Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function

Abstract

Whereas cell cycle arrest, apoptosis, and senescence are traditionally thought of as the major functions of the tumor suppressor p53, recent studies revealed two unique functions for this protein: p53 regulates cellular energy metabolism and antioxidant defense mechanisms. Here, we identify glutaminase 2 (GLS2) as a previously uncharacterized p53 target gene to mediate these two functions of the p53 protein. GLS2 encodes a mitochondrial glutaminase catalyzing the hydrolysis of glutamine to glutamate. p53 increases the GLS2 expression under both nonstressed and stressed conditions. GLS2 regulates cellular energy metabolism by increasing production of glutamate and alpha-ketoglutarate, which in turn results in enhanced mitochondrial respiration and ATP generation. Furthermore, GLS2 regulates antioxidant defense function in cells by increasing reduced glutathione (GSH) levels and decreasing ROS levels, which in turn protects cells from oxidative stress (e.g., H(2)O(2))-induced apoptosis. Consistent with these functions of GLS2, the activation of p53 increases the levels of glutamate and alpha-ketoglutarate, mitochondrial respiration rate, and GSH levels and decreases reactive oxygen species (ROS) levels in cells. Furthermore, GLS2 expression is lost or greatly decreased in hepatocellular carcinomas and the overexpression of GLS2 greatly reduced tumor cell colony formation. These results demonstrated that as a unique p53 target gene, GLS2 is a mediator of p53's role in energy metabolism and antioxidant defense, which can contribute to its role in tumor suppression.

Fig. 1.

The human GLS2 gene contains a p53 DNA consensus binding element in the promoter region. (A) Putative p53 consensus binding elements in the human GLS2 gene predicted by the p53MH program. N, any nucleotide; Pu, purine; Py, pyramidine. (B) p53 binds to the p53 consensus binding element in the human GLS2 promoter region as detected by ChIP assays. The V138/H1299 cells were shifted to 32 °C for 24 h before assays. The p53-binding element in the MDM2 promoter serves as a positive control. DO-1: p53 antibody. (C) p53 activates luciferase activity of a reporter vector containing the p53-binding element in the GLS2 promoter region. The p53-null H1299 and HCT116 p53^−/− cells were cotransfected with the luciferase reporter vectors and vectors expressing either wild-type (pRC p53) or mutant p53 protein (pRC 273H) 24 h before measuring luciferase activities.

Fig. 2.

p53 increases GLS2 transcription under both stressed and nonstressed conditions. p53 induces GLS2 expression at mRNA (A) and protein levels (B) in V138/H1299 cells at 32 °C and in LN-2024 cells treated with Dox for different hours. The GLS2 mRNA levels were determined by real-time PCR and normalized with Actin. Exogenous GLS2 protein was detected using whole cell extracts from cells transfected with GLS2 expression vectors (B, panel 1). Endogenous GLS2 protein was detected using mitochondrial extracts (B, panels 2–4). GLS2 siRNA oligo was employed to knock down endogenous GLS2 expression in HepG2 cells (B, panel 2). NS: nonspecific band. (C and D) IR (10 Gy) induces GLS2 mRNA levels in a p53-dependent fashion in HCT116 and HepG2 cells. (E) Etoposide (20 μM) and H₂O₂ (150 μM) induce GLS2 mRNA levels in a p53-dependent fashion in HCT116 cells. (F) p53 regulates GLS2 basal expression levels.

Fig. 3.

GLS2 expression increases the levels of intracellular glutamate and α-ketoglutarate. (A) The mitochondrial localization of exogenous GLS2 protein in cells transfected with GLS2 expression vectors. Mitochondria were stained with MitoTracker. (B) Exogenous GLS2 expression increases glutamate levels (Left), whereas GLS2 knockdown by siRNA (GLS2-siRNA) decreases glutamate levels in cells (Right) (P < 0.05). Glutamate levels were determined at 24 h after transfection. (C) (Left) Exogenous GLS2 expression increases α-ketoglutarate levels. (Right) GLS2 knockdown decreases α-ketoglutarate levels (P < 0.05). (D and E) p53 increases the levels of glutamate and α-ketoglutarate in cells (P < 0.05).

Fig. 4.

GLS2 promotes oxygen consumption, mitochondrial respiration, and ATP generation. Cells were transfected with GLS2 expression vectors or GLS2 siRNA oligo. Cells were seeded in 96-well plates at 24 h after transfection and oxygen consumption was measured every 2 h after seeding (0 h). (A) Exogenous GLS2 expression promotes oxygen consumption. (B) GLS2 knockdown decreases oxygen consumption. (C) p53 increases oxygen consumption in cells. (D) Exogenous GLS2 expression increases ATP levels in cells (P < 0.05). (E) GLS2 knockdown decreases ATP levels in cells (P < 0.05). ATP levels were measured at 24 h after transfection.

Fig. 5.

GLS2 increases intracellular levels of GSH and NADH. Cells were transfected with GLS2 expression vector or GLS2 siRNA 24 h before assays. (A) Exogenous GLS2 expression increases GSH levels and relative GSH/GSSG ratio (P < 0.05). (B) GLS2 knockdown decreases GSH levels and GSH/GSSH ratio (P < 0.05). (C) Exogenous GLS2 expression increases NADH levels, and GLS2 knockdown decreases NADH levels (P < 0.05). (D) p53 increases GSH levels, GSH/GSSH ratio, and NADH levels (P < 0.05). LN-2024 cells were treated with Dox for 24 h.

Fig. 6.

GLS2 decreases ROS levels in cells and protects cells from H₂O₂-induced apoptosis. Cells were transfected with GLS2 expression vectors or siRNA oligo for 24 h and then treated with H₂O₂ (400 μM) for 4 h before measuring ROS or for 24 h before measuring apoptosis. (A) Exogenous GLS2 expression decreases ROS levels in cells (P < 0.05). (B) GLS2 knockdown increases ROS levels in cells (P < 0.05). (C) p53 decreases ROS levels in cells (P < 0.05). LN-2024 cells were treated with Dox for 24 h. (D) GLS2 overexpression protects cells from H₂O₂-induced apoptosis (P < 0.05). (E) GLS2 knockdown sensitizes cells to H₂O₂-induced apoptosis (P < 0.05).

Fig. 7.

Loss of GLS2 expression in hepatocellular carcinomas and the inhibition of colony formation by GLS2 in tumor cells. (A) Loss or significant decrease of mRNA expression of GLS2 in human hepatocellular carcinomas. HCC, hepatocellular carcinoma; N, normal liver. Tumor adjacent tissues: L1–5, tissues with cirrhosis; L6–10, tissues with fatty changes; L11–13, tissues with chronic hepatitis. The GLS2 expression was detected by real-time PCR and normalized with Actin. GLS2 levels of N1 are designated as 1. Samples were provided by Origene Techonologies. P < 0.0001 (normal vs. tumor), P < 0.0001 (tumor adjacent tissues vs. tumor), and P = 0.2367 (normal vs. tumor adjacent tissue). (B) Exogenous GLS2 expression reduces cell colony formation in tumor cells, including H1299, HepG2, and HTB15 cells (P < 0.05).