National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, United States of America.
Section of Orthodontics and Dentofacial Orthopedics, Faculty of Dental Science, Kyushu University, Fukuoka-shi, Fukuoka, Japan.
PLoS One. 2021 Nov 15;16(11):e0259966. doi: 10.1371/journal.pone.0259966. eCollection 2021.
Amelogenins, major extra cellular matrix proteins of developing tooth enamel, are predominantly expressed by ameloblasts and play significant roles in the formation of enamel. Recently, amelogenin has been detected in various epithelial and mesenchymal tissues, implicating that it might have distinct functions in various tissues. We have previously reported that leucine rich amelogenin peptide (LRAP), one of the alternate splice forms of amelogenin, regulates receptor activator of NF-kappa B ligand (RANKL) expression in cementoblast/periodontal ligament cells, suggesting that the amelogenins, especially LRAP, might function as a signaling molecule in bone metabolism. The objective of this study was to identify and define LRAP functions in bone turnover. We engineered transgenic (TgLRAP) mice using a murine 2.3kb α1(I)-collagen promoter to drive expression of a transgene consisting of LRAP, an internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) to study functions of LRAP in bone formation and resorption. Calvarial cell cultures from the TgLRAP mice showed increased alkaline phosphatase (ALP) activity and increased formation of mineralized nodules compared to the cells derived from wild-type (WT) mice. The TgLRAP calvarial cells also showed an inhibitory effect on osteoclastogenesis in vitro. Gene expression comparison by quantitative polymerase chain reaction (Q-PCR) in calvarial cells indicated that bone formation makers such as Runx2, Alp, and osteocalcin were increased in TgLRAP compared to the WT cells. Meanwhile, Rankl expression was decreased in the TgLRAP cells in vitro. The ovariectomized (OVX) TgLRAP mice resisted bone loss induced by ovariectomy resulting in higher bone mineral density in comparison to OVX WT mice. The quantitative analysis of calcein intakes indicated that the ovariectomy resulted in increased bone formation in both WT and TgLRAP mice; OVX TgLRAP appeared to show the most remarkably increased bone formation. The parameters for bone resorption in tissue sections showed increased number of osteoclasts in OVX WT, but not in OVX TgLRAP over that of sham operated WT or TgLRAP mice, supporting the observed bone phenotypes in OVX mice. This is the first report identifying that LRAP, one of the amelogenin splice variants, affects bone turnover in vivo.
釉原蛋白是牙釉质中主要的细胞外基质蛋白，主要由成釉细胞表达，在釉质形成中发挥重要作用。最近，釉原蛋白已在各种上皮和间充质组织中被检测到，这表明它可能在不同组织中具有不同的功能。我们之前曾报道，富含亮氨酸的釉原蛋白肽（LRAP）是釉原蛋白的一种剪接变体，可调节成牙骨质细胞/牙周韧带细胞中核因子 κB 配体受体激活剂（RANKL）的表达，这表明釉原蛋白，特别是 LRAP，可能作为一种信号分子在骨代谢中发挥作用。本研究的目的是鉴定并定义 LRAP 在骨转换中的功能。我们使用小鼠 2.3kb α1(I)-胶原启动子工程转基因（TgLRAP）小鼠，该启动子驱动由 LRAP、内部核糖体进入位点（IRES）和增强型绿色荧光蛋白（EGFP）组成的转基因表达，以研究 LRAP 在骨形成和吸收中的功能。与来自野生型（WT）小鼠的细胞相比，TgLRAP 小鼠的颅骨细胞培养物显示碱性磷酸酶（ALP）活性增加，矿化结节形成增加。TgLRAP 颅骨细胞在体外还显示出对破骨细胞形成的抑制作用。颅骨细胞的定量聚合酶链反应（Q-PCR）基因表达比较表明，与 WT 细胞相比，骨形成标志物如 Runx2、Alp 和骨钙素在 TgLRAP 中增加。同时，TgLRAP 细胞中的 Rankl 表达在体外减少。去卵巢（OVX）TgLRAP 小鼠抵抗去卵巢引起的骨丢失，导致骨密度高于 OVX WT 小鼠。钙黄绿素摄取的定量分析表明，去卵巢导致 WT 和 TgLRAP 小鼠的骨形成增加；OVX TgLRAP 似乎表现出最明显的骨形成增加。组织切片中骨吸收参数显示，与假手术 WT 或 TgLRAP 小鼠相比，OVX WT 小鼠的破骨细胞数量增加，但 OVX TgLRAP 小鼠没有，这支持了 OVX 小鼠观察到的骨表型。这是首次报道鉴定釉原蛋白的一种剪接变体 LRAP 影响体内骨转换。
