Calcium Dyshomeostasis Alters CCL5 Signaling in Differentiated PC12 Cells.

Tomasz Radzik,Ludmila Zylinska,Lukasz Pulaski,Bozena Ferenc,Tomasz Boczek,Maciej Studzian

doi:10.1155/2019/9616248

Abstract

Background Plasma membrane Ca2+-ATPase (PMCA) is the most sensitive cellular calcium detector. It exists in four main isoforms (PMCA1-4), among which PMCA2 and PMCA3 are considered as fast-acting neuron-specific forms. In the brain, PMCA function declines progressively during aging; thereby impaired calcium homeostasis may contribute to some neurodegenerative diseases. These destructive processes can be propagated by proinflammatory chemokines, including chemokine CCL5, which causes phospholipase C-mediated liberation of Ca2+ from endoplasmic reticulum by IP3-gated channels. Methods To mimic the changes in aged neurons we used stable transfected differentiated PC12 cells with downregulated PMCA2 or PMCA3 and analyzed the effect of CCL5 on calcium transients with Fluo-4 reagent. Chemokine receptors were evaluated using Western blot, and IP3 receptors expression level was assessed using qRT-PCR and Western blot. Results In PMCA-reduced cell lines, CCL5 released more Ca2+ by IP3-sensitive receptors, and the time required for Ca2+ clearance was significantly longer. Also, in these lines we detected altered expression level of CCR5 and IP3 receptors. Conclusion Although modification of PMCAs composition could provide some protection against calcium overload, reduction of PMCA2 appeared to be more detrimental to the cells than deficiency of PMCA3. Under pathological conditions, including inflammatory CCL5 action and long-lasting Ca2+ dyshomeostasis, insufficient cell protection may result in progressive degeneration and death of neurons.

Highlights

Growing body of evidence suggests that disrupted calcium homeostasis plays a detrimental role in triggering neurodegeneration
We have previously developed stable transfected lines of PC12 cells with downregulated expression of neuron-specific PMCA2 ( 2 line) or PMCA3 ( 3 line), which have been validated in our several other studies [36, 37]
Stable transfected cell lines with reduced PMCA2 ( 2) or PMCA3 ( 3) protein level were achieved using an antisense RNA cloned into pcDNA3.1(+) vector transfected to naive PC12 cells, as described previously [36]

Summary

Introduction

Growing body of evidence suggests that disrupted calcium homeostasis plays a detrimental role in triggering neurodegeneration This process can be propagated by repeated inflammatory reactions, including local production of chemokines. Plasma membrane Ca2+-ATPase (PMCA) is the most sensitive cellular calcium detector It exists in four main isoforms (PMCA1-4), among which PMCA2 and PMCA3 are considered as fast-acting neuron-specific forms. PMCA function declines progressively during aging; thereby impaired calcium homeostasis may contribute to some neurodegenerative diseases These destructive processes can be propagated by proinflammatory chemokines, including chemokine CCL5, which causes phospholipase C-mediated liberation of Ca2+ from endoplasmic reticulum by IP3-gated channels. In PMCA-reduced cell lines, CCL5 released more Ca2+ by IP3-sensitive receptors, and the time required for Ca2+ clearance was significantly longer In these lines we detected altered expression level of CCR5 and IP3 receptors. Under pathological conditions, including inflammatory CCL5 action and long-lasting Ca2+ dyshomeostasis, insufficient cell protection may result in progressive degeneration and death of neurons

Methods

Discussion

Conclusion