Histone deacetylase 6–mediated selective autophagy regulates COPD-associated cilia dysfunction
Hilaire C. Lam, … , Stefan W. Ryter, Augustine M.K. Choi
Hilaire C. Lam, … , Stefan W. Ryter, Augustine M.K. Choi
Published November 8, 2013
Citation Information: J Clin Invest. 2013;123(12):5212-5230. https://doi.org/10.1172/JCI69636.
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Research Article Pulmonology

Histone deacetylase 6–mediated selective autophagy regulates COPD-associated cilia dysfunction

Abstract

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) that are associated with epithelial cell dysfunction, cilia shortening, and mucociliary clearance disruption. Exposure to CS reduced cilia length and induced autophagy in vivo and in differentiated mouse tracheal epithelial cells (MTECs). Autophagy-impaired (Becn1+/– or Map1lc3B–/–) mice and MTECs resisted CS-induced cilia shortening. Furthermore, CS increased the autophagic turnover of ciliary proteins, indicating that autophagy may regulate cilia homeostasis. We identified cytosolic deacetylase HDAC6 as a critical regulator of autophagy-mediated cilia shortening during CS exposure. Mice bearing an X chromosome deletion of Hdac6 (Hdac6–/Y) and MTECs from these mice had reduced autophagy and were protected from CS-induced cilia shortening. Autophagy-impaired Becn1–/–, Map1lc3B–/–, and Hdac6–/Y mice or mice injected with an HDAC6 inhibitor were protected from CS-induced mucociliary clearance (MCC) disruption. MCC was preserved in mice given the chemical chaperone 4-phenylbutyric acid, but was disrupted in mice lacking the transcription factor NRF2, suggesting that oxidative stress and altered proteostasis contribute to the disruption of MCC. Analysis of human COPD specimens revealed epigenetic deregulation of HDAC6 by hypomethylation and increased protein expression in the airways. We conclude that an autophagy-dependent pathway regulates cilia length during CS exposure and has potential as a therapeutic target for COPD.

Authors

Hilaire C. Lam, Suzanne M. Cloonan, Abhiram R. Bhashyam, Jeffery A. Haspel, Anju Singh, J. Fah Sathirapongsasuti, Morgan Cervo, Hongwei Yao, Anna L. Chung, Kenji Mizumura, Chang Hyeok An, Bin Shan, Jonathan M. Franks, Kathleen J. Haley, Caroline A. Owen, Yohannes Tesfaigzi, George R. Washko, John Quackenbush, Edwin K. Silverman, Irfan Rahman, Hong Pyo Kim, Ashfaq Mahmood, Shyam S. Biswal, Stefan W. Ryter, Augustine M.K. Choi

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Figure 1

Cigarette smoke causes cilia loss and shortening and increased autophagy in primary cultured epithelial cells.

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Cigarette smoke causes cilia loss and shortening and increased autophagy...
(A) Cells exposed to 50 or 100 mg/m3 were harvested 24 hours after exposure and analyzed for the cilia marker acetylated α-tubulin, F-actin, and Hoechst staining by confocal xy- or z-stack analysis. Scale bar: 10 μm. Arrows indicate intact cilia; arrowheads indicate shortened cilia. Dara are representative of three independent experiments. Quantification of ciliated cells (right) was performed based on the morphological appearance of acetylated α-tubulin–positive cells normalized to total nuclei. (B) Distribution of cilia lengths by SEM in MTECs 24 hours after exposure to 50 mg/m3 CS. Distributions were constructed by creating histograms from 0.2 AU bins, equally weighting each sample. (C) Average cilia length and change in the number of cells with cilia lengths of 0.6 AU or more in MTECs exposed to 50 or 100 mg/m3 CS. Cilia lengths were calculated from 10 SEM images/MTEC culture (n = 3). The number of MTECs with cilia lengths of 0.6 AU or more were calculated from 5 SEM images per sample (n = 4 cultures/group) normalized to controls. (D) Ciliated MTECs were analyzed for ultrastructural changes 24 hours after exposure to CS at the indicated doses. Red arrows indicate autophagosomes/autophagolysosomes. a, axoneme; bb, basal body; m, mitochondria. Scale bar: 500 nm. Image is representative of 45 images (n = 1 MTEC culture). (E) Autophagosome-associated GFP-LC3 puncta were imaged and quantified (right) by confocal microscopy in control- and CS-treated (50 mg/m3) MTECs (5 images/culture from 3 MTEC cultures). All data are the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA followed by a Bonferroni’s post test.