Primary Genes Promoting Cementoblast Differentiaton

描述(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。开发更好的再生治疗方法需要对成牙骨质细胞、成骨细胞和PDL成纤维细胞生物学有更深入的了解。新近建立的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宝贵的研究工具。我们发现前列腺素(PG)E_2和氟前列腺素(FP受体激动剂)促进OC-CM的矿化和分化，而IL-1抑制OC-CM的矿化和分化。这些效应在原代人类成牙骨质细胞中是可以重现的。在机制上，受体结合的Flup和IL-1诱导初级基因，这些基因调控下游基因，最终控制细胞功能。一些主要基因是由Flup和IL-I共同诱导的，而另一些基因则是由每种处理优先诱导的。我们假设，优先诱导的主要基因决定了Flup和IL-L调节的成牙骨质细胞功能。为了确定主要基因，我们用0.1(M Flup)或10 ng/mlIL-1处理OC-CM细胞90min进行消减杂交。进行两次消减：(A)Flup-IL-L，用于鉴定Flup诱导的基因；(B)IL-1-Flup，用于鉴定IL-L诱导的基因。确定了几个优先诱导的初级基因。我们的目的是研究转录因子Nur77和Egr1以及MAPK信号调节因子Mkp1在成牙骨质细胞功能中的作用。在初步研究中，在OC-CM细胞中，这三个基因的mRNA都能被Flup快速而短暂地诱导，但不能被IL-1诱导。这种Nur77、Egr1和mkp1mRNA表达的时间模式在原代人牙骨质成骨细胞和原代小鼠成骨细胞中被复制。我们提出了三个特定的目标：1)研究前列腺素对成牙本质细胞中Nur77、Egr1和Mkp1基因表达的调控；2)研究NUR77、Egr1和Mkp1蛋白对成牙本质细胞基因表达和分化的调控作用；3)检测PGE2和PGF2α对成牙骨质细胞中Nur77、Egr1和Mkp1基因表达的诱导作用。我们预计Nur77、Egr1和Mkp1将是成牙骨质细胞分化的关键调节因子。这些研究将有助于我们理解调控成牙本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.