PARATHYROID HORMONE INDUCED ICER IN BONE

甲状旁腺激素引起的骨冰

• 批准号: 6628103
• 负责人: BARBARA E KREAM
• 金额: $ 23.94万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-08 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6628103
• 关键词: 3T3 cells; antisense nucleic acid; cyclic AMP; gene expression; gene induction /repression; genetic transcription; genetically modified animals; hormone regulation /control mechanism; immunofluorescence technique; in situ hybridization; laboratory mouse; northern blottings; organ culture; osteoblasts; osteogenesis; parathyroid hormones; physiologic bone resorption; polymerase chain reaction; radioimmunoassay; transcription factor; western blottings

This proposal is based on the hypothesis that ICER, the inducible cAMP early repressor, represents a novel mechanism for regulating parathyroid hormone (PTH)-induced gene transcription in osteoblastic cells. PTH, the major calcium-regulating hormone in humans, stimulates bone resorption and can increase or decrease bone formation depending on its mode of administration. PTH elicits these responses by regulating gene expression in osteoblastic cells. Inducible cAMP early repressor (ICER) is a member of the ATF/CREB transcription factor family. ICER is transcribed from an intronic promoter of the cAMP response element modulator (CREM) gene and acts as a dominant negative repressor of cAMP-dependent gene transcription. We recently discovered that PTH and cAMP induced ICER in osteoblastic cell lines and mouse calvariae. WE also found that overexpression of ICER repressed PTH-dependent transcription of a primary response gene in osteoblastic cells. However, the lowest concentration of overexpressed ICER also enhanced transcription, suggesting that ICER is not strictly a repressor. Based on our preliminary data, we hypothesize that the expression of ICER in osteoblasts may not only mediate the attenuation PTH-dependent gene expression but may also augment the induction phase of transcription. We predict that perturbation of ICER levels in osteoblastic cells will alter the pattern of PTH-dependent gene expression and result in deregulation of PTH bioactivities. To address these hypotheses, we propose the following specific aims: (1) To determine the role of ICER in modulating the expression of selected PTH-inducible primary response genes in cultured osteoblastic MC3T3-E1 cells using overexpression and antisense strategies; (2) To determine the temporal and spatial pattern of ICER expression in vivo in response to injections and infusion of PTH; and (3) To determine the function of ICER in vivo by generating transgenic mice that have bone-directed ICER overexpression and by examining CREM knockout mice that lack ICER expression. The experiments proposed here are a first step in understanding the role of ICER in modulating PTH-dependent gene expression and function in bone.

这一建议是基于一种假设，即诱导性cAMP早期抑制因子ICER代表了一种调节成骨细胞中甲状旁腺激素（PTH）诱导的基因转录的新机制。甲状旁腺激素是人体中主要的钙调节激素，它能刺激骨吸收，并根据给药方式增加或减少骨形成。甲状旁腺激素通过调节成骨细胞的基因表达引发这些反应。诱导型cAMP早期抑制因子（ICER）是ATF/CREB转录因子家族的一员。ICER是从cAMP反应元件调节（CREM）基因的内含子启动子转录而来，并作为cAMP依赖基因转录的显性负抑制因子。我们最近发现PTH和cAMP在成骨细胞系和小鼠颅骨中诱导ICER。我们还发现，在成骨细胞中，ICER的过表达抑制了一种主要应答基因的pth依赖性转录。然而，最低浓度的过表达ICER也增强了转录，这表明ICER并不是严格意义上的抑制因子。基于我们的初步数据，我们假设ICER在成骨细胞中的表达可能不仅介导pth依赖性基因表达的衰减，还可能增加转录的诱导阶段。我们预测，成骨细胞中ICER水平的扰动将改变甲状旁腺激素依赖性基因表达的模式，并导致甲状旁腺激素生物活性的解除。为了解决这些假设，我们提出了以下具体目标：(1)通过过表达和反义策略确定ICER在培养的成骨细胞MC3T3-E1中调节pth诱导的主要反应基因表达的作用；(2)测定注射和输注PTH后体内ICER表达的时空格局；(3)通过产生骨导向ICER过表达的转基因小鼠和检测缺乏ICER表达的CREM敲除小鼠来确定ICER在体内的功能。本文提出的实验是了解ICER在骨中调节甲状旁腺激素依赖性基因表达和功能中的作用的第一步。