Christner P J, Hitraya E G, Peters J, McGrath R, Jiménez S A
Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107-5541, USA.
Arthritis Rheum. 1998 Dec;41(12):2132-42. doi: 10.1002/1529-0131(199812)41:12<2132::AID-ART8>3.0.CO;2-W.
To investigate the levels of expression of type I and type III collagen genes in dermal fibroblasts from tight skin 2 (Tsk2) and normal mice and to examine the transcriptional regulation of the alpha1(I) procollagen gene (COL1A1) in these cells.
Dermal fibroblasts from Tsk2 mice and from normal age- and sex-matched control mice were studied. Steady-state levels of alpha1(I) and alpha1(III) procollagen messenger RNA (mRNA) were evaluated by Northern and dot-blot hybridization analyses. The transcriptional regulation of COL1A1 was examined by transient transfection experiments with deletion constructs containing portions of the COL1A1 promoter ligated to the chloramphenicol acetyltransferase reporter gene. To identify DNA binding proteins that interact with regulatory elements within the COL1A1 promoter, gel mobility shift assays were performed with nuclear extracts prepared from normal and Tsk2 dermal fibroblasts.
Synthesis of collagen was almost 100% higher in Tsk2 dermal fibroblasts than in control fibroblasts. Up-regulation of mRNA for 2 extracellular matrix proteins was observed in the Tsk2 dermal fibroblasts compared with the normal cells: the alpha1(I) procollagen mRNA steady-state levels were 50% higher, and those of the alpha1(III) procollagen mRNA 100% higher, in Tsk2 cells. The results of transient transfection experiments with COL1A1 promoter constructs demonstrated that the elevated levels of alpha1(I) collagen mRNA in Tsk2 cells were largely due to increased transcriptional activity of the corresponding gene. Electrophoretic mobility shift assays performed with a probe encompassing a relevant COL1A1 promoter region revealed increased DNA-protein binding activities in nuclear extracts prepared from Tsk2 fibroblasts compared with normal fibroblasts. Competition experiments using consensus Spl and nuclear factor 1 (NF-1) oligonucleotides and supershift experiments using anti-Sp1 and anti-NF-1 antibodies indicated that at least 2 transcription factors, Sp1 and NF-1, or their homologs are involved in the up-regulated transcriptional activity of the COL1A1 promoter in Tsk2 fibroblasts.
Dermal fibroblasts from Tsk2 mice display increased collagen synthesis and up-regulation of alpha1(I) and alpha1(III) procollagen mRNA in vitro. The data also directly demonstrate the transcriptional activation of COL1A1 in dermal fibroblasts from Tsk2 mice and suggest that the transcription factors Sp1 and NF-1 or their homologs play an important role in the upregulated expression of this gene in Tsk2 fibroblasts. These findings are similar to those described for fibroblasts from humans with systemic sclerosis and validate the use of Tsk2 as a model for the study of the connective tissue alterations in this disease.
研究紧皮2（Tsk2）小鼠和正常小鼠真皮成纤维细胞中I型和III型胶原基因的表达水平，并检测这些细胞中α1（I）前胶原基因（COL1A1）的转录调控。
研究了Tsk2小鼠以及年龄和性别匹配的正常对照小鼠的真皮成纤维细胞。通过Northern和斑点杂交分析评估α1（I）和α1（III）前胶原信使核糖核酸（mRNA）的稳态水平。通过瞬时转染实验检测COL1A1的转录调控，该实验使用含有与氯霉素乙酰转移酶报告基因连接的COL1A1启动子部分的缺失构建体。为了鉴定与COL1A1启动子内调控元件相互作用的DNA结合蛋白，用从正常和Tsk2真皮成纤维细胞制备的核提取物进行凝胶迁移率变动分析。
Tsk2真皮成纤维细胞中胶原的合成比对照成纤维细胞高出近100%。与正常细胞相比，在Tsk2真皮成纤维细胞中观察到2种细胞外基质蛋白的mRNA上调：Tsk2细胞中α1（I）前胶原mRNA的稳态水平高出50%，α1（III）前胶原mRNA高出100%。用COL1A1启动子构建体进行的瞬时转染实验结果表明，Tsk2细胞中α1（I）胶原mRNA水平的升高主要是由于相应基因转录活性的增加。用包含COL1A1启动子相关区域的探针进行的电泳迁移率变动分析显示，与正常成纤维细胞相比，从Tsk2成纤维细胞制备的核提取物中DNA-蛋白质结合活性增加。使用共有Sp1和核因子1（NF-1）寡核苷酸的竞争实验以及使用抗Sp1和抗NF-1抗体的超迁移实验表明，至少2种转录因子Sp1和NF-1或其同源物参与了Tsk2成纤维细胞中COL1A1启动子转录活性的上调。
Tsk2小鼠的真皮成纤维细胞在体外表现出胶原合成增加以及α1（I）和α1（III）前胶原mRNA上调。数据还直接证明了Tsk2小鼠真皮成纤维细胞中COL1A1的转录激活，并表明转录因子Sp1和NF-1或其同源物在Tsk2成纤维细胞中该基因的上调表达中起重要作用。这些发现与系统性硬化症患者成纤维细胞的描述相似，并验证了使用Tsk2作为研究该疾病结缔组织改变的模型。
