Frenkel B, Capparelli C, Van Auken M, Baran D, Bryan J, Stein J L, Stein G S, Lian J B
Department of Cell Biology and Cancer Center, University of Massachusetts Medical Center, Worcester 01655, USA.
Endocrinology. 1997 May;138(5):2109-16. doi: 10.1210/endo.138.5.5105.
The bone-specific osteocalcin gene is a well established marker of osteoblast activity. We have studied osteocalcin transcription in transgenic mice carrying rat osteocalcin promoter-chloramphenicol acetyltransferase (CAT) reporter constructs. Transgenic lines carrying each of the 1.7-, 1.1-, 0.72-, or 0.35-kilobase promoter constructs expressed the reporter gene in a tissue-specific manner. However, each of these constructs was sensitive to site of integration effects, reflected by a high frequency of nonexpressing transgenic lines. High expression of the 1.7-kilobase promoter in osseous tissues was accompanied by low ectopic expression in the brain. Analysis of CAT expression in femurs, calvariae, and lumbar vertebrae of this line indicated considerable variability in promoter activity among individual transgenic animals. Analysis of the variance in CAT activity demonstrated a linkage between promoter activities in these distant skeletal sites. Promoter activity was inversely correlated with age, and females exhibited severalfold higher activity than age-matched males. Bone marrow stromal cells from these animals, cultured under conditions that support osteoblast differentiation, exhibited the expected postproliferative onset of osteocalcin promoter activity, as assessed by CAT assay. The ex vivo CAT activity was not dependent on the sex or the age of the donor transgenic mouse. Taken together, our results are consistent with the hypothesis that a common, probably humoral, factor(s) regulates osteocalcin transcription in distant skeletal sites. We suggest that the abundance of this factor(s) is different between males and females and among individual mice at a given time point, and that ex vivo culturing of osteoblasts reduces the variation in osteocalcin promoter activity by eliminating the physiological contribution of this factor.
骨特异性骨钙素基因是成骨细胞活性的一个公认标志物。我们研究了携带大鼠骨钙素启动子-氯霉素乙酰转移酶（CAT）报告基因构建体的转基因小鼠中的骨钙素转录情况。携带1.7、1.1、0.72或0.35千碱基启动子构建体的转基因品系以组织特异性方式表达报告基因。然而，这些构建体中的每一个都对整合位点效应敏感，这表现为非表达转基因品系的高频率出现。1.7千碱基启动子在骨组织中的高表达伴随着在脑中的低异位表达。对该品系的股骨、颅骨和腰椎中的CAT表达分析表明，各个转基因动物之间启动子活性存在相当大的变异性。对CAT活性方差的分析表明，这些远距离骨骼部位的启动子活性之间存在联系。启动子活性与年龄呈负相关，并且雌性比年龄匹配的雄性表现出高几倍的活性。在支持成骨细胞分化的条件下培养的这些动物的骨髓基质细胞，通过CAT测定评估，表现出预期的骨钙素启动子活性的增殖后起始。体外CAT活性不依赖于供体转基因小鼠的性别或年龄。综上所述，我们的结果与以下假设一致：一种共同的、可能是体液性的因子调节远距离骨骼部位的骨钙素转录。我们认为，在给定时间点，这种因子在雄性和雌性之间以及个体小鼠之间的丰度不同，并且成骨细胞的体外培养通过消除这种因子的生理贡献而减少了骨钙素启动子活性的变化。
