Identification of the functional activity of the [A-4] amelogenin gene splice product in newborn mouse ameloblasts
Identification of the functional activity of the [A-4] amelogenin gene splice product in newborn mouse ameloblasts
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DOI：
10.1016/j.bone.2008.01.023
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发表时间：
2008-06-01
期刊：
影响因子：
4.1
通讯作者：
Veis, Arthur
中科院分区：
文献类型：
--
作者：
Iacob, Stanca;Veis, Arthur
在小鼠牙齿器官中，出生后不久，成釉细胞获得其分泌表型，其特征在于釉原蛋白的两种亚型M180和M59（LRAP，[A-4]）的显著表达和随后的分泌。釉原蛋白沉积到成釉细胞外基质中促进釉质生物矿化。一组复杂的细胞间信号传导事件，即发育中的口腔上皮和其下面的牙齿间充质之间的相互通信，引导釉原蛋白mRNA的表达，并将其限制在牙齿发育的限定时期。在牙胚器官培养中，添加缺乏釉原蛋白外显子4和外显子6片段a、B、c的[A-4]同种型显示影响成釉细胞发育。为了了解这种调节活性的基础，我们研究了r[A-4]对成釉细胞样LS 8细胞的影响，以及推定的[A-4]细胞表面受体LAMP 1和相关受体LAMP 3的作用。在LS 8细胞中，通过RTPCR鉴定釉原蛋白、LAMP 1和LAMP 3的剪接异构体的表达，并且实时PCR半定量分析评估M180信息的调节。M180 mRNA被外源性[A-4]上调，并且通过阻断LAMP 1进一步增加，表明由离散激动剂激活的细胞内信号传导途径之间的累加效应。免疫荧光染色鉴定了[A-4]和LAMP 1在LS 8细胞中的定位模式。内化的r[A-4]与LAMP 1共定位于晚期内体/溶酶体隔室中。因此，LAMP 1和[A-4]细胞内分选途径是相互关联的。外源性[A-4]可激活一氧化氮（NO）信号通路。[A-4]调节诱导型一氧化氮合酶（iNOS，NOS 2）和内皮型一氧化氮合酶（eNOS，NOS 3）的表达，尽管程度不同。NOS 2在4 h后表达显著上调，而NOS 3在24 h后表达略有上调。用r[A-4]和抗LAMP 1抗体共处理LS 8细胞进一步增强了NOS 2表达。抗-LAMP 1抗体没有消除用r[A-4]处理4小时的LS 8细胞中的NO产生，但是iNOS抑制剂L-Nil下调NO产生和M180 mRNA的表达。这些数据表明，[A-4]调节M180 mRNA的表达，部分，通过NO信号通路。(C)2008年爱思唯尔公司All rights reserved.
In the mouse tooth organ, shortly after birth, ameloblasts acquire their secretory phenotype, which is characterized by the prominent expression and subsequent secretion of two isoforms of amelogenin, M180 and M59 (LRAP, [A-4]). Amelogenin deposition into the ameloblast extracellular matrix promotes enamel biomineralization. A complex set of intercellular signaling events, reciprocal communications between the developing oral epithelium and its underlying dental mesenchyme, guide the expression of amelogenin mRNA, and limit it to a defined period of tooth development. In tooth germ organ culture, addition of the [A-4] isoform, lacking amelogenin exon 4 and exon 6 segments a, b, c, was shown to affect ameloblast development. To understand the basis for this regulatory activity, we have studied the effects of r[A-4] on ameloblast-like LS8 cells, and the role of the putative [A-4] cell surface receptor, LAMP1, as well as the related receptor LAMP3. In the LS8 cells, the expression of the spliced isoforms of amelogenin, LAMP1, and LAMP3 were identified by RTPCR, and real-time PCR semi-quantitative analysis assessed the modulation of M180 message. M180 mRNA was up-regulated by exogenous [A-4], and this was further increased by blockade of LAMP1, suggesting additive effects between the intracellular signaling pathways activated by the discrete agonists. Immunofluorescence staining identified the patterns of [A-4] and LAMP1 localization in LS8 cells. Internalized r[A-4] was co-localized with LAMP1 in late endosomal/lysosomal compartments. Thus, the LAMP1 and [A-4] intracellular sorting pathways are interrelated. The nitric oxide (NO) signaling pathway was activated by exogenous [A-4]. [A-4] modulated inducible nitric oxide synthase (iNOS, NOS2) and endothelial nitric oxide synthase (eNOS, NOS3) expression, albeit, to different extents. NOS2 was significantly up-regulated after 4 h, while NOS3 increased slightly after 24 h. Co-treatment of LS8 cells with r[A-4] and anti-LAMP1 antibodies further enhanced NOS2 expression. Anti-LAMP1 antibodies did not abrogate NO production in LS8 cells treated for 4 h with r[A-4], but the iNOS inhibitor, L-Nil, down-regulated both NO production and the expression of M180 mRNA. These data suggest that [A-4] modulates M180 mRNA expression, partly, via the NO signaling pathway. (C) 2008 Elsevier Inc. All rights reserved.