵育外植体导致MMP-2和MMP-9的表达和活化显著降低。TNF-α和C2-神经酰胺显著增加蛋白多糖释放到培养基中，这也被2-氨基嘌呤抑制。当用TNF-α和C2-神经酰胺处理外植体时，观察到细胞活力的丧失，发现这是由PKR调节的。我们已经表明，C2-神经酰胺和TNF-α治疗关节软骨导致MMPs的合成和活化增加，蛋白多糖的释放增加，细胞死亡增加。这些作用通过用PKR抑制剂2-氨基嘌呤治疗而消除。总的来说，这些结果表明PKR在MMP的合成和激活中的新作用，并支持我们的假设，即PKR及其激活剂PACT与关节炎疾病中发生的软骨降解有关。
We investigated the role of the proinflammatory cytokine TNF-α, the second messenger C2-ceramide, and protein kinase R (PKR) in bovine articular cartilage degradation. Bovine articular cartilage explants were stimulated with C2-ceramide or TNF-α for 24 hours. To inhibit the activation of PKR, 2-aminopurine was added to duplicate cultures. Matrix metalloproteinase (MMP) expression and activation in the medium were analysed by gelatin zymography, proteoglycan release by the dimethylmethylene blue assay, and cell viability by the Cytotox 96® assay. C2-ceramide treatment of cartilage explants resulted in a significant release of both pro- and active MMP-2 into the medium. Small increases were also seen with TNF-α treatment. Incubation of explants with 2-aminopurine before TNF-α or C2-ceramide treatment resulted in a marked reduction in expression and activation of both MMP-2 and MMP-9. TNF-α and C2-ceramide significantly increased proteoglycan release into the medium, which was also inhibited by cotreatment with 2-aminopurine. A loss of cell viability was observed when explants were treated with TNF-α and C2-ceramide, which was found to be regulated by PKR. We have shown that C2-ceramide and TNF-α treatment of articular cartilage result in the increased synthesis and activation of MMPs, increased release of proteoglycan, and increased cell death. These effects are abrogated by treatment with the PKR inhibitor 2-aminopurine. Collectively, these results suggest a novel role for PKR in the synthesis and activation of MMPs and support our hypothesis that PKR and its activator, PACT, are implicated in the cartilage degradation that occurs in arthritic disease.
登录
查看更多内容
影响因子：
5
作者：
Goldring, M B
通讯作者：
Goldring, M B
影响因子：
2.8
作者：
Aizawa, T;Kon, T;Gerstenfeld, LC
通讯作者：
Gerstenfeld, LC
影响因子：
4.1
作者：
Little, CB;Flannery, CR;Caterson, B
通讯作者：
Caterson, B
影响因子：
15.9
作者：
DEAN, DD;MARTELPELLETIER, J;WOESSNER, JF
通讯作者：
WOESSNER, JF
影响因子：
4.8
作者：
Curtis, CL;Hughes, CE;Caterson, B
通讯作者：
Caterson, B