Collagen, type V, α1 (COL5A1) is regulated by TGF-β in osteoblasts
Collagen, type V, α1 (COL5A1) is regulated by TGF-β in osteoblasts
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DOI：
10.1016/j.matbio.2004.09.004
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发表时间：
2004-11-01
期刊：
影响因子：
6.9
通讯作者：
Seth, A
中科院分区：
文献类型：
--
作者：
Kahai, S;Vary, CPH;Seth, A
骨基质含有高浓度的生长因子，已知其在骨生成过程中发挥重要的调节作用，特别是转化生长因子-β（TGF-β）。根据实验条件、所用细胞及其成熟阶段，在体外和体内均报道了TGF-β对骨形成的不同作用。在这项研究中，我们使用了克隆成骨细胞系MC 3 T3-E1来自新生小鼠颅骨，作为骨发育的体外模型。这些细胞进行有序的。时间依赖性发育序列，其特征在于三个阶段（增殖、分化和矿化），超过30-35天。在这项研究中，cDNA微阵列技术被用来研究8470个基因的表达谱，在TGF-β 1的存在下，在成骨细胞的发展。微阵列分析揭示了120个cDNA在用TGF-β 1处理的MC 3 T3-E1成骨细胞中差异表达。从120个差异表达的基因中，我们选择胶原蛋白，V型，α 1（COL 5A 1）{差异表达=+4.9}进行进一步研究，因为它代表了以前未表征的骨基质组分。利用北方印迹，我们发现，当MC 3 T3-E1细胞用TGF-β 1处理时，在成骨的增殖和分化阶段，COL 5A 1被上调。同时，通过RNA原位杂交和北方印迹杂交，我们发现在E17.5小鼠胚胎的颅骨和发育中的骨中有COL 5A 1 mRNA的表达。最后，通过免疫组织化学观察到E17.5小鼠胚胎发育骨中COL 5A 1蛋白的显著表达。总之，通过使用体外和体内方法，我们发现COL 5A 1基因是成骨过程中TGF-β的靶点。(C)2004 Elsevier B.V/国际基质生物学学会。All rights reserved.
Bone matrix contains high concentrations of growth factors that are known to play important regulatory roles during osteogenesis, particularly transforming growth factor-beta (TGF-beta). Divergent effects of TGF-beta on bone formation have been reported both in vitro and in vivo depending upon experimental conditions, cells employed and their stage of maturation. In this study, we have used a clonal osteoblastic cell line MC3T3-E1 derived from newborn mouse calvaria, as an in vitro model of bone development. These cells undergo an ordered. time-dependent developmental sequence characterized by three stages (proliferation, differentiation and mineralization), over a 30-35-day period. In this study, cDNA microarray technology was used to study the expression profile of 8470 genes, in the presence of TGF-beta1 during osteoblast development. Microarray analysis revealed 120 cDNAs to be differentially expressed in MC3T3-E1 osteoblasts that had been treated with TGF-betal. From the 120 differentially expressed genes, we selected Collagen, type V, alpha1 (COL5A1) {differential expression=+4.9} for further studies since it represents a previously uncharacterized component of the bone matrix. Using Northern blotting, we found that, when MC3T3-E1 cells were treated with TGF-beta1 COL5A1 was up-regulated during the proliferation and differentiation phases of osteogenesis. Furthermore, by a combination of RNA in situ hybridization and Northern blotting, we found COL5A1 mRNA to be expressed in the calvaria and developing bone of the E17.5 mouse embryos. Lastly, significant COL5A1 protein expression was observed by immunohistochemistry in the developing bone of the E17.5 mouse embryos. In conclusion, by the use of in vitro and in vivo approaches, we have discovered that the COL5A1 gene is a target of TGF-beta during osteogenesis. (C) 2004 Elsevier B.V/International Society of Matrix Biology. All rights reserved.