Primary Genes Promoting Cementoblast Differentiaton

描述（由申请人提供）：牙周膜再生是牙科中一个重要但无法实现的治疗目标。开发更好的再生治疗需要更深入地了解成牙骨质细胞，成骨细胞和PDL成纤维细胞生物学。最近建立的OC-CM成牙骨质细胞系为研究成牙骨质细胞分子生物学提供了一个宝贵的研究工具。我们发现，前列腺素（PG）E2和氟前列醇（flup;一种特异性FP受体激动剂）促进，而白细胞介素-1（IL-1）抑制，OC-CM矿化和分化。这些作用在原代人成牙骨质细胞中可重现。在机制上，受体结合的flup和IL-1诱导初级基因，其调节下游基因并最终控制细胞功能。一些主要基因被flup和IL-1诱导，而其他基因被每种处理优先诱导。我们假设优先诱导的初级基因决定flup和IL-1调节的成牙骨质细胞功能。为了鉴定初级基因，我们使用用0.1 μ M flup或10 ng/ml IL-1处理90分钟的OC-CM细胞进行消减杂交。进行两次cDNA消减：（a）flup -IL-1，以鉴定flup诱导的基因，和（B）IL-1 - flup，以鉴定IL-1诱导的基因。几个优先诱导的主要基因进行了鉴定。我们的目的是研究转录因子Nur 77和egr 1以及MAP激酶信号调节因子mkp 1在成牙骨质细胞功能中的作用。在初步研究中，来自所有三个基因的mRNA在OC-CM细胞中被flup快速和瞬时诱导，但不被IL-1诱导。Nur 77，egr 1和mkp 1 mRNA表达的时间模式在用flup处理的原代人成牙骨质细胞和原代小鼠成骨细胞中复制。我们提出了三个具体目标：1）表征成牙骨质细胞中前列腺素类对Nur 77、egr 1和mkp 1基因的调控，2）表征Nur 77、egr 1和mkp 1蛋白参与调控成牙骨质细胞基因表达和分化，3）研究体内PGE 2和PGF 2 α对成牙骨质细胞中Nur 77、egr 1和mkp 1基因表达的诱导作用。我们预期Nur 77、egr 1和mkp 1将是成牙骨质细胞分化的关键介质。这些研究将有助于我们了解调节成牙骨质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.