PARATHYROID HORMONE INDUCED ICER IN BONE

这一建议是基于这样的假设，ICER，诱导型cAMP早期阻遏物，代表了一种新的机制，调节甲状旁腺激素（PTH）诱导的基因转录成骨细胞。 PTH是人体内主要的钙调节激素，刺激骨吸收，并可根据其给药方式增加或减少骨形成。 PTH通过调节成骨细胞中的基因表达来增强这些反应。 诱导型cAMP早期阻遏物（ICER）是ATF/CREB转录因子家族的成员。 ICER由cAMP反应元件调节因子（CREM）基因的内含子启动子转录，并作为cAMP依赖性基因转录的显性负阻遏物。 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ICER的过度表达抑制了成骨细胞中一个初级反应基因的PTH依赖性转录。 然而，最低浓度的过表达ICER也增强转录，表明ICER不是严格的阻遏物。 基于我们的初步数据，我们假设ICER在成骨细胞中的表达不仅可以介导PTH依赖性基因表达的衰减，而且还可以增加转录的诱导期。 我们预测成骨细胞中ICER水平的扰动将改变PTH依赖性基因表达的模式，并导致PTH生物活性的失调。 为了解决这些假设，我们提出了以下具体目标：（1）确定ICER在培养的成骨细胞MC 3 T3-E1中使用过表达和反义策略调节选定的PTH诱导的初级反应基因表达中的作用;（2）确定ICER在体内响应于PTH注射和输注的时间和空间模式;和（3）通过产生具有骨定向ICER过表达的转基因小鼠和通过检查缺乏ICER表达的CREM敲除小鼠来确定ICER的体内功能。 这里提出的实验是了解ICER在调节骨中PTH依赖性基因表达和功能中的作用的第一步。

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.