决定骨骼发育的转录因子的转录调控机制的研究
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摘要
骨是一个极其复杂、不断变化的组织,它的生长过程涉及诸多因素。转录因子是影响骨生长非常重要的因素。Runx2和Osterix是成骨细胞分化和功能及骨形成的两个重要的转录因子。在成骨细胞分化途径中,Osterix处于Runx2下游,但Osterix是否受Runx2的直接调控目前仍不清楚。
通过RT-PCR技术,我们发现在许多非成骨细胞系(包括已分化细胞和间质细胞)和成骨细胞系中瞬时表达Runx2能够诱导Osterix的表达。我们克隆了3.2kb人的Osterix基因5’侧翼区,并将启动子连接到报告基因上进行功能分析。结果显示Runx2能够上调该启动子的活性,并且进一步的实验又证明了在转录起始位点上游1.1kb和0.5kb之间的区域是维持启动子基本活性所必需,同时该区间也同Runx2的上调活性密切相关。在此区间内我们确定了一个Runx2的功能性结合位点“AGTGGTT”,此外还有另一个弱调节位点“TGTGGT”。以上实验数据支持Runx2参与调控Osterix基因的表达的假说。此外,在非成骨细胞中,瞬时转染和荧光素酶双报告实验显示Osterix能上调I-胶原蛋白的2.3kb启动子活性,但Runx2不能。这点差别暗示了在成骨细胞分化中处于Runx2下游的Osterix是激活I-胶原蛋白表达所必需的。
Sox9、L-Sox5和Sox6这三种转录因子是软骨细胞分化和软骨形成所必需的。它们共同作用足以诱导永久性软骨的形成。在软骨细胞分化过程中,这三种转录因子更为精细的调控机制还不为所知。我们利用反转录病毒系统建立了以ATDC5细胞为基础的,过表达L-Sox5、Sox6、Sox9和L-Sox5+Sox9的稳转细胞系,并通过Alcian blue、RT-PCR等方法检测软骨细胞分化程度。用抑制剂来确定与Stat1有关的各个稳转细胞的增殖情况以及MAPK所调节的分化情况。结果表明过表达L-Sox5和Sox6能加快软骨细胞增殖,抑制分化;过表达Sox9显著抑制增殖,但却能提高软骨特异性基因的表达。如果外源L-Sox5与Sox9的比例接1:1,便能使增殖和分化在很大程度上接近正常。此外Sox转录因子表达状态的变化能够调控Stat1的表达变化,也使MAPK途径产生不同应答。这些实验结果说明Sox转录因子之间的比例变化能够调控软骨细胞的增殖和分化。在软骨中,胞外信号之所以造就了不同类型的软骨细胞(增殖或分化)至少部分是通过建立不同的Sox比例实现的。
The skeleton is a very complicated and continuously changing tissue. Multiple factors influence skeletal development. Among these factors, transcription factors should play a central regulatory role. Runx2 and Osterix are both key transcription factors involved in osteoblast differentiation and bone formation. However, whether Runx2 positively regulats Osterix is not known.
By using RT-PCR, we found that transient expression of Runx2 in a number of non-osteoblastic cell lines, either pluripotent or differentiated, and in the osteoblastic cell lines was able to induce the expression of Osterix. We cloned the 3.2 kb 5’-flanking region of human Osterix gene and analyzed the promoter linked to a firefly luciferase reporter gene. We found that Runx2 could upregulate the promoter activity. The fragment between 1.1 kb and 0.5 kb upstream of the first translation start codon was needed for basal promoter activity. A main functional Runx2 binding site“AGTGGTT”was then identified within the promoter, whereas, another Runx2 binding site“TGTGGT”was canonical, but not conserved. Moreover, the transient transfection and dual-luciferase assay showed Osterix up-regulated the activity of the 2.3kb type I collagen promoter in the non-osteoblastic cells, but Runx2 did not. This defference implies that Osterix, the down stream transcription factor of Runx2 during osteoblast differentiation, is needed to stimulate the osteoblast-specific gene expression of type I collagen.
The three Sox transcription factors, L-Sox5, Sox6, and Sox9, are essentially required for chondrocyte differentiation and cartilage development. They are sufficient to induce permanent cartilage. However, their precise mode of action is still poorly understood when they function together in chondrocytes. By using a retroviral system, we generate several stable cell lines overexpressing L-Sox5, Sox6, Sox9, and L-Sox5 and Sox9, respectively. Chondrogenic differentiation was examined by Alcian Blue staining, RT-PCR etc. Specific inhibitors were used to characterize the Stat1-related proliferation or the MAPK-mediated differentiation of the cell lines. The aberrant overexpression of L-Sox5 or Sox6 in ATDC5 dramatically accelerated proliferation and inhibited differentiation during chondrogenesis. In contrast, the aberrant overexpression of Sox9 markedly inhibited proliferation but enhanced the expression of cartilage-specific genes. Exogenous L-Sox5 and Sox9 in the ratio near 1:1 largely secured normal proliferation and differentiation. In addition, the altered expression statuses of the Sox transcription factors differently regulated Stat1 expression. And these alterations also resulted in different responses to the MAPK pathway. The alterations in the starting ratio of the Sox transcription factors modulate proliferation and differentiation during chondrogenesis. Extracellular signals generate different chondrocytes at least in part through establishing varied ratios of the Sox transcription factors in the cartilage.
通过RT-PCR技术,我们发现在许多非成骨细胞系(包括已分化细胞和间质细胞)和成骨细胞系中瞬时表达Runx2能够诱导Osterix的表达。我们克隆了3.2kb人的Osterix基因5’侧翼区,并将启动子连接到报告基因上进行功能分析。结果显示Runx2能够上调该启动子的活性,并且进一步的实验又证明了在转录起始位点上游1.1kb和0.5kb之间的区域是维持启动子基本活性所必需,同时该区间也同Runx2的上调活性密切相关。在此区间内我们确定了一个Runx2的功能性结合位点“AGTGGTT”,此外还有另一个弱调节位点“TGTGGT”。以上实验数据支持Runx2参与调控Osterix基因的表达的假说。此外,在非成骨细胞中,瞬时转染和荧光素酶双报告实验显示Osterix能上调I-胶原蛋白的2.3kb启动子活性,但Runx2不能。这点差别暗示了在成骨细胞分化中处于Runx2下游的Osterix是激活I-胶原蛋白表达所必需的。
Sox9、L-Sox5和Sox6这三种转录因子是软骨细胞分化和软骨形成所必需的。它们共同作用足以诱导永久性软骨的形成。在软骨细胞分化过程中,这三种转录因子更为精细的调控机制还不为所知。我们利用反转录病毒系统建立了以ATDC5细胞为基础的,过表达L-Sox5、Sox6、Sox9和L-Sox5+Sox9的稳转细胞系,并通过Alcian blue、RT-PCR等方法检测软骨细胞分化程度。用抑制剂来确定与Stat1有关的各个稳转细胞的增殖情况以及MAPK所调节的分化情况。结果表明过表达L-Sox5和Sox6能加快软骨细胞增殖,抑制分化;过表达Sox9显著抑制增殖,但却能提高软骨特异性基因的表达。如果外源L-Sox5与Sox9的比例接1:1,便能使增殖和分化在很大程度上接近正常。此外Sox转录因子表达状态的变化能够调控Stat1的表达变化,也使MAPK途径产生不同应答。这些实验结果说明Sox转录因子之间的比例变化能够调控软骨细胞的增殖和分化。在软骨中,胞外信号之所以造就了不同类型的软骨细胞(增殖或分化)至少部分是通过建立不同的Sox比例实现的。
The skeleton is a very complicated and continuously changing tissue. Multiple factors influence skeletal development. Among these factors, transcription factors should play a central regulatory role. Runx2 and Osterix are both key transcription factors involved in osteoblast differentiation and bone formation. However, whether Runx2 positively regulats Osterix is not known.
By using RT-PCR, we found that transient expression of Runx2 in a number of non-osteoblastic cell lines, either pluripotent or differentiated, and in the osteoblastic cell lines was able to induce the expression of Osterix. We cloned the 3.2 kb 5’-flanking region of human Osterix gene and analyzed the promoter linked to a firefly luciferase reporter gene. We found that Runx2 could upregulate the promoter activity. The fragment between 1.1 kb and 0.5 kb upstream of the first translation start codon was needed for basal promoter activity. A main functional Runx2 binding site“AGTGGTT”was then identified within the promoter, whereas, another Runx2 binding site“TGTGGT”was canonical, but not conserved. Moreover, the transient transfection and dual-luciferase assay showed Osterix up-regulated the activity of the 2.3kb type I collagen promoter in the non-osteoblastic cells, but Runx2 did not. This defference implies that Osterix, the down stream transcription factor of Runx2 during osteoblast differentiation, is needed to stimulate the osteoblast-specific gene expression of type I collagen.
The three Sox transcription factors, L-Sox5, Sox6, and Sox9, are essentially required for chondrocyte differentiation and cartilage development. They are sufficient to induce permanent cartilage. However, their precise mode of action is still poorly understood when they function together in chondrocytes. By using a retroviral system, we generate several stable cell lines overexpressing L-Sox5, Sox6, Sox9, and L-Sox5 and Sox9, respectively. Chondrogenic differentiation was examined by Alcian Blue staining, RT-PCR etc. Specific inhibitors were used to characterize the Stat1-related proliferation or the MAPK-mediated differentiation of the cell lines. The aberrant overexpression of L-Sox5 or Sox6 in ATDC5 dramatically accelerated proliferation and inhibited differentiation during chondrogenesis. In contrast, the aberrant overexpression of Sox9 markedly inhibited proliferation but enhanced the expression of cartilage-specific genes. Exogenous L-Sox5 and Sox9 in the ratio near 1:1 largely secured normal proliferation and differentiation. In addition, the altered expression statuses of the Sox transcription factors differently regulated Stat1 expression. And these alterations also resulted in different responses to the MAPK pathway. The alterations in the starting ratio of the Sox transcription factors modulate proliferation and differentiation during chondrogenesis. Extracellular signals generate different chondrocytes at least in part through establishing varied ratios of the Sox transcription factors in the cartilage.
引文
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