Platelet Activating Factor Receptor Activation Improves siRNA Uptake and RNAi Responses in Well-differentiated Airway Epithelia

Well-differentiated human airway epithelia present formidable barriers to efficient siRNA delivery. We previously reported that treatment of airway epithelia with specific small molecules improves oligonucleotide uptake and facilitates RNAi responses. Here, we exploited the platelet activating facto...

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Main Authors:	Krishnamurthy, Sateesh, Behlke, Mark A, Apicella, Michael A, McCray, Paul B, Davidson, Beverly L
Format:	Online
Language:	English
Published:	Nature Publishing Group 2014
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121516/

id	pubmed-4121516
recordtype	oai_dc
spelling	pubmed-41215162014-08-14 Platelet Activating Factor Receptor Activation Improves siRNA Uptake and RNAi Responses in Well-differentiated Airway Epithelia Krishnamurthy, Sateesh Behlke, Mark A Apicella, Michael A McCray, Paul B Davidson, Beverly L Original Article Well-differentiated human airway epithelia present formidable barriers to efficient siRNA delivery. We previously reported that treatment of airway epithelia with specific small molecules improves oligonucleotide uptake and facilitates RNAi responses. Here, we exploited the platelet activating factor receptor (PAFR) pathway, utilized by specific bacteria to transcytose into epithelia, as a trigger for internalization of Dicer-substrate siRNAs (DsiRNA). PAFR is a G-protein coupled receptor which can be engaged and activated by phosphorylcholine residues on the lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae and the teichoic acid of Streptococcus pneumoniae as well as by its natural ligand, platelet activating factor (PAF). When well-differentiated airway epithelia were simultaneously treated with either nontypeable Haemophilus influenzae LOS or PAF and transduced with DsiRNA formulated with the peptide transductin, we observed silencing of both endogenous and exogenous targets. PAF receptor antagonists prevented LOS or PAF-assisted DsiRNA silencing, demonstrating that ligand engagement of PAFR is essential for this process. Additionally, PAF-assisted DsiRNA transfection decreased CFTR protein expression and function and reduced exogenous viral protein levels and titer in human airway epithelia. Treatment with spiperone, a small molecule identified using the Connectivity map database to correlate gene expression changes in response to drug treatment with those associated with PAFR stimulation, also induced silencing. These results suggest that the signaling pathway activated by PAFR binding can be manipulated to facilitate siRNA entry and function in difficult to transfect well-differentiated airway epithelial cells. Nature Publishing Group 2014-07 2014-07-15 /pmc/articles/PMC4121516/ /pubmed/25025465 http://dx.doi.org/10.1038/mtna.2014.26 Text en Copyright © 2014 American Society of Gene & Cell Therapy http://creativecommons.org/licenses/by-nc-nd/3.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Krishnamurthy, Sateesh Behlke, Mark A Apicella, Michael A McCray, Paul B Davidson, Beverly L
spellingShingle	Krishnamurthy, Sateesh Behlke, Mark A Apicella, Michael A McCray, Paul B Davidson, Beverly L Platelet Activating Factor Receptor Activation Improves siRNA Uptake and RNAi Responses in Well-differentiated Airway Epithelia
author_facet	Krishnamurthy, Sateesh Behlke, Mark A Apicella, Michael A McCray, Paul B Davidson, Beverly L
author_sort	Krishnamurthy, Sateesh
title	Platelet Activating Factor Receptor Activation Improves siRNA Uptake and RNAi Responses in Well-differentiated Airway Epithelia
title_short	Platelet Activating Factor Receptor Activation Improves siRNA Uptake and RNAi Responses in Well-differentiated Airway Epithelia
title_full	Platelet Activating Factor Receptor Activation Improves siRNA Uptake and RNAi Responses in Well-differentiated Airway Epithelia
title_fullStr	Platelet Activating Factor Receptor Activation Improves siRNA Uptake and RNAi Responses in Well-differentiated Airway Epithelia
title_full_unstemmed	Platelet Activating Factor Receptor Activation Improves siRNA Uptake and RNAi Responses in Well-differentiated Airway Epithelia
title_sort	platelet activating factor receptor activation improves sirna uptake and rnai responses in well-differentiated airway epithelia
description	Well-differentiated human airway epithelia present formidable barriers to efficient siRNA delivery. We previously reported that treatment of airway epithelia with specific small molecules improves oligonucleotide uptake and facilitates RNAi responses. Here, we exploited the platelet activating factor receptor (PAFR) pathway, utilized by specific bacteria to transcytose into epithelia, as a trigger for internalization of Dicer-substrate siRNAs (DsiRNA). PAFR is a G-protein coupled receptor which can be engaged and activated by phosphorylcholine residues on the lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae and the teichoic acid of Streptococcus pneumoniae as well as by its natural ligand, platelet activating factor (PAF). When well-differentiated airway epithelia were simultaneously treated with either nontypeable Haemophilus influenzae LOS or PAF and transduced with DsiRNA formulated with the peptide transductin, we observed silencing of both endogenous and exogenous targets. PAF receptor antagonists prevented LOS or PAF-assisted DsiRNA silencing, demonstrating that ligand engagement of PAFR is essential for this process. Additionally, PAF-assisted DsiRNA transfection decreased CFTR protein expression and function and reduced exogenous viral protein levels and titer in human airway epithelia. Treatment with spiperone, a small molecule identified using the Connectivity map database to correlate gene expression changes in response to drug treatment with those associated with PAFR stimulation, also induced silencing. These results suggest that the signaling pathway activated by PAFR binding can be manipulated to facilitate siRNA entry and function in difficult to transfect well-differentiated airway epithelial cells.
publisher	Nature Publishing Group
publishDate	2014
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121516/
_version_	1613120846742683648

Platelet Activating Factor Receptor Activation Improves siRNA Uptake and RNAi Responses in Well-differentiated Airway Epithelia

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