Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients.

Francesco Lodola,Cinzia Bottino,Paolo Pedrazzoli,Elisa Bonetti,Mariangela Manzoni,Germano Guerra,Umberto Laforenza,Margherita Massa,Silvia Dragoni,Armando A Genazzani,Indu S Ambudkar,Vittorio Rosti,Franco Tanzi,Hwei Ling Ong,Camillo Porta,Dmitry Lim,Francesco Moccia,Carlo Ganini

doi:10.1371/journal.pone.0042541

Abstract

BackgroundEndothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca2+ entry (SOCE), which is activated by a depletion of the intracellular Ca2+ pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca2+-sensor, Stim1, and the plasmalemmal Ca2+ channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca2+ influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients.Methodology/Principal FindingsThe present study employed Ca2+ imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La3+ and Gd3+. Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca2+ release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca2+ buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs.ConclusionsSOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis.

Highlights

An increase in intracellular Ca2+ concentration ([Ca2+]i) occurs in all cell types following the activation of either G-protein coupled receptors or tyrosine kinase receptors [1,2]
Store-operated Ca2+ entry (SOCE) is remodelled in Endothelial progenitor cells (EPCs) from renal cellular carcinoma (RCC) patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis
We have recently shown that human EPCs isolated from peripheral blood (PB) of healthy donors express a SOCE pathway mediated by Stromal Interaction Molecule-1 (Stim1) and Orai1 [26,27]

Summary

Introduction

An increase in intracellular Ca2+ concentration ([Ca2+]i) occurs in all cell types following the activation of either G-protein coupled receptors or tyrosine kinase receptors [1,2]. A number of studies have, shown that transient receptor potential channel 1 (TRPC1) may either serve as store-dependent channels upon binding to Stim or participate to SOCE by forming a ternary complex with Stim and Orai. A number of studies have, shown that transient receptor potential channel 1 (TRPC1) may either serve as store-dependent channels upon binding to Stim or participate to SOCE by forming a ternary complex with Stim and Orai1 In the latter case, Orai is essential for TRPC1 to become store-sensitive [3]. Store-operated Ca2+ entry (SOCE), which is activated by a depletion of the intracellular Ca2+ pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca2+-sensor, Stim, and the plasmalemmal Ca2+ channel, Orai. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca2+ influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis in tumor patients

Results

Discussion

Conclusion