Twigg Stephen M, Joly Alison H, Chen Michelle M, Tsubaki Junko, Kim Ho-Seong, Hwa Vivian, Oh Youngman, Rosenfeld Ron G
Department of Pediatrics, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Endocrinology. 2002 Apr;143(4):1260-9. doi: 10.1210/endo.143.4.8741.
Expansion of extracellular matrix with fibrosis occurs in many tissues, including skin, as part of the end-organ complications in diabetes. Advanced glycosylation end-products (AGEs) have been implicated as a pathogenic factor in diabetic tissue fibrosis. Connective tissue growth factor (CTGF), also known as IGF-binding protein-related protein-2, induces extracellular matrix. We have recently shown that CTGF mRNA and protein are up-regulated by AGE treatment of cultured human dermal fibroblasts. The aim of this study was to determine whether CTGF is an autocrine mediator in the induction of fibronectin (FN) by AGE. Primary cultures of nonfetal human dermal fibroblasts in confluent monolayer were treated with synthesized soluble AGE BSA, 0-200 microg/ml. Analysis of mRNA, by quantitative real-time RT-PCR and conditioned media from treated cultures, showed that FN mRNA was increased by approximately 4-fold at 48 h, and FN protein levels by Western immunoblot and FN ELISA were doubled, compared with control. In the same system, added recombinant human CTGF (0-500 ng/ml) induced FN mRNA and protein levels dose dependently and in a rapid time course. To test whether AGE BSA acts through cell-derived CTGF to induce FN, a CTGF neutralizing antibody was shown to significantly attenuate, but not fully inhibit, the AGE induction of FN mRNA. A pan-specific PKC inhibitor, GF109203X, at 0.2 microM, inhibited the induction of FN mRNA by AGE BSA. Although the same inhibitor did not significantly affect the induction of CTGF mRNA by AGE, it blocked the induction of FN mRNA by recombinant human CTGF. In summary, the induction of FN by AGE is partly mediated by the AGE-induced up-regulation of cell-derived CTGF and is dependent on PKC activity. These results have potential implications for the expansion of extracellular matrix in diabetes mellitus by advanced glycosylation end products.
作为糖尿病终末器官并发症的一部分，细胞外基质随纤维化在包括皮肤在内的许多组织中发生扩张。晚期糖基化终产物（AGEs）被认为是糖尿病组织纤维化的致病因素。结缔组织生长因子（CTGF），也称为胰岛素样生长因子结合蛋白相关蛋白-2，可诱导细胞外基质生成。我们最近发现，经AGE处理的培养人皮肤成纤维细胞中CTGF mRNA和蛋白表达上调。本研究的目的是确定CTGF是否是AGE诱导纤连蛋白（FN）的自分泌介质。将汇合单层的非胎儿人皮肤成纤维细胞原代培养物用合成的可溶性AGE BSA（0 - 200μg/ml）处理。通过定量实时RT-PCR分析mRNA以及处理后培养物的条件培养基，结果显示，与对照相比，48小时时FN mRNA增加约4倍，通过Western免疫印迹和FN ELISA检测的FN蛋白水平增加一倍。在同一系统中，添加重组人CTGF（0 - 500 ng/ml）可剂量依赖性且快速诱导FN mRNA和蛋白水平。为了测试AGE BSA是否通过细胞来源的CTGF诱导FN，一种CTGF中和抗体可显著减弱但不能完全抑制AGE对FN mRNA的诱导。一种泛特异性PKC抑制剂GF109203X，浓度为0.2μM时，可抑制AGE BSA对FN mRNA的诱导。虽然相同抑制剂对AGE诱导CTGF mRNA没有显著影响，但它可阻断重组人CTGF对FN mRNA的诱导。总之，AGE对FN的诱导部分由AGE诱导的细胞来源CTGF上调介导，且依赖于PKC活性。这些结果对晚期糖基化终产物在糖尿病中细胞外基质扩张具有潜在意义。
