Activation of multiple receptors stimulates extracellular vesicle release from trophoblast cells

Abstract

Reports of the stimulated release of extracellular vesicles (EVs) are few, and the mechanisms incompletely understood. To our knowledge, the possibility that the activation of any one of the multitudes of G‐protein‐coupled receptors (GPCRs) expressed by a single cell‐type might increase EV release has not been explored. Recently, we identified the expression of cholecystokinin (CCK), gastrin, gastrin/cholecystokinin types A and/or B receptors (CCKAR and/or –BR), and the bitter taste receptor, TAS2R14 in the human and mouse placenta. specifically, trophoblast. These GPCR(s) were also expressed in four different human trophoblast cell lines. The current objective was to employ two of these cell lines—JAR choriocarcinoma cells and HTR‐8/SVneo cells derived from first‐trimester human villous trophoblast—to investigate whether CCK, TAS2R14 agonists, and other GPCR ligands would each augment EV release. EVs were isolated from the cell‐culture medium by filtration and ultracentrifugation. The preparations were enriched in small EVs (<200 nm) as determined by syntenin western blot before and after sucrose gradient purification, phycoerythrin (PE)‐ADAM10 antibody labeling, and electron microscopy. Activation of TAS2R14, CCKBR, cholinergic muscarinic 1 & 3, and angiotensin II receptors, each increased EV release by 4.91‐, 2.79‐, 1.87‐, and 3.11‐fold, respectively (all p < .05 versus vehicle controls), without significantly changing EV diameter. A progressive increase of EV concentration in conditioned medium was observed over 24 hr consistent with the release of preformed EVs and de novo biogenesis. Compared to receptor‐mediated stimulation, EV release by the calcium ionophore, A23187, was less robust (1.63‐fold, p = .08). Diphenhydramine, a TAS2R14 agonist, enhanced EV release in JAR cells at a concentration 10‐fold below that required to increase intracellular calcium. CCK activation of HTR‐8/SVneo cells, which did not raise intracellular calcium, increased EV release by 2.06‐fold (p < .05). Taken together, these results suggested that other signaling pathways may underlie receptor‐stimulated EV release besides, or in addition to, calcium. To our knowledge, the finding that the activation of multiple GPCRs can stimulate EV release from a single cell‐type is unprecedented and engenders a novel thesis that each receptor may orchestrate intercellular communication through the release of EVs containing a subset of unique cargo, thus mobilizing a specific integrated physiological response by a network of neighboring and distant cells.

Mechanisms of Extracellular Vesicle Biogenesis, Cargo Loading, and Release

“…Activation of CCKBR, TAS2R14, cholinergic muscarinic 1 and 3, and angiotensin II receptors, each increased EV release without affecting the overall size of the EVs [17]. Also, EV release by the calcium ionophore, A23187, was less robust when compared to receptor-mediated stimulation [17]. These finding warrant the investigation on whether activation of other GPCRs can mediate EV release.…”

“…The size of EVs may or may not depend on the amount and type of cargo enriched in the vesicles. Some studies have shown an increase in EV release without a change in EV size, while other studies have shown the contrary [17,18]. Apoptotic bodies represent the largest type of EVs and contain organelles, in addition to nucleic acids, proteins, and lipids.…”

“…Pharmacological activation of specific G-protein coupled receptors (GPCRs) from trophoblast cells was shown to trigger the release of EVs [17]. Activation of CCKBR, TAS2R14, cholinergic muscarinic 1 and 3, and angiotensin II receptors, each increased EV release without affecting the overall size of the EVs [17]. Also, EV release by the calcium ionophore, A23187, was less robust when compared to receptor-mediated stimulation [17].…”

A Comparison of Blood Plasma Exosome Enrichment Strategies for Proteomic Analysis

“…Similarly, EX release from cells in response to DNA damage is under the control of p53-driven gene expression, and EX-derived miRNA are essential for mutant p53-associated oncogenesis (49). Furthermore, the stimulation of cholecystokinin receptors (CCKR) have been shown to increase the release of EVs by more than 2-fold in human and mouse trophoblast cell lines (50). The increased presence of proteins associated with these pathways in EX-enriched samples suggests that it is possible to isolate these EX-populations in peripheral blood plasma, which could be of utility in clinical studies.…”

Might proton pump or sodium‐hydrogen exchanger inhibitors be of value to ameliorate SARs‐CoV‐2 pathophysiology?

“…As mentioned earlier, if the predominant role of EVs is to impede viral infection, then increasing the release of EVs may prove beneficial. Although stimulated release of EVs has not been extensively and systematically investigated (Alonso et al., 2005; Pironti et al., 2015; Verweij et al., 2018), activation of several G‐protein‐coupled receptors (GPCRs) was recently found to increase EV release from a single cell type (trophoblast‐derived cells) (Conrad et al., 2020). It was speculated that, because there are as many as 800 GPCRs in the human genome, many of which can be expressed by one given cell‐type (Kroeze et al., 2003), perhaps each GPCR (or cohort of functionally related GPCRs) can affect EV release in a cell‐type‐specific manner containing a unique subset of cargo (e.g., RNAs, proteins, lipids) that, in turn, coordinates specific physiological responses among neighboring and distant cells (Conrad et al., 2020).…”