TERMINATION OF PTH RESPONSES IN OSTEOBLASTS

描述(申请人提供)：甲状旁腺激素和甲状旁腺素受体在许多疾病中是钙稳态和骨转换的重要调节者。甲状旁腺素、甲状旁腺素受体和其他GPCR激动剂对基因表达的许多影响是短暂的。我们最近发现，甲状旁腺素或肾上腺素能激动剂刺激后终止基因调控的主要机制(S)位于受体脱敏、腺苷环化酶激活和cAMP降解的下游。甲状旁腺素和β-肾上腺素能激动剂也能短暂地刺激PKA活性和转录因子的磷酸化。因此，PKA活性的终止很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，蛋白激酶抑制物(PKI)会使PKA失活。因此，我们推测，在PTH或肾上腺素能激动剂的刺激下，PKI终止PKA活性是终止基因调控的主要原因。尽管有大量研究检测纯化或过度表达的PKI，但对这些分子在细胞中的生理作用知之甚少。我们提出的实验将首次确定内源性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)检验内源性PKI在PTH或异丙肾上腺素刺激后与PKA相互作用的假说；2)检验内源性PKI在PTH或异丙肾上腺素刺激后终止PKA活性和基因调控的假说；3)检验内源性PKI终止PTH诱导的基因调控在生理上重要的假说。这些实验将大大增加我们对PKI在PKA信号和间充质细胞基因调控中的作用以及平衡PTH分解代谢效应的机制的理解。我们的发现也将作为其他类型细胞中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.