IN VIVO REGULATION OF cAMP/PKA SIGNALING BY PKIgamma

PKIgamma 对 cAMP/PKA 信号传导的体内调节

• 批准号: 7297123
• 负责人: EDWARD M. GREENFIELD
• 金额: $ 19.93万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7297123
• 关键词: Adrenergic Agonists; Anabolic Agents; Apoptosis; Apoptotic; Cell Line; Cell Nucleus; Cultured Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic AMP-Responsive DNA-Binding Protein; Cytoplasm; Development; Effectiveness; Exploratory/Developmental Grant; Exposure to; Figs - dietary; Future; Gene Expression; Genes; Immediate-Early Genes; Isoproterenol; Lead; Left; Measures; Mediating; Monitor; Mus; Nuclear Protein; Nuclear Proteins; Osteoblasts; Osteoporosis; Parathyroid Hormones; Patients; Phosphorylation; Physiological; Protein Kinase Inhibitors; Qualifying; Regulation; Risk; Side; Signal Transduction; Testing; Transcriptional Activation; United States Food and Drug Administration; Up-Regulation; base; bone; bone turnover; hormone therapy; human PTH protein; improved; in vivo; prevent; protein kinase inhibitor; recombinase; research study; response; transcription factor

DESCRIPTION (provided by applicant): PTH exerts both anabolic and catabolic actions on bone turnover primarily by activating cAMP/PKA signaling in osteoblasts. The catabolic action is primarily mediated by genes that are regulated in a sustained fashion. In contrast, the anabolic action is primarily mediated by immediate-early (I-E) genes that are rapidly, but transiently, up-regulated by PTH. Our overall hypothesis is that PKI( inhibits the transient anabolic action of cAMP/PKA signaling in osteoblasts. This overall hypothesis is based on our recent demonstration that endogenous PKI( inhibits the transient effects of PTH in an osteoblastic cell line. This inactivation of PKA terminates both expression of I-E genes and the anti-apoptotic effect of PTH. The anti-apoptotic effect of PTH was used as an indicator of the anabolic action of PTH since otherwise the anabolic action is difficult to study in cell culture. Since these results were obtained in an osteoblastic cell line, it is important to test their relevance in a more physiological setting. We are therefore proposing to determine whether endogenous PKI( regulates the anabolic actions of PTH in vivo and in primary osteoblast cultures. We have also demonstrated that the (-adrenergic agonist, isoproterenol, acts like PTH in osteoblastic cell lines to transiently induce nuclear PKA activity and I-E gene expression and to transiently inhibit apoptosis. These results provide the underpinning for our secondary hypothesis that intermittent administration of (-adrenergic agonists is anabolic for bone. Also like PTH, the transient effects of isoproterenol are terminated by endogenous PKI(, which further supports our overall hypothesis. Each of the Specific Aims will contribute to testing of our overall hypothesis by measuring the ability of endogenous PKI( to inhibit responses to PTH or (-adrenergic agonists in mice and in primary osteoblast cultures. The (-adrenergic agonist portions of the Specific Aims will also test our secondary hypothesis. Aim 1 will determine whether endogenous PKI( inhibits PKA signaling induced by PTH or by (-adrenergic agonists in mice and in primary osteoblast cultures. Aim 2 will determine whether endogenous PKI( inhibits I-E gene expression induced by PTH or by (-adrenergic agonists in mice and in primary osteoblast cultures. Aim 3 will determine whether endogenous PKI( inhibits the anabolic action of PTH or by (-adrenergic agonists in mice and in primary osteoblast cultures. Intermittent PTH is the only anabolic agent approved by the FDA for treatment of osteoporosis. However, a substantial proportion of patients are unresponsive to intermittent PTH. A better understanding of the mechanisms responsible for regulating the anabolic and catabolic actions of PTH might allow (1) identification of patients who are likely to be unresponsive, (2) improved monitoring of the effectiveness of PTH therapy, or (3) development of co-therapies that increase the anabolic actions of PTH, especially for patients who respond poorly to PTH by itself.

描述(申请人提供)：甲状旁腺素主要通过激活成骨细胞中的cAMP/PKA信号，在骨转换过程中发挥合成代谢和分解代谢作用。分解代谢的作用主要是由以持续方式调节的基因介导的。相反，合成代谢作用主要由即刻早期(I-E)基因介导，这些基因迅速但短暂地被甲状旁腺素上调。我们的总体假设是，PKI()抑制成骨细胞中cAMP/PKA信号的瞬时合成代谢作用。这一总体假设是基于我们最近证明内源性PKI(抑制甲状旁腺素在成骨细胞系中的瞬时效应)。这种PKA的失活既终止了I-E基因的表达，也终止了PTH的抗凋亡作用。甲状旁腺素的抗凋亡作用被认为是甲状旁腺素合成代谢作用的指标，否则在细胞培养中很难研究其合成代谢作用。由于这些结果是在成骨细胞系中获得的，因此在更具生理性的环境中测试它们的相关性是很重要的。因此，我们建议确定内源性PKI(在体内和原代成骨细胞培养中是否调节甲状旁腺素的合成代谢作用)。我们还证明了-肾上腺素能激动剂异丙肾上腺素在成骨细胞系中的作用类似于甲状旁腺素，可瞬时诱导核PKA活性和I-E基因表达，并瞬时抑制细胞凋亡。这些结果为我们的第二个假设提供了支持，即间歇性给予肾上腺素能激动剂是骨的合成代谢。与甲状旁腺激素一样，异丙肾上腺素的短暂作用被内源性PKI()终止，这进一步支持了我们的总体假设。每一个特定的目标都将有助于通过测量内源性PKI(在小鼠和原代成骨细胞培养中抑制甲状旁腺素或肾上腺素能激动剂的反应)的能力来检验我们的总体假设。特定目标的(肾上腺素能激动剂)部分也将检验我们的第二个假设。目的1确定内源性PKI是否抑制甲状旁腺激素或肾上腺素能激动剂在小鼠和原代成骨细胞培养中诱导的PKA信号转导。目的2确定内源性PKI是否抑制甲状旁腺激素或肾上腺素能激动剂诱导的小鼠和原代成骨细胞的I-E基因表达。目的3将确定在小鼠和原代成骨细胞培养中，内源性PKI()是否抑制甲状旁腺素或肾上腺素能激动剂的合成代谢作用。间歇性甲状旁腺素是FDA批准的唯一用于治疗骨质疏松症的合成代谢药。然而，相当大比例的患者对间歇性甲状旁腺激素无反应。更好地了解调节甲状旁腺素合成代谢和分解代谢作用的机制可能有助于(1)识别可能无反应的患者，(2)改进对甲状旁腺素治疗效果的监测，或(3)开发增加甲状旁腺素合成代谢作用的联合疗法，特别是对于本身对甲状旁腺素反应较差的患者。