Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Box 9037, Grand Forks, ND 58202, USA.
Osteoarthritis Cartilage. 2009 Oct;17(10):1385-92. doi: 10.1016/j.joca.2009.03.024. Epub 2009 Apr 17.
Since fibronectin fragments (Fn-fs) enhance cartilage damage through integrins, the objective was to investigate the role of integrin linked kinases, focal adhesion kinase (FAK) and a soluble form of FAK, proline rich tyrosine kinase 2 (Pyk2) and cellular src kinase (c-src) and the transcription factor, nuclear factor kappaB (NF-kappaB) in cartilage damage.
Bovine chondrocytes were cultured with various concentrations of three different Fn-fs, an amino-terminal 29 kDa, a gelatin binding 50 kDa and a central 140-kDa Fn-fs, each with progressively weaker cartilage damaging activity, or with native fibronectin (Fn), and lysates probed for activation of the selected kinases. Confocal microscopy was used to visualize intracellular location of activated kinases and NF-kappaB. Various kinase inhibitors were tested for their effects on Fn-f mediated upregulation of matrix metalloproteinase (MMP)-3 and -13 and cartilage proteoglycan (PG) depletion.
The Fn-fs kinetically enhanced phosphorylation of FAK but did not show a clear dose-response effect. The 29-kDa and 50-kDa Fn-fs enhanced phosphorylation of Pyk2, c-src and NF-kappaB to a much greater extent than the 140-kDa Fn-f and native Fn and did so as a function of dose. The 29-kDa Fn-f enhanced the phosphorylation of nuclear Pyk2 as compared with no treatment or native Fn. Inhibitors of Pyk2, c-src, NF-kappaB and protein kinase Cdelta (PKCdelta) decreased MMP upregulation and decreased Fn-f mediated damage to cartilage.
These studies enhance our knowledge of crucial factors in Fn-f mediated signaling in MMP upregulation and cartilage damage and because of the potential physiologic relevance of Fn-fs, provide a better knowledge of cartilage degeneration in general.
由于纤连蛋白片段（Fn-fs）通过整合素增强软骨损伤，本研究旨在探讨整合素连接激酶、粘着斑激酶（FAK）和 FAK 的可溶性形式、富含脯氨酸的酪氨酸激酶 2（Pyk2）和细胞Src 激酶（c-src）以及转录因子核因子κB（NF-κB）在软骨损伤中的作用。
用不同浓度的三种不同的 Fn-fs（氨基端 29kDa、胶凝结合 50kDa 和中心 140kDa Fn-fs）培养牛软骨细胞，每种 Fn-fs 的软骨损伤活性逐渐减弱，或用天然纤连蛋白（Fn）培养，然后用所选激酶的激活物探测裂解物。共聚焦显微镜用于观察激活激酶和 NF-κB 的细胞内位置。测试了各种激酶抑制剂对 Fn-f 介导的基质金属蛋白酶（MMP）-3 和 -13 以及软骨蛋白聚糖（PG）耗竭的上调作用。
Fn-fs 动力学增强了 FAK 的磷酸化，但没有显示出明显的剂量反应效应。29kDa 和 50kDa Fn-fs 增强了 Pyk2、c-src 和 NF-κB 的磷酸化，比 140kDa Fn-f 和天然 Fn 要强得多，而且呈剂量依赖性。29kDa Fn-f 增强了核 Pyk2 的磷酸化，而与未处理或天然 Fn 相比。Pyk2、c-src、NF-κB 和蛋白激酶 C 德尔塔（PKCδ）抑制剂降低了 MMP 的上调，并降低了 Fn-f 介导的软骨损伤。
这些研究增强了我们对 Fn-f 介导的信号转导中 MMP 上调和软骨损伤的关键因素的认识，并且由于 Fn-fs 的潜在生理相关性，提供了对软骨退变的更好认识。
