Neurokinin 1 Receptor Mediates Membrane Blebbing and Sheer Stress-Induced Microparticle Formation in HEK293 Cells

Panpan Chen,Florin Tuluc,John Meshki,Steven D Douglas,Ilya Ulasov

doi:10.1371/journal.pone.0045322

Panpan Chen, Florin Tuluc + Show 3 more

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https://doi.org/10.1371/journal.pone.0045322

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Abstract

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2–10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.

Highlights

The mechanisms of intercellular communication involve the release in the extracellular medium of messenger molecules that bind to receptors on target cells
In the present study we examined microparticle generation induced by substance P (SP) in HEK293 cells expressing the neurokinin 1 receptor (NK1R) receptor
Two distinct populations of microparticles were detected by flow cytometry in HEK293 cells expressing NK1R, based on forward and side light scattering properties recorded, and based on the data recorded in fluorescent channels after staining the cells with cytosolic and nuclear dyes

Summary

Introduction

The mechanisms of intercellular communication involve the release in the extracellular medium of messenger molecules that bind to receptors on target cells. It has been described that microparticles affect other cells in various ways, from activating intracellular signaling pathways to transferring genetic material or proteins [1]. Cell-derived microparticles are heterogeneous and have diameters ranging from 50 to 2,000 nm [2,3,4]. Their formation is associated with three major cellular events: release of exosomes from late endosomes, cellular apoptotic breakdown, and membrane blebbing [5]. Exosomes isolated from cells infected with various intracellular pathogens, including Mycobacterium tuberculosis and Toxoplasma gondii, contain microbial components and can promote antigen presentation and macrophage activation, strongly suggesting that exosomes may function in immune surveillance [10,11]

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