Primary Genes Promoting Cementoblast Differentiaton

描述（由申请人提供）：牙周再生是一个重要的，但无法实现的，牙科治疗目标。发展更好的再生治疗需要对成水泥细胞、成骨细胞和PDL成纤维细胞生物学有更深入的了解。最近建立的OC-CM成水泥细胞系为研究成水泥细胞分子生物学提供了宝贵的研究工具。我们发现前列腺素（PG） E2和氟前列腺醇（flup；一种特异性FP受体激动剂）促进OC-CM的矿化和分化，而白细胞介素-1 （IL-1）抑制OC-CM的分化。这些效应在原代人成水泥细胞中可重现。从机制上讲，受体结合flup和IL-1诱导原代基因调控下游基因，最终控制细胞功能。一些原代基因可同时被flup和il - 1诱导，而另一些则优先被每种治疗诱导。我们假设优先诱导的原代基因决定了flup和il - 1调节的成水泥细胞功能。为了鉴定原代基因，我们使用0.1 (M flup或10 ng/ml IL-1处理90分钟的OC-CM细胞进行减法杂交。进行两项cDNA消减：(a) flup- IL-1，鉴定flup诱导的基因；(b) IL-1 - flup，鉴定IL-1诱导的基因。鉴定出几个优先诱导的原代基因。我们的目的是研究转录因子Nur77和egr1以及MAP激酶信号调节因子mkp1在成水泥细胞功能中的作用。在初步研究中，这三个基因的mRNA在OC-CM细胞中被flup快速而短暂地诱导，而不是被IL-1诱导。这种Nur77、egr1和mkp1 mRNA表达的时间模式在经flup处理的人原代成水泥细胞和小鼠原代成骨细胞中被复制。我们提出了三个特定目的：1)表征成水泥细胞中prostanoids对Nur77、egr1和mkp1基因的调控；2)表征Nur77、egr1和mkp1蛋白参与成水泥细胞基因表达和分化的调控；3)在体内检测PGE2和PGF2alpha诱导成水泥细胞中Nur77、egr1和mkp1基因表达的情况。我们预计Nur77、egr1和mkp1将是成水泥细胞分化的关键介质。这些研究将有助于我们理解调节成水泥细胞功能的分子信号。

DESCRIPTION (provided by applicant): Periodontal regeneration is an important, yet unattainable, treatment objective in dentistry. Developing better regenerative treatments requires a deeper understanding of cementoblast, osteoblast, and PDL fibroblast biology. The recently established OC-CM cementoblastic cell line provides an invaluable research tool for studying cementoblast molecular biology. We found that prostaglandin (PG) E2 and fluprostenol (flup; a specific FP receptor agonist) promoted, while interleukin-1 (IL-1) inhibited, OC-CM mineralization and differentiation. These effects were reproducible in primary human cementoblasts. Mechanistically, receptor bound flup and IL-1 induce primary genes, which regulate downstream genes and ultimately control cell function. Some primary genes are induced by both flup and IL-I, while others are preferentially induced by each treatment. We hypothesize that preferentially induced primary genes dictate flup- and IL-l-regulated cementoblast function. To identify primary genes we performed subtraction hybridization using OC-CM cells treated with 0.1 (M flup or 10 ng/ml IL-1 for 90 min. Two cDNA subtractions were performed: (a) flup - IL-l, to identify flup-induced genes and (b) IL-1 - flup, to identify IL-l-induced genes. Several preferentially induced primary genes were identified. Our objective is to study the role of the transcription factors Nur77 and egr1, and the MAP kinase signaling regulator mkp1 in cementoblast function. In preliminary studies, mRNA from all three genes was rapidly and transiently induced by flup, but not by IL-1, in OC-CM cells. This temporal pattern of Nur77, egr1, and mkp1 mRNA expression was replicated in primary human cementoblasts and primary mouse osteoblasts treated with flup. We propose three Specific Aims: 1) to characterize Nur77, egr1, and mkp1 gene regulation by prostanoids in cementoblasts, 2) to characterize Nur77, egr1, and mkp1 protein involvement in regulating cementoblast gene expression and differentiation, and 3) to examine PGE2 and PGF2alpha induction of Nur77, egr1, and mkp1 gene expression in cementoblasts in vivo. We anticipate that Nur77, egr1, and mkp1 will be key mediators of cementoblast differentiation. These studies will contribute to our understanding of the molecular signals that regulate cementoblast function.