FBXL19 Targeted STK11 Degradation Enhances Cigarette Smoke-Induced Airway Epithelial Cell Cytotoxicity

Abstract

Objective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11’s role in CS-induced airway epithelial cell cytotoxicity.Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs–human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits.Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner.Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death.

HIGHLIGHTS

• Lung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression.

• STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity.

• STK11 depletion augments CS-induced airway epithelial cell cytotoxicity.

• CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.

Graphical Abstract

Keywords:

• Serine-Threonine Kinase 11
• bronchial airway epithelial cells
• cigarette smoke
• FBXL19
• chronic obstructive pulmonary disease

Acknowledgments

All frozen lung tissue samples used in this study were provided by the tissue core of the Lovelace Respiratory Research Institute and the Lung Tissue Research Consortium (LTRC).

Consent for publication

All authors in this manuscript consent to the publication in the Journal.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval

The University of Pittsburgh Institutional Review Board (IRB) approved the use of human subject samples was approved by the University of Pittsburgh Institutional Review Board (IRB) in this study.

Data availability statement

The datasets used and analyzed in this study are available from the corresponding author upon reasonable request.

Additional information

Funding

This study was supported by funding from a Merit Review award CX001048 and CX000105 (TN) from the US Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development; and grants from the American Heart Association (TN); NIH grants HL149719 (TN) and GM132127 (YJ).