Fibronectin fragments and blocking antibodies to α2β1 and α5β1 integrins stimulate mitogen-activated protein kinase signaling and increase collagenase 3 (matrix metalloproteinase 13) production by human articular chondrocytes
Fibronectin fragments and blocking antibodies to α2β1 and α5β1 integrins stimulate mitogen-activated protein kinase signaling and increase collagenase 3 (matrix metalloproteinase 13) production by human articular chondrocytes
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DOI：
10.1002/art.10502
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发表时间：
2002-09-01
影响因子：
--
通讯作者：
Loeser, RF
中科院分区：
文献类型：
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作者：
Forsyth, CB;Pulai, J;Loeser, RF
目标。确定整合素介导的信号传导是否导致软骨细胞有丝分裂原激活蛋白（MAP）激酶的激活，从而导致基质金属蛋白酶13 （MMP-13；胶原酶3）的表达增加，基质金属蛋白酶13是软骨基质降解的有效介质。从正常踝关节和膝关节软骨中分离的人关节软骨细胞被培养成单层。用120-kd的纤维连接蛋白片段（FN-f）处理细胞，该片段可以结合alpha5beta1整合素或特异性整合素受体的抗体。磷酸特异性抗体免疫印迹法检测MAP激酶的活化。明胶酶谱法测定MMP的产生，免疫印迹法和荧光肽法测定MMP-13的产生和激活。研究发现，人关节软骨细胞对120-kd FN-f和针对alpha2beta1和alpha5beta1整合素的粘附阻断抗体有反应，细胞外信号调节激酶1 (ERK1)/ERK2、c-Jun n末端激酶（JNK）和p38 MAP激酶的磷酸化增加。完整的FN和针对alpha1、alpha3和alphaVbeta3的整合素阻断抗体以及非阻断性alpha5抗体没有作用。MAP激酶激活后，c-Jun和核因子κ b抑制剂的磷酸化增加，随后条件培养基中促和活化的MMP-13增加。丝裂原活化蛋白激酶激酶、p38和JNK的抑制剂都能抑制MMP-13的增加，而白细胞介素-1受体拮抗剂（IL-1Ra）蛋白则不能。然而，IL-1Ra部分抑制fn -f诱导的mmp -13的激活。FN-f刺激的整合素介导的MAP激酶信号传导与前体和活性MMP-13的产生和释放增加有关。自分泌IL-1的产生似乎导致额外的MMP-13激活。这些过程可能在关节炎中负责进行性软骨退化的反馈回路中起关键作用。
Objective. To determine if integrin-mediated signaling results in activation of chondrocyte mitogenactivated protein (MAP) kinases that lead to increased expression of matrix metalloproteinase 13 (MMP-13; collagenase 3), a potent mediator of cartilage matrix degradation.Methods. Human articular chondrocytes isolated from normal ankle and knee cartilage obtained from tissue donors were cultured in monolayers. The cells were treated with a 120-kd fibronectin fragment (FN-f) that binds the alpha5beta1 integrin or with antibodies to specific integrin receptors. Activation of MAP kinases was determined by immunoblotting with phosphospecific antibodies. MMP production was measured by gelatin zymography, and MMP-13 production and activation were determined by immunoblotting and by a fluorogenic peptide assay.Results. Human articular chondrocytes were found to respond to the 120-kd FN-f and to adhesion-blocking antibodies to the alpha2beta1 and alpha5beta1 integrins with increased phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2, c-Jun N-terminal kinase (JNK), and p38 MAP kinases. Intact FN and integrin-blocking antibodies to alpha1, alpha3, and alphaVbeta3 and a nonblocking alpha5 antibody had no effect. After MAP kinase activation, increased phosphorylation of c-Jun and the nuclear factor kappaB inhibitor was noted, followed by increased pro- and activated MMP-13 in the conditioned media. Inhibitors of mitogen-activated protein kinase kinase, p38, and JNK were each able to inhibit increased MMP-13 production, while the interleukin-1 receptor antagonist (IL-1Ra) protein did not. However, the IL-1Ra partially inhibited FN-f-induced activation of MMP-13.Conclusion. Integrin-mediated MAP kinase signaling stimulated by FN-f is associated with increased production and release of pro- and active MMP-13. Autocrine production of IL-1 appears to result in additional MMP-13 activation. These processes may play a key role in feedback loops responsible for progressive cartilage degradation in arthritis.