Graduation Date

Spring 5-5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Cellular & Integrative Physiology

First Advisor

Joseph H. Sisson

Abstract

Alcohol abuse, which can impair clearance of pathogens and debris from the airway, is associated with an increase in complications and a higher mortality rate during the progression of pneumonia. With prolonged alcohol exposure, mucociliary clearance (MCC), which depends on the coordinated beating of cilia, is dysfunctional. Effective MCC relies on cAMP-dependent protein kinase (PKA) activation of ciliary beat frequency (CBF). PKA activation and stimulation of CBF are blunted with chronic exposure to alcohol. This phenomenon is known as alcohol-induced ciliary dysfunction (AICD). Previous studies have shown that concomitant feeding of mice with alcohol and antioxidants prevents AICD, suggesting a key role for oxidant signaling in AICD. We hypothesized that increased cilia-associated oxidant driven phosphatase activity plays a key role in the link between alcohol-driven oxidant production and cilia dysfunction. To test this hypothesis we assessed phosphatase activity and key checkpoints of ciliary function, such as CBF-and PKA responsiveness in isolated cilia, intact cell, tissue and animal models of AICD. Alcohol increased S-nitrosation in bronchoalveolar fluid and protein phosphatase 1 (PP1) activity in tracheal rings from mice drinking alcohol for 6 weeks. Furthermore, alcohol-stimulation of PP1 activity and S-nitrosation persisted at the tissue, cell and isolated cilia organelle level. Inhibition of PP1 reversed AICD, demonstrating a key role for PP1 in AICD. Structure based studies indicate that PP1 contains an oxidant sensitive active site. We identified PP1 cysteine 155 (PP1C155) as a key S-nitrosated residue in AICD and generated and expressed PP1C155 mutants in human airway epithelial cells. We then exposed cells expressing mutant PP1 to alcohol and assessed PP1 activity, S-nitrosation, and responsiveness of PKA and CBF. Importantly, mutagenesis of PP1C155A prevented PP1 activation, PKA desensitization and CBF desensitization in the context of alcohol exposure. These studies provide new evidence that S-nitrosation and activation of PP1 are involved in the signaling mechanism of AICD in the axoneme and offer new insight to how dysregulation of redox mechanisms in the axoneme may lead to the mucociliary dysfunction in the context of alcohol exposure.


Download

Share

COinS