The peroxisome proliferator activated receptor gamma agonist pioglitazone increases functional expression of the glutamate transporter excitatory amino acid transporter 2 (EAAT2) in human glioblastoma cells.

Jared Ching,Thomas Zheng,Stephanie Amiridis,Rodney B Luwor,Andrew P Morokoff,Nicole Kountouri,Terence J O’Brien,Lucy Paradiso,Stanley S Stylli,Andrew R Bjorksten,Andrew H Kaye

doi:10.18632/oncotarget.4019

Abstract

Glioma cells release glutamate through expression of system xc-, which exchanges intracellular glutamate for extracellular cysteine. Lack of the excitatory amino acid transporter 2 (EAAT2) expression maintains high extracellular glutamate levels in the glioma microenvironment, causing excitotoxicity to surrounding parenchyma. Not only does this contribute to the survival and proliferation of glioma cells, but is involved in the pathophysiology of tumour-associated epilepsy (TAE). We investigated the role of the peroxisome proliferator activated receptor gamma (PPARγ) agonist pioglitazone in modulating EAAT2 expression in glioma cells. We found that EAAT2 expression was increased in a dose dependent manner in both U87MG and U251MG glioma cells. Extracellular glutamate levels were reduced with the addition of pioglitazone, where statistical significance was reached in both U87MG and U251MG cells at a concentration of ≥ 30 μM pioglitazone (p < 0.05). The PPARγ antagonist GW9662 inhibited the effect of pioglitazone on extracellular glutamate levels, indicating PPARγ dependence. In addition, pioglitazone significantly reduced cell viability of U87MG and U251MG cells at ≥ 30 μM and 100 μM (p < 0.05) respectively. GW9662 also significantly reduced viability of U87MG and U251MG cells with 10 μM and 30 μM (p < 0.05) respectively. The effect on viability was partially dependent on PPARγ activation in U87MG cells but not U251MG cells, whereby PPARγ blockade with GW9662 had a synergistic effect. We conclude that PPARγ agonists may be therapeutically beneficial in the treatment of gliomas and furthermore suggest a novel role for these agents in the treatment of tumour associated seizures through the reduction in extracellular glutamate.

Highlights

Glutamate is the most abundant neurotransmitter in the mammalian central nervous system and mediates its excitatory physiological effect by binding to ionotropic and metabotropic receptors. [1, 2] Glioma cells release glutamate through expression of the xc- exchanger, which exchanges intracellular glutamate for extracellular cysteine. [3] Intracellular uptake of cysteine via system xc- permits glioma cell survival through glutathione formation
Excitatory amino acid transporter 2 (EAAT2) is one of 5 subtypes of sodium dependent plasma membrane glutamate transporters that accounts for up to 90% of extracellular glutamate uptake, with Glutamate Aspartate Transporter (GLAST-1) accounting for the largest remaining proportion. [4, 5] Glioma cell lines www.impactjournals.com/oncotarget lack expression of both EAAT2 and GLAST-1, which is associated with impaired glutamate uptake. [3, 6] Together, these features result in abnormally high extracellular glutamate concentrations resulting in excitotoxicity, causing necrosis of the adjacent parenchyma, which creates space permitting proliferation. [7, 8] cysteine uptake provides a precursor for the formation of glutathione, which is protective against endogenous reactive oxygen species and further promotes glioma growth
We established the baseline expression of the glutamate transporters EAAT2 and GLAST-1 (EAAT1) in a number of glioma cell lines (U87MG, U251MG and GSC #35) relative to rodent normal brain tissue as a control

Summary

Introduction

Glutamate is the most abundant neurotransmitter in the mammalian central nervous system and mediates its excitatory physiological effect by binding to ionotropic and metabotropic receptors. [1, 2] Glioma cells release glutamate through expression of the xc- exchanger, which exchanges intracellular glutamate for extracellular cysteine. [3] Intracellular uptake of cysteine via system xc- permits glioma cell survival through glutathione formation. [11] Expression of PPARγ in the brain has been found in multiple cell types including microglia, astrocytes, oligodendrocytes, and neurons It has been shown in rat cortical cultures incubated with the commercially available PPARγ agonist rosiglitazone, that this drug increases the expression levels of EAAT2 at both the protein and mRNA levels. We describe a novel mechanism of the PPARγ agonist, pioglitazone, in which we demonstrate its ability to increase EAAT2 expression and extracellular glutamate levels in glioma cells. In addition we show that this agent alters cellular morphology, whilst reducing the viability of human glioblastoma cell lines These results warrant further investigation into the potential role of this class of agents in both anti-neoplastic and anti-convulsant therapy in gliomas

Methods

Results

Discussion

Conclusion