Lin C W, Robbins P D, Georgescu H I, Evans C H
Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
Exp Cell Res. 1996 Feb 25;223(1):117-26. doi: 10.1006/excr.1996.0064.
Induction of stromelysin and collagenase mRNAs in response to phorbol myristate acetate (PMA) and autocrine factors (CAF) was compared in primary cultures of lapine synovial fibroblasts and an immortalized line of these cells known as HIG-82. In both cell types, message induction was quicker for CAF than for PMA. Appearance of both stromelysin and collagenase mRNAs occurred earlier in HIG-82 cells and, unlike primary cells, HIG-82 cells partially resisted inhibition by cycloheximide. To determine whether differences in AP-1 activity could account for these observations, the induction of c-fos and c-jun mRNAs was studied in conjunction with gel shift assays for AP-1 binding. Both inducers increased the abundance of c-fos mRNA, although the response was weaker in HIG-82 cells. However, the increase in c-jun mRNA was more marked in HIG-82 cells; furthermore, this increase was sustained for over 6 h. Gel shift assays confirmed that in both types of cells PMA and CAF increased AP-1 binding activity. In primary cells, this activity was sensitive to cycloheximide, but in HIG-82 cells, there was only partial sensitivity to cycloheximide. The gel shift analyses and data from experiments using an AP-1-CAT reporter construct revealed, in many cultures, constitutive AP-1 activity in the absence of stromelysin and collagenase expression, suggesting that AP-1 alone is insufficient for matrix metalloproteinase induction. Antisense oligonucleotides to c-fos and c-jun strongly inhibited the induction of stromelysin mRNA in primary cells treated with PMA, but was only weakly active against message induction in HIG-82 cells. In neither primary cells nor HIG-82 cells did antisense oligonucleotides strongly inhibit stromelysin induction in response to CAF. These data suggest there may exist an AP-1-independent route to message induction or that factors other than c-FOS and c-JUN may be used in certain circumstances. Western blot analyses detected no marked difference between HIG-82 cells and primary cells in their resting levels of c-FOS and c-JUN. Thus the differences reported here between HIG-82 cells and primary cells in their resting levels of c-FOS and c-JUN. Thus the differences reported here between HIG-82 cells and primary cells in the kinetics and cycloheximide sensitivity of MMP induction may reside in their abilities to modify posttranslationally the relevant transcription factors.
在兔滑膜成纤维细胞原代培养物和一种名为HIG-82的这些细胞的永生化细胞系中，比较了佛波酯肉豆蔻酸酯（PMA）和自分泌因子（CAF）诱导基质溶解素和胶原酶mRNA的情况。在这两种细胞类型中，CAF诱导信息的速度都比PMA快。基质溶解素和胶原酶mRNA在HIG-82细胞中出现得更早，并且与原代细胞不同，HIG-82细胞对放线菌酮的抑制有部分抗性。为了确定AP-1活性的差异是否可以解释这些观察结果，结合AP-1结合的凝胶迁移试验研究了c-fos和c-jun mRNA的诱导情况。两种诱导剂都增加了c-fos mRNA的丰度，尽管在HIG-82细胞中的反应较弱。然而，c-jun mRNA的增加在HIG-82细胞中更为明显；此外，这种增加持续了6小时以上。凝胶迁移试验证实，在两种细胞类型中，PMA和CAF都增加了AP-1结合活性。在原代细胞中，这种活性对放线菌酮敏感，但在HIG-82细胞中，对放线菌酮只有部分敏感性。凝胶迁移分析以及使用AP-1-CAT报告构建体的实验数据显示，在许多培养物中，在没有基质溶解素和胶原酶表达的情况下存在组成型AP-1活性，这表明单独的AP-1不足以诱导基质金属蛋白酶。针对c-fos和c-jun的反义寡核苷酸强烈抑制了用PMA处理的原代细胞中基质溶解素mRNA的诱导，但对HIG-82细胞中的信息诱导只有微弱的活性。在原代细胞和HIG-82细胞中，反义寡核苷酸都没有强烈抑制对CAF的基质溶解素诱导。这些数据表明，可能存在一条不依赖AP-1的信息诱导途径，或者在某些情况下可能使用除c-FOS和c-JUN之外的其他因子。蛋白质印迹分析未检测到HIG-82细胞和原代细胞在c-FOS和c-JUN的静息水平上有明显差异。因此，这里报道的HIG-82细胞和原代细胞在MMP诱导的动力学和放线菌酮敏感性方面的差异可能在于它们对相关转录因子进行翻译后修饰的能力。
