Xia Wei, Kong Wuyi, Wang Zhen, Phan Toan-Thang, Lim Ivor J, Longaker Michael T, Yang George P
Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305, USA.
Ann Surg. 2007 Nov;246(5):886-95. doi: 10.1097/SLA.0b013e318070d54f.
We examined the transcriptional response to serum stimulation as an in vitro model of wound healing in keloid fibroblasts to identify molecular mechanisms leading to their aberrant growth.
Keloids are proliferative dermal growths representing a pathologic wound healing response. Although several groups have shown increased expression of profibrotic factors in keloids, there is little known about why they are expressed at higher levels than normal.
Fibroblasts derived from keloids and normal scar were subjected to serum stimulation as an in vitro model to mimic a component of the wound microenvironment to examine differential gene expression in keloid derived fibroblasts versus normal human fibroblasts. A promoter analysis was performed to identify specific enhancers involved in mediating the differential response of connective tissue growth factor (CTGF, CCN2). Point mutations in the enhancers were performed to confirm their role. Finally, we examined activation of transcription factors known to bind the targeted enhancers.
Transcription of CCN2 after serum stimulation was significantly higher in keloid versus normal fibroblasts. Promoter analysis demonstrates the fragment from -625/-140 conferred increased serum responsiveness. Mutational analysis showed an AP-1 and SMAD binding site were both necessary for serum responsiveness. Preventing activation of either transcriptional complex will block CCN2 transcription. Additional experiments suggest that a single complex that includes components of the AP-1 and SMAD binding complexes is responsible for transactivation in response to serum. The key difference between keloid and normal fibroblasts appears to be the degree of activation of c-Jun.
We suggest that altered responsiveness to cellular stress, based upon current data using serum stimulation and past data on response to mechanical strain, is a key defect leading to keloid formation.
我们研究了瘢痕疙瘩成纤维细胞对血清刺激的转录反应，以此作为伤口愈合的体外模型，以确定导致其异常生长的分子机制。
瘢痕疙瘩是增生性皮肤肿物，代表病理性伤口愈合反应。尽管有几个研究小组已表明瘢痕疙瘩中促纤维化因子的表达增加，但对于它们为何比正常水平表达更高却知之甚少。
将瘢痕疙瘩和正常瘢痕来源的成纤维细胞作为体外模型进行血清刺激，以模拟伤口微环境的一个组成部分，从而检测瘢痕疙瘩来源的成纤维细胞与正常人成纤维细胞之间的差异基因表达。进行启动子分析以确定参与介导结缔组织生长因子（CTGF，CCN2）差异反应的特定增强子。对增强子进行点突变以证实其作用。最后，我们检测了已知与靶向增强子结合的转录因子的激活情况。
血清刺激后，瘢痕疙瘩成纤维细胞中CCN2的转录显著高于正常成纤维细胞。启动子分析表明，来自-625 / -140的片段赋予了增强的血清反应性。突变分析表明，AP-1和SMAD结合位点对于血清反应性都是必需的。阻止任何一种转录复合物的激活都会阻断CCN2转录。进一步的实验表明，一个包含AP-1和SMAD结合复合物成分的单一复合物负责对血清的反式激活。瘢痕疙瘩成纤维细胞与正常成纤维细胞之间的关键差异似乎在于c-Jun的激活程度。
基于目前使用血清刺激的数据以及过去对机械应变反应的数据，我们认为对细胞应激反应性的改变是导致瘢痕疙瘩形成的关键缺陷。
Zotero 插件