High‐dose estrogen administration is known to induce new bone formation in mouse long bones. To study the role of regulatory proteins in this response, we examined associated changes in femoral messenger RNA (mRNA) for candidate factors. 17β‐estradiol (E2) 0.5 mg was administered to intact female mice by weekly injection, and Northern blot analysis was performed 1, 2, 4, 8, 12, and 16 days after the first injection. In contrast to other factors, an increase was observed in mRNA for bone morphogenetic protein‐6 (BMP‐6), which reached significance at day 8 and subsequent time‐points. Estrogen‐induced changes in BMP‐6 protein expression were assessed by immunocytochemistry in longitudinal femoral sections. In untreated animals, BMP‐6 was expressed by a significant proportion of growth plate chondrocytes and a subpopulation of bone marrow cells. In contrast, osteoblasts were consistently BMP‐6 negative. From as early as 4 days after starting estrogen, clusters of slightly elongated BMP‐6‐positive cells were observed within the marrow cavity; the majority were close to active bone formation surfaces. Double immunolabeling studies revealed that only approximately 10% of BMP‐6‐positive bone marrow cells co‐expressed the osteoblast transcription factor Cbfa1 suggesting that they are largely distinct from the osteoblast precursor population generated concurrently. BMP‐6‐positive cells expressed neither leukocyte nor erythroid markers (CD45 and TER‐119, respectively), consistent with a stromal origin. We conclude that estrogen‐induced osteogenesis in female mice is associated with increased levels of BMP‐6 mRNA in mouse femurs, which seems to reflect the emergence of clusters of BMP‐6 positive stromal cells adjacent to active bone formation surfaces. These findings raise the possibility that BMP‐6 serves as a paracrine mediator of estrogen's osteogenic action in mice.
众所周知,大剂量
雌激素可诱导小鼠长骨中新骨的形成。为了研究调节蛋白在这一反应中的作用,我们研究了股骨信使 RNA(mRNA)中候选因子的相关变化。每周给完整的雌性小鼠注射 0.5 毫克 17β-estradiol (E2),并在首次注射后 1、2、4、8、12 和 16 天进行 Northern 印迹分析。与其他因素不同的是,观察到骨形态发生蛋白-6(
BMP-6)的 mRNA 增加,在第 8 天及其后的时间点达到显著
水平。
雌激素诱导的
BMP-6 蛋白表达变化是通过免疫细胞
化学法在股骨纵切片中进行评估的。在未经处理的动物中,相当大比例的生长板软骨细胞和骨髓细胞亚群表达
BMP-6。相比之下,成骨细胞的
BMP-6 表达一直呈阴性。早在开始使用
雌激素 4 天后,骨髓腔内就观察到了成群的略微拉长的
BMP-6 阳性细胞;其中大部分靠近活跃的骨形成面。双重免疫标记研究显示,只有约 10% 的
BMP-6 阳性骨髓细胞同时表达成骨细胞转录因子 Cbfa1,这表明它们在很大程度上有别于同时产生的成骨细胞前体。
BMP-6阳性细胞既不表达白细胞标记,也不表达红细胞标记(分别为C
D45和TER-119),与基质来源一致。我们的结论是,
雌激素诱导的雌性小鼠骨生成与小鼠股骨中
BMP-6 mRNA
水平的升高有关,这似乎反映了在骨形成活跃面附近出现了
BMP-6 阳性基质细胞群。这些发现提出了一种可能性,即
BMP-6 是
雌激素在小鼠体内成骨作用的旁分泌介质。