TY - JOUR
T1 - Primary cilia and the exocyst are linked to urinary extracellular vesicle production and content
AU - Zuo, Xiaofeng
AU - Kwon, Sang Ho
AU - Janech, Michael G.
AU - Dang, Yujing
AU - Lauzon, Steven D.
AU - Fogelgren, Ben
AU - Polgar, Noemi
AU - Lipschutz, Joshua H.
N1 - Funding Information:
This work was supported in part by Veterans Affairs Merit Award I01 BX000820 (to J. H. L), National Institutes of Health Grant P30DK074038 (to J. H. L.), the Dialysis Clinic, Inc grant (to M. G. J. and J. H. L.), American Heart Association AWRP Winter 2017 Collaborative Sciences Award (to J. H. L.), and National Institutes of Health Grant 3UL1 TR001450 (to S. D. L.) to the South Carolina Clinical and Translational Research Insti-tute. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
This work was supported in part by Veterans Affairs Merit Award I01 BX000820 (to J. H. L), National Institutes of Health Grant P30DK074038 (to J. H. L.), the Dialysis Clinic, Inc grant (to M. G. J. and J. H. L.), American Heart Association AWRP Winter 2017 Collaborative Sciences Award (to J. H. L.), and National Institutes of Health Grant 3UL1 TR001450 (to S. D. L.) to the South Carolina Clinical and Translational Research Institute. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
Acknowledgments—The UAB P30 Hepatorenal Fibrocystic Disease Core Center, supported by National Institutes of Health Grant P30DK074038, is acknowledged for generating the Exoc5fl/fl mouse line. Dr. Ben Humphreys is gratefully acknowledged for providing the SLC34A-CreERT2 mice. The Medical University of South Carolina Proteomics Center is acknowledged for performing MS. Dr. E. Starr Hazard is gratefully acknowledged for help with the proteomics data analysis.
Publisher Copyright:
© 2019 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.
PY - 2019/12/13
Y1 - 2019/12/13
N2 - The recently proposed idea of “urocrine signaling” hypothesizes that small secreted extracellular vesicles (EVs) contain proteins that transmit signals to distant cells. However, the role of renal primary cilia in EV production and content is unclear. We previously showed that the exocyst, a highly conserved trafficking complex, is necessary for ciliogenesis; that it is present in human urinary EVs; that knockdown (KD) of exocyst complex component 5 (EXOC5), a central exocyst component, results in very short or absent cilia; and that human EXOC5 overexpression results in longer cilia. Here, we show that compared with control Madin-Darby canine kidney (MDCK) cells, EXOC5 overexpression increases and KD decreases EV numbers. Proteomic analyses of isolated EVs from EXOC5 control, KD, and EXOC5-overexpressing MDCK cells revealed significant alterations in protein composition. Using immunoblotting to specifically examine the expression levels of ADP-ribosylation factor 6 (ARF6) and EPS8-like 2 (EPS8L2) in EVs, we found that EXOC5 KD increases ARF6 levels and decreases EPS8L2 levels, and that EXOC5 overexpression increases EPS8L2. Knockout of intraflagellar transport 88 (IFT88) confirmed that the changes in EV number/content were due to cilia loss: similar to EXOC5, the IFT88 loss resulted in very short or absent cilia, decreased EV numbers, increased EV ARF6 levels, and decreased Eps8L2 levels compared with IFT88-rescued EVs. Compared with control animals, urine from proximal tubule-specific EXOC5-KO mice contained fewer EVs and had increased ARF6 levels. These results indicate that perturbations in exocyst and primary cilia affect EV number and protein content.
AB - The recently proposed idea of “urocrine signaling” hypothesizes that small secreted extracellular vesicles (EVs) contain proteins that transmit signals to distant cells. However, the role of renal primary cilia in EV production and content is unclear. We previously showed that the exocyst, a highly conserved trafficking complex, is necessary for ciliogenesis; that it is present in human urinary EVs; that knockdown (KD) of exocyst complex component 5 (EXOC5), a central exocyst component, results in very short or absent cilia; and that human EXOC5 overexpression results in longer cilia. Here, we show that compared with control Madin-Darby canine kidney (MDCK) cells, EXOC5 overexpression increases and KD decreases EV numbers. Proteomic analyses of isolated EVs from EXOC5 control, KD, and EXOC5-overexpressing MDCK cells revealed significant alterations in protein composition. Using immunoblotting to specifically examine the expression levels of ADP-ribosylation factor 6 (ARF6) and EPS8-like 2 (EPS8L2) in EVs, we found that EXOC5 KD increases ARF6 levels and decreases EPS8L2 levels, and that EXOC5 overexpression increases EPS8L2. Knockout of intraflagellar transport 88 (IFT88) confirmed that the changes in EV number/content were due to cilia loss: similar to EXOC5, the IFT88 loss resulted in very short or absent cilia, decreased EV numbers, increased EV ARF6 levels, and decreased Eps8L2 levels compared with IFT88-rescued EVs. Compared with control animals, urine from proximal tubule-specific EXOC5-KO mice contained fewer EVs and had increased ARF6 levels. These results indicate that perturbations in exocyst and primary cilia affect EV number and protein content.
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U2 - 10.1074/jbc.RA119.009297
DO - 10.1074/jbc.RA119.009297
M3 - Article
C2 - 31694916
AN - SCOPUS:85076502668
SN - 0021-9258
VL - 294
SP - 19099
EP - 19110
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 50
ER -