A study on the function of Sphingomyelin Synthases

骨细胞被认为在骨代谢中起着至关重要的作用，尽管关于其特定产物和生理功能的信息有限。为了分离在骨细胞中表达的基因，我们采用了从骨细胞富集的骨片段中提取RNA的减法抑制PCR方法。通过该方法分离的一个基因编码一种参与磷脂代谢的新酶，该酶被发现是鞘磷脂合成酶(SMS2: Huitema, et al.(2004)；Yamaoka等人（2004）)。为了确定Sms2在体内的表达，我们将LacZ基因引入小鼠Sms2位点，监测Sms2的表达。在E13.5时，通过X-gal染色检测，仅在骨骼元件中检测到表达，特别是在成骨细胞富集的区域。在E16.5胚胎成骨细胞和骨细胞中均有表达。Sms2在长骨的部分分化软骨细胞中表达，但在未钙化的肋骨和关节软骨中未检测到。在成人中，虽然在非骨骼组织中检测到表达，但在骨细胞中明显表达。在基质囊泡中检测到鞘磷脂酶活性，鞘磷脂在基质矿化过程中起关键作用，鞘磷脂在矿化过程中被水解。此外，携带SmpdS（一种编码中性鞘磷脂酶的基因）突变的小鼠表现出骨骼畸形和延迟钙化。事实上，这促使我们假设鞘磷脂可能对矿化有一定的抑制作用。事实上，当Sms2基因在成骨细胞中过表达时，矿化明显受到抑制。因此，Sms2可能在成骨细胞的终末分化和/或骨基质矿化中发挥作用。

Osteocytes are thought to play crucial roles in bone metabolism, although limited information has been available on their specific products and physiological function. To isolate genes expressed in osteocytes, we employed subtractive suppression PCR method using RNA from osteocyte-enriched bone fraction. A genes isolated by the method encodes a novel enzyme involved in phospholipid metabolism, which has been found as a sphingomyelin synthase(SMS2 : Huitema, et. al.(2004) ; Yamaoka, et. al.(2004)). To determine the expression of Sms2 in vivo, LacZ gene was introduced into mouse Sms2 locus to monitor the expression of Sms2. At E13.5, the expression, as monitored by X-gal staining, was detected only in skeletal elements, especially in the areas enriched in osteoblasts. The expression was observed not only in osteoblasts but also in osteocytes of E16.5 embryo. Sms2 was expressed in some differentiated chondrocytes of long bones, but not detected in rib or articular cartilages which remain uncalcified. In adult, although the expression was detected in non-skeletal tissues, it is markedly expressed in osteocytes. It has been reported that sphingomyelinase activity was detected in matrix vesicles, which play a pivotal role in matrix mineralization, and that sphingomyelin was hydrolyzed upon mineralization. Furthermore, mice carrying mutations in SmpdS, a gene encoding neutral sphingomyelinase, exhibited skeletal malformation and delayed calcification. Indeed, it prompted us to hypothesize that sphingomyelin may have some inhibitory effects on mineralization. In fact, when Sms2 gene was overexpressed in osteoblasts, mineralization was significantly suppressed. Thus, Sms2 may play a role in terminal differentiation of osteoblast and/or matrix mineralization in bone.