Paz J, Wade K, Kiyoshima T, Sodek J, Tang J, Tu Q, Yamauchi M, Chen J
Division of Oral Biology, Tufts University School of Dental Medicine, Boston, MA 02111, USA.
Matrix Biol. 2005 Aug;24(5):341-52. doi: 10.1016/j.matbio.2005.05.009.
Bone sialoprotein (BSP) is a phosphorylated glycoprotein that is expressed almost exclusively in mineralizing connective tissues. In bone, expression of BSP correlates with the differentiation of osteoblasts and the onset of mineralization. To determine how the tissue- and differentiation-specific transcription of BSP is regulated, various lengths of promoter sequence were ligated to a luciferase reporter and stably transfected into a rat stromal bone marrow cell line, RBMC-D8 and undifferentiated C3H10T1/2 cells. Luciferase transcription of reporter constructs including 5.4 kb (mBSP5.4Luc) and 9.0 kb (mBSP9.0Luc) of the BSP promoter was strongly up-regulated in parallel with endogenous BSP mRNA in differentiating SBMCs, but not in C3H10T1/2 cells. In contrast, 0.1 kb and 1.4 kb BSP promoter constructs did not show selective expression. To determine tissue-specific expression in vivo, transgenic mice expressing reporter constructs for the 9.0 kb promoter and a 4.8 kb promoter lacking two upstream Cbfa1/Runx2 elements (mBSP9.0Luc and mBSP4.8Luc, respectively) were generated. Analysis of various tissues collected from 1-, 4-, 7-, 14-, and 42-day-old mice revealed extremely high levels of luciferase activity in calvaria, mandible, and tibia of the mBSP9.0Luc mice. In contrast, soft tissues showed negligible luciferase expression. Mice harboring the 4.8 kb transgene also showed selective luciferase expression but displayed a significantly lower activity in mineralized tissues. Northern hybridization of endogenous BSP mRNA and immunostaining of BSP in mBSP9.0Luc mice showed a temporo-spatial expression pattern consistent with the luciferase activity. These results indicate that regulatory elements within the 9.0 kb region of the promoter are required for strong, tissue- and differentiation-specific expression of BSP.
骨唾液蛋白（BSP）是一种磷酸化糖蛋白，几乎只在矿化结缔组织中表达。在骨组织中，BSP的表达与成骨细胞的分化及矿化的开始相关。为了确定BSP的组织特异性和分化特异性转录是如何调控的，将不同长度的启动子序列与荧光素酶报告基因连接，并稳定转染到大鼠骨髓基质细胞系RBMC-D8和未分化的C3H10T1/2细胞中。包含BSP启动子5.4 kb（mBSP5.4Luc）和9.0 kb（mBSP9.0Luc）的报告基因构建体的荧光素酶转录，在分化的骨髓基质细胞（SBMCs）中与内源性BSP mRNA平行强烈上调，但在C3H10T1/2细胞中则不然。相比之下，0.1 kb和1.4 kb的BSP启动子构建体未显示出选择性表达。为了确定体内的组织特异性表达，构建了分别表达9.0 kb启动子和缺少两个上游Cbfa1/Runx2元件的4.8 kb启动子的报告基因构建体的转基因小鼠（分别为mBSP9.0Luc和mBSP4.8Luc）。对1日龄、4日龄、7日龄、14日龄和42日龄小鼠采集的各种组织进行分析，发现mBSP9.0Luc小鼠的颅骨、下颌骨和胫骨中荧光素酶活性极高。相比之下，软组织中的荧光素酶表达可忽略不计。携带4.8 kb转基因的小鼠也显示出选择性荧光素酶表达，但在矿化组织中的活性明显较低。mBSP9.0Luc小鼠中内源性BSP mRNA的Northern杂交和BSP的免疫染色显示出与荧光素酶活性一致的时空表达模式。这些结果表明，启动子9.0 kb区域内的调控元件是BSP强大的、组织特异性和分化特异性表达所必需的。
