PARATHYROID HORMONE INDUCED ICER IN BONE

这一建议是基于一种假设，即ICER是可诱导的cAMP早期抑制因子，代表了调节甲状旁腺激素(PTH)诱导的成骨细胞基因转录的新机制。甲状旁腺素是人体内主要的钙调节激素，可刺激骨吸收，并可根据其给药方式的不同而增加或减少骨形成。甲状旁腺激素通过调节成骨细胞中的基因表达来引起这些反应。诱导型cAMP早期抑制因子(ICER)是ATF/CREB转录因子家族的成员。ICER由cAMP反应元件调节器(CREM)基因的内含子启动子转录而来，是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PTH依赖的初级反应基因的转录。然而，过表达的ICER的最低浓度也促进了转录，这表明ICER并不是严格意义上的抑制子。根据我们的初步数据，我们假设ICER在成骨细胞中的表达不仅可能介导了PTH依赖的基因表达的减弱，而且还可能增强了转录的诱导阶段。我们预测，成骨细胞ICER水平的改变将改变PTH依赖的基因表达模式，并导致PTH生物活性的解除调节。为了解决这些假说，我们提出了以下具体目标：(1)确定ICER在使用过表达和反义策略调控成骨细胞MC3T3-E1中选定的PTH诱导的初级反应基因表达中的作用；(2)确定注射和注入PTH后ICER在体内表达的时空模式；以及(3)通过产生具有骨定向ICER过表达的转基因小鼠和通过检测缺乏ICER表达的CREM基因敲除小鼠来确定ICER在体内的功能。这里提出的实验是了解ICER在调节骨骼中PTH依赖的基因表达和功能中所起的作用的第一步。

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.