PTH INDUCES RGS2, NURR1 & NFIL3/E4BP4 EXPRESSION IN BONE

描述(摘自《调查者摘要》)：骨量和骨质是决定牙科手术后临床结果的主要因素。改善骨骼新陈代谢和促进骨细胞适应治疗要求的合成代谢剂可以改善牙科治疗和预后。甲状旁腺激素(PTH)是血钙的主要调节剂，其间歇或连续给药对骨代谢具有合成代谢和分解代谢的作用。了解甲状旁腺素这些相反作用的机制可能会导致更有效的骨合成代谢治疗。甲状旁腺激素激活的信号迅速诱导几个成骨基因的转录。这些主要反应基因是甲状旁腺素治疗后最先受到影响的基因。首席调查员已经确定了三个主要的反应基因，它们是甲状旁腺素在成骨细胞中快速和瞬时诱导的。它们是G蛋白信号的调节因子RGS2、孤儿核受体NURR1和转录因子NFIL3/E4BP4。他假设这些基因介导甲状旁腺素对骨代谢的影响。为了解决这一假设，首席研究者提出了三个具体的目标：(1)确定介导PTH诱导的RGS2、NURR1和NFIL3/E4BP4基因表达的信号通路(S)。成骨细胞和小鼠颅骨将被PTH激活的信号通路的激动剂和抑制剂处理。RGS2、NURR1和NFIL3/E4BP4的基因转录和蛋白水平将分别通过Northern印迹和/或RT-PCR、核连续实验和Western印迹分析进行检测。(2)探讨RGS2、NURR1和NFIL3/E4BP4蛋白在成骨细胞功能和甲状旁腺激素对成骨细胞基因表达调控中的作用。RGS2、NURR1和NFIL3/E4BP4蛋白将被反义策略过度表达或阻断，并将研究其对成骨细胞增殖和分化的影响。甲状旁腺激素在过表达或缺乏RGS2、NURR1和NFIL3/E4BP4蛋白的细胞中调节基因表达的能力也将被测试。(3)体内检测甲状旁腺激素诱导的RGS2、NURR1和NFIL3/E4BP4基因表达。小鼠将接受间歇性或连续性甲状旁腺激素治疗，已知分别为合成代谢和分解代谢，并将在骨骼和其他甲状旁腺素靶组织中进行原位杂交。这些实验将有助于了解甲状旁腺素诱导RGS2、NURR1和NFIL3/E4BP4基因表达的机制，并揭示这些基因在甲状旁腺素调控成骨细胞基因表达中的作用。

DESCRIPTION (adapted from the Investigator's abstract): Bone quantity and quality are major determinants of clinical outcome following dental surgery. Anabolic agents that improve bone metabolism and promote bone-cell adaptation to treatment requirements improve dental treatment and prognosis. Parathyroid hormone (PTH), the major regulator of serum calcium, has anabolic and catabolic effects on bone metabolism depending on its intermittent or continuous administration. Understanding the mechanisms that underlie these opposing actions of PTH might lead to more effective anabolic treatments for bone. PTH-activated signaling rapidly induces transcription of several osteoblastic genes. These primary response genes are the first genes to be affected by PTH treatment. The Principal Investigator has identified three primary response genes that are rapidly and transiently induced by PTH in osteoblasts. They are the regulator of G-protein signaling, RGS2, the orphan nuclear receptor NURR1, and the transcription factor NFIL3/E4BP4. He hypothesizes that these genes mediate PTH effects on bone metabolism. To address this hypothesis, the Principal Investigator proposes three specific aims: (1) to identify the signaling pathway(s) that mediate PTH-induced RGS2, NURR1 and NFIL3/E4BP4 gene expression. Osteoblastic cells and mouse calvariae will be treated with agonists and inhibitors of the signaling pathways activated by PTH. RGS2, NURR1 and NFIL3/E4BP4 mRNA gene transcription and protein levels will be measured by Northern blot and/or RT-PCR, nuclear run-on assays and Western blot analysis, respectively. (2) To identify the importance of RGS2, NURR1 and NFIL3/E4BP4 proteins in osteoblastic function and PTH regulation of osteoblastic gene expression. RGS2, NURR1 and NFIL3/E4BP4 proteins will be overexpressed or blocked by antisense strategies and the effects on osteoblastic proliferation and differentiation will be studied. The ability of PTH to regulate gene expression in cells overexpressing or lacking RGS2, NURR1 and NFIL3/E4BP4 proteins will also be tested. (3) To examine PTH-induced RGS2, NURR1 and NFIL3/E4BP4 gene expression in vivo. Mice will be treated with intermittent or continuous PTH, known to be anabolic and catabolic, respectively and in situ hybridization will be performed in bone and other PTH-target tissues. The experiments proposed will help understand the mechanisms of PTH-induction of RGS2, NURR1 and NFIL3/E4BP4 gene expression and unveil the role of these genes in the PTH regulation of osteoblastic gene expression.