TERMINATION OF PTH RESPONSES IN OSTEOBLASTS

描述（由申请人提供）：PTH和PTHrP是许多疾病中钙稳态和骨转换的重要调节剂。 PTH、PTHrP和其他GPCR激动剂对基因表达的许多影响是短暂的。 我们最近的研究表明，PTH或β-肾上腺素能激动剂刺激后终止基因调控的主要机制是受体脱敏、腺苷酸环化酶激活和cAMP降解的下游。 甲状旁腺素和肾上腺素能激动剂也刺激PKA活性和转录因子的磷酸化在一个短暂的方式。 因此，PKA活性的终止可能是下游事件的终止，包括转录因子磷酸化和基因调控。 当过表达时，蛋白激酶抑制剂（PKI）使PKA失活。 因此，我们假设PKA活性的终止PKI是主要负责终止基因调控后的PTH或<$-肾上腺素能激动剂的刺激。 尽管大量的研究检查纯化或过表达的PKI，很少有人知道这些分子在细胞中的生理作用。 我们提出的实验将是第一个确定内源性PKI水平是否足以降低PKA活性或下游基因调控。 我们的具体目标是：1）检验在PTH或异丙肾上腺素刺激后，内源性PKI与PKA相互作用的假设; 2）检验在PTH或异丙肾上腺素刺激后，内源性PKI主要负责PKA活性和基因调节的终止的假设; 3）检验内源性PKI终止PTH诱导的基因调节的假设生理上重要。 这些实验将大大增加我们对PKI在PKA信号传导和间充质细胞基因调控中的作用以及负责平衡PTH分解代谢作用的机制的理解。我们的发现也将作为其他细胞类型中PKA信号调节的范例。

DESCRIPTION (provided by applicant): PTH and PTHrP are important regulators of calcium homeostasis and bone turnover in many diseases. Many of the effects of PTH, PTHrP, and other GPCR agonists on gene expression are transient. We have recently shown that the primary mechanism(s) responsible for termination of gene regulation following stimulation by PTH or ¿-adrenergic agonists is(are) downstream of receptor desensitization, adenyl cyclase activation, and cAMP degradation. PTH and ¿-adrenergic agonists also stimulate PKA activity and transcription factor phosphorylation in a transient fashion. Thus, it is likely that termination of PKA activity is responsible for termination of downstream events, including transcription factor phosphorylation and gene regulation. When overexpressed, protein kinase inhibitor (PKI) inactivates PKA. We therefore hypothesize that termination of PKA activity by PKI is primarily responsible for termination of gene regulation following stimulation by PTH or ¿-adrenergic agonists. Despite the large number of studies examining purified or overexpressed PKI, little is known about the physiological role of these molecules in cells. Our proposed experiments will be the first to determine whether endogenous levels of PKI are sufficient to reduce PKA activity or downstream gene regulation. Our specific aims are: 1) Test the hypothesis that endogenous PKI interacts with PKA following stimulation by PTH or isoproterenol; 2) Test the hypothesis that endogenous PKI is primarily responsible for termination of both PKA activity and gene regulation following stimulation by PTH or isoproterenol; 3) Test the hypothesis that termination of PTH-induced gene regulation by endogenous PKI is physiologically important. These experiments will substantially increase our understanding of the role of PKI in both PKA signaling and gene regulation in mesenchymal cells as well as the mechanisms responsible for balancing the catabolic effects of PTH. Our findings will also serve as paradigm for regulation of PKA signaling in other cell types.