Prostate cancer cells stimulated by calcium-mediated activation of protein kinase C undergo a refractory period before re-releasing calcium-bearing microvesicles

https://doi.org/10.1016/j.bbrc.2015.03.061Get rights and content

Highlights

  • Prostate cancer cells release microvesicles upon stimulation with sublytic C5b-9.

  • Stimulated cells have a 20 min refractory period before restimulation is possible.

  • Microvesicles from C5b-9 activated PCa cells carry calcium from the cell.

Abstract

MVs are released in response to several stress agents, in an attempt to prevent continued cellular damage. After an initial stimulus of prostate cancer cells with sublytic C5b-9 and activation of MV release through PKC, cells take at least 20 min to fully recover their ability to microvesiculate. This release of MVs through activation of sublytic C5b-9 was inhibited by the PKC inhibitor bisindoylmaleimide I but not the Rho kinase inhibitor, Y27632. After stimulus there is a rise of 79 nMs−1 over 11 s, reaching a peak [Ca2+]i of 920 nM. The concentration of cytosolic calcium then falls steadily at 2.4 nMs−1 over 109 s reaching baseline levels (50–100 nM) within 10–15 min. In PC3 cells the rate of release of MVs from stimulated cells also reaches a minimum within 10–15 min. Using fura-2 AM-loaded cells, upon stimulation, cells were found to release MVs with a concentration of intravesicular calcium estimated at ∼430 nM.

Introduction

Cells typically produce a variety of vesicles, some of which including lysosomes, endosomes, multivesicular bodies and various transport and secretory vehicles remain within the cell. Of those that are released, exosomes are distinguished from microvesicles (MVs) by their smaller size, 50–100 nm, lower expression of phosphatidylserine (PtSer) and by expression of the surface markers Alix, CD63 and TSG101 [1], [2]. The established MV characteristics include size, ∼100 nm μm in diameter, and for all MVs, the expression of negatively charged phospholipids such as phosphatidylserine (PtSer) and phosphotidylcholine translocated on to the outer leaflet of the plasma membrane [1] as seen during early apoptosis [3].

The formation of MVs as a characteristic feature of cells undergoing early stage apoptosis [4] is typified by PtSer externalisation and the calpain-mediated cleavage of the actin cytoskeleton due to a rise in intracellular Ca2+ [1]. This rise in [Ca2+]i originates mostly from external sources and enters the cell through ion channels [3], membrane pores, including membrane attack complex (MAC) or through certain types of cell damage [1] and even interaction with intracellular pathogens including certain protozoan parasites [5] and viruses [6].

We report here insights into the pathway of MV release following sublytic MAC stimulation and whether cells can effectively receive repeated stimuli. We also investigate the intravesicular content of [Ca2+]i in MVs and begin to look at the potential for calcium to be released from cells, speculating on any possible roles ranging from calcium homoeostasis and transport to cell protection.

Section snippets

Stimulation of cells with sublytic complement (NHS) or BzATP

To stimulate MV release with sublytic complement, PC3 cells (1 × 105 cells/ml) presensitized (for 30 min/4 °C) in rabbit anti-PC3 cell membrane antiserum (5%, v/v) in pre-warmed RPMI and 2 mM CaCl2 were treated with exosome-/MV-free 5% NHS (human serum type AB; Sigma) (0.22 μm filtered and spun at 100,000 g/16 h). Cells were also stimulated with 200 μM BzATP (37 °C/30 min) or 5 μg/ml LPS, for release of MVs, in the presence of 2 mM CaCl2. Where needed cells were also treated with calcium

Cells stimulated through sublytic C5b-9 release MVs through calcium-mediated activation of protein kinase C

Sublytic complement stimulated a greater release of MVs from metastatic PCa, PC3, cells than from an equivalent number of benign prostate PNT2 cells (Fig. 1A) but the MV release increased marginally as the cell number was increased up to 10-fold. We also found that extracellular calcium in the range 0.5–4.0 mM was required for sublytic C5b-9-mediated MV release with a peak occurring at 1.0 mM. This approximates to the typical levels found in extracellular fluid of about 1.2 mM [11], [12]; the

Discussion

Microvesiculation may be triggered from cells upon deposition with sublytic complement (Membrane Attack Complex, C5b-9) by increasing [Ca2+]i, as it rises to ≥300 nM. We found that a repeated stimulus, in this case with sublytic C5b-9 of PCa cells, PC3, LNCaP and Du-145, is unable to allow microvesiculation to re-occur until 20 min have elapsed. After this, the cells are able to take a repeat stimulus and to release MVs once more. Refractory periods where cells lose their sensitivity to calcium

Acknowledgments

This work was funded by HEFCE QR funding (RAE2008). We are indebted to members of CMIRC for critically reading the manuscript and to Maria McCrossan for assisting with the electron microscopy.

References (26)

  • J.M. Inal et al.

    Microvesicles in health and disease

    Arch. Immunol. Ther. Exp.

    (2012)
  • I. Cestari et al.

    Trypanosoma cruzi immune evasion mediated by host cell-derived microvesicles

    J. Immunol.

    (2012)
  • J.M. Inal et al.

    Interplay of host-pathogen microvesicles and their role in infectious disease

    Biochem. Soc. Trans.

    (2013)
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