磷酸二酯酶 8A 调节气道上皮细胞中的 CFTR 活性。

Phosphodiesterase 8A Regulates CFTR Activity in Airway Epithelial Cells.

机构信息

Department of Physiology, McGill University, Montréal, QC, Canada,

Cystic Fibrosis Translational Research Centre, McGill University, Montréal, QC, Canada.

出版信息

Cell Physiol Biochem. 2021 Dec 23;55(6):784-804. doi: 10.33594/000000477.

DOI:10.33594/000000477

PMID:34936285

Abstract

BACKGROUND/AIMS: Cystic fibrosis transmembrane conductance regulator (CFTR), the anion channel that is defective in cystic fibrosis (CF), is phosphorylated and activated by cAMP-dependent protein kinase (PKA). cAMP levels are downregulated by a large family of phosphodiesterases that have variable expression in different cell types. We have previously observed high levels of PDE8A expression in well-differentiated primary human bronchial epithelial (pHBE) cells and thus aimed to assess whether it played a role in cAMP-dependent regulation of CFTR activity.

METHODS

We assessed the effect of the selective PDE8 inhibitor PF-04957325 (PF) on intracellular cAMP levels ([cAMP]) in well differentiated pHBE cells from non-CF or CF donors and also in CFBE41o- cells that stably express wild-type CFTR (CFBE41o- WT) using ELISA and FRET-FLIM microscopy. CFTR channel function was also measured using electrophysiological recordings from pHBE and CFBE41o- WT cells mounted in Ussing Chambers.

RESULTS

PDE8 inhibition elevated [cAMP] in well-differentiated pHBE cells and stimulated wild-type CFTR-dependent ion transport under basal conditions or after cells had been pre-stimulated with physiological cAMP-elevating agents. The response to PDE8 inhibition was larger than to PDE3 or PDE5 inhibition but smaller and synergistic with that elicited by PDE4 inhibition. CRISPR Cas9-mediated knockdown of PDE8A enhanced CFTR gene and protein expression yet reduced the effect of PDE8 inhibition. Acute pharmacological inhibition PDE8 increased CFTR activity in CF pHBE cells (F508del/F508del and F508del/R117H-5T) treated with clinically-approved CFTR modulators.

CONCLUSION

These results provide the first evidence that PDE8A regulates CFTR and identifies PDE8A as a potential target for adjunct therapies to treat CF.

摘要

背景/目的：囊性纤维化跨膜电导调节因子（CFTR）是囊性纤维化（CF）中缺陷的阴离子通道，可被 cAMP 依赖性蛋白激酶（PKA）磷酸化和激活。cAMP 水平受磷酸二酯酶（PDE）家族的下调，该家族在不同细胞类型中的表达不同。我们之前观察到在分化良好的原代人支气管上皮（pHBE）细胞中 PDE8A 的高表达水平，因此旨在评估其是否在 CFTR 活性的 cAMP 依赖性调节中发挥作用。

方法

我们评估了选择性 PDE8 抑制剂 PF-04957325（PF）对来自非 CF 或 CF 供体的分化良好的 pHBE 细胞以及稳定表达野生型 CFTR（CFBE41o-WT）的 CFBE41o-细胞中细胞内 cAMP 水平（[cAMP]）的影响，使用 ELISA 和 FRET-FLIM 显微镜。还使用 pHBE 和 CFBE41o-WT 细胞在 Ussing 室中进行的电生理记录测量 CFTR 通道功能。

结果

PDE8 抑制在分化良好的 pHBE 细胞中升高了[cAMP]，并在基础条件下或在细胞预先用生理升高 cAMP 的激动剂刺激后刺激了野生型 CFTR 依赖性离子转运。对 PDE8 抑制的反应大于对 PDE3 或 PDE5 抑制的反应，但小于并与 PDE4 抑制的反应协同。CRISPR Cas9 介导的 PDE8A 敲低增强了 CFTR 基因和蛋白表达，但降低了 PDE8 抑制的作用。急性药理学抑制 PDE8 增加了用临床批准的 CFTR 调节剂治疗的 CF pHBE 细胞（F508del/F508del 和 F508del/R117H-5T）中 CFTR 的活性。