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Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia

Background: Cilia are actin based cellular protrusions conserved from algae to complex multicellular organisms like Homo sapiens. Respiratory motile cilia line epithelial cells of the tracheobronchial tree, beat in a synchronous, metachronal wave, moving inhaled pollutants and pathogens cephalad. Th...

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Autores principales: Zahid, Maliha, Feinstein, Timothy N., Oro, Anthony, Schwartz, Molly, Lee, Alex D., Lo, Cecilia W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464104/
https://www.ncbi.nlm.nih.gov/pubmed/32823934
http://dx.doi.org/10.3390/biom10081182
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author Zahid, Maliha
Feinstein, Timothy N.
Oro, Anthony
Schwartz, Molly
Lee, Alex D.
Lo, Cecilia W.
author_facet Zahid, Maliha
Feinstein, Timothy N.
Oro, Anthony
Schwartz, Molly
Lee, Alex D.
Lo, Cecilia W.
author_sort Zahid, Maliha
collection PubMed
description Background: Cilia are actin based cellular protrusions conserved from algae to complex multicellular organisms like Homo sapiens. Respiratory motile cilia line epithelial cells of the tracheobronchial tree, beat in a synchronous, metachronal wave, moving inhaled pollutants and pathogens cephalad. Their role in both congenital disorders like primary ciliary dyskinesia (PCD) to acquired disorders like chronic obstructive pulmonary disease (COPD) continues to evolve. In this current body of work we outline a protocol optimized to reciliate human nasal epithelial cells and mouse tracheal cells in vitro. Using this protocol, we knocked down known cilia genes, as well as use a small molecule inhibitor of Notch, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl Ester (DAPT), to assess the effect of these on ciliogenesis in order to show the validity of our protocol. Methods: Tracheas were harvested from wild-type, adult C57B6 mice, pronase digested and sloughed off epithelial cells grown to confluence in stationary culture on rat-tail collagen coated wells. Upon reaching confluence, collagen was digested and cells placed suspension culture protocol to reciliate the cells. Using this suspension culture protocol, we employed siRNA gene knockdown to assay gene functions required for airway ciliogenesis. Knock down of Dynein axonemal heavy chain 5 (Dnah5), a ciliary structural protein, was confirmed using immunostaining. Mouse tracheal cells were treated in suspension with varying doses of DAPT, an inhibitor of Notch, with the purpose of evaluating its effect and dose response on ciliogenesis. The optimum dose was then used on reciliating human nasal epithelial cells. Results: siRNA knockdown of Foxj1 prevented ciliation, consistent with its role as a master regulator of motile cilia. Knockdown of Dnai1 and Dnah5 resulted in immotile cilia, and Cand1 knockdown, a centrosome protein known to regulate centrosome amplification, inhibited airway ciliogenesis. Dnah5 knockdown was confirmed with significantly decreased immunostaining of cilia for this protein. Inhibiting Notch signaling by inhibiting gamma secretase with DAPT enhanced the percentage of ciliation, and resulted in longer cilia that beat with higher frequency in both mouse and human airway epithelia. Conclusions: Modifying existing reciliation protocols to suit both human nasal epithelial and mouse tracheal tissue, we have shown that knockdown of known cilia-related genes have the expected effects. Additionally, we have demonstrated the optimal dosage for significantly improving reciliation of airway epithelia using DAPT. Given that cilia length and function are significantly compromised in COPD, these findings open up interesting avenues for further exploration.
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spelling pubmed-74641042020-09-04 Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia Zahid, Maliha Feinstein, Timothy N. Oro, Anthony Schwartz, Molly Lee, Alex D. Lo, Cecilia W. Biomolecules Article Background: Cilia are actin based cellular protrusions conserved from algae to complex multicellular organisms like Homo sapiens. Respiratory motile cilia line epithelial cells of the tracheobronchial tree, beat in a synchronous, metachronal wave, moving inhaled pollutants and pathogens cephalad. Their role in both congenital disorders like primary ciliary dyskinesia (PCD) to acquired disorders like chronic obstructive pulmonary disease (COPD) continues to evolve. In this current body of work we outline a protocol optimized to reciliate human nasal epithelial cells and mouse tracheal cells in vitro. Using this protocol, we knocked down known cilia genes, as well as use a small molecule inhibitor of Notch, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl Ester (DAPT), to assess the effect of these on ciliogenesis in order to show the validity of our protocol. Methods: Tracheas were harvested from wild-type, adult C57B6 mice, pronase digested and sloughed off epithelial cells grown to confluence in stationary culture on rat-tail collagen coated wells. Upon reaching confluence, collagen was digested and cells placed suspension culture protocol to reciliate the cells. Using this suspension culture protocol, we employed siRNA gene knockdown to assay gene functions required for airway ciliogenesis. Knock down of Dynein axonemal heavy chain 5 (Dnah5), a ciliary structural protein, was confirmed using immunostaining. Mouse tracheal cells were treated in suspension with varying doses of DAPT, an inhibitor of Notch, with the purpose of evaluating its effect and dose response on ciliogenesis. The optimum dose was then used on reciliating human nasal epithelial cells. Results: siRNA knockdown of Foxj1 prevented ciliation, consistent with its role as a master regulator of motile cilia. Knockdown of Dnai1 and Dnah5 resulted in immotile cilia, and Cand1 knockdown, a centrosome protein known to regulate centrosome amplification, inhibited airway ciliogenesis. Dnah5 knockdown was confirmed with significantly decreased immunostaining of cilia for this protein. Inhibiting Notch signaling by inhibiting gamma secretase with DAPT enhanced the percentage of ciliation, and resulted in longer cilia that beat with higher frequency in both mouse and human airway epithelia. Conclusions: Modifying existing reciliation protocols to suit both human nasal epithelial and mouse tracheal tissue, we have shown that knockdown of known cilia-related genes have the expected effects. Additionally, we have demonstrated the optimal dosage for significantly improving reciliation of airway epithelia using DAPT. Given that cilia length and function are significantly compromised in COPD, these findings open up interesting avenues for further exploration. MDPI 2020-08-14 /pmc/articles/PMC7464104/ /pubmed/32823934 http://dx.doi.org/10.3390/biom10081182 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zahid, Maliha
Feinstein, Timothy N.
Oro, Anthony
Schwartz, Molly
Lee, Alex D.
Lo, Cecilia W.
Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia
title Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia
title_full Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia
title_fullStr Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia
title_full_unstemmed Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia
title_short Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia
title_sort rapid ex-vivo ciliogenesis and dose-dependent effect of notch inhibition on ciliogenesis of respiratory epithelia
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464104/
https://www.ncbi.nlm.nih.gov/pubmed/32823934
http://dx.doi.org/10.3390/biom10081182
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