Primary cilia and the exocyst are linked to urinary extracellular vesicle production and content

Xiaofeng Zuo,Sang-Ho Kwon,Michael G Janech,Yujing Dang,Steven D Lauzon,Ben Fogelgren,Noemi Polgar,Joshua H Lipschutz

doi:10.1074/jbc.ra119.009297

Abstract

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.

Highlights

The recently proposed idea of “urocrine signaling” hypothesizes that small secreted extracellular vesicles (EVs) contain proteins that transmit signals to distant cells
Knockout of intraflagellar transport 88 (IFT88) confirmed that the changes in EV number/content were due to cilia loss: similar to exocyst complex component 5 (EXOC5), the IFT88 loss resulted in very short or absent cilia, decreased EV numbers, increased EV ADP-ribosylation factor 6 (ARF6) levels, and decreased Eps8L2 levels compared with IFT88-rescued EVs
We showed in renal tubule cells that exocyst components are localized to primary cilia [26], that the exocyst is required for ciliogenesis [27], that Exoc5-containing vesicles are seen by electron microscopy (EM) gold microscopy at the tip and sides of primary cilia [27] (Fig. S1), that the exocyst genetically interacts with polycystin-2 in zebrafish [28, 29], and that kidney-specific knockout of Exoc5 leads to renal cystogenesis [30, 31]

Summary

Introduction

The recently proposed idea of “urocrine signaling” hypothesizes that small secreted extracellular vesicles (EVs) contain proteins that transmit signals to distant cells. Proteomic analyses of isolated EVs from EXOC5 control, KD, and EXOC5-overexpressing MDCK cells revealed significant alterations in protein composition. 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. H. L.), and National Institutes of Health Grant 3UL1 TR001450 The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. These results indicate that perturbations in exocyst and primary cilia affect EV number and protein content. PKD is the fourth leading cause of end-stage kidney disease, accounting for ϳ5% of all end-stage kidney disease cases in the United States [10], the molecular mechanisms linking ciliary mutations to the cystic phenotype remain unclear

Methods

Results

Conclusion