IN VIVO REGULATION OF cAMP/PKA SIGNALING BY PKIgamma

描述（由申请人提供）：甲状旁腺激素主要通过激活成骨细胞中的cAMP/PKA信号，对骨转换产生合成代谢和分解代谢作用。分解代谢作用主要是由基因介导的，这些基因以一种持续的方式被调节。相反，合成代谢作用主要是由立即早期（I-E）基因介导的，这些基因被甲状旁腺激素迅速但短暂地上调。我们的总体假设是PKI抑制了成骨细胞中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的研究，即内源性PKI抑制成骨细胞系中甲状旁腺激素的瞬时效应。PKA的失活终止了I-E基因的表达和PTH的抗凋亡作用。PTH的抗凋亡作用作为PTH合成代谢作用的指标，否则很难在细胞培养中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，因此在更生理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是否调节体内和原代成骨细胞中PTH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与甲状旁腺激素类似，可短暂诱导核PKA活性和I-E基因表达，并短暂抑制细胞凋亡。这些结果为我们的第二个假设提供了基础，即间歇性给药（-肾上腺素能激动剂）对骨骼是合成代谢的。与甲状旁腺激素一样，异丙肾上腺素的短暂效应也会被内源性PKI终止，这进一步支持了我们的总体假设。通过测量内源性PKI抑制小鼠和初代成骨细胞对甲状旁腺激素或肾上腺素能激动剂反应的能力，每个特定目标都将有助于测试我们的总体假设。特异性目标的肾上腺素能激动剂部分也将检验我们的次要假设。目的1将在小鼠和原代成骨细胞培养中确定内源性PKI是否抑制PTH或-肾上腺素能激动剂诱导的PKA信号。目的2将在小鼠和原代成骨细胞培养中确定内源性PKI是否抑制PTH或-肾上腺素能激动剂诱导的I-E基因表达。目的3将在小鼠和原代成骨细胞培养中确定内源性PKI是否抑制PTH或-肾上腺素能激动剂的合成代谢作用。间歇性甲状旁腺激素是唯一被FDA批准用于治疗骨质疏松症的合成代谢药物。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PTH合成代谢和分解代谢作用的机制可能允许(1)识别可能无反应的患者，(2)改进PTH治疗有效性的监测，或(3)开发增加PTH合成代谢作用的联合疗法，特别是对PTH本身反应较差的患者。

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.