Glutaminase-1 stimulates the proliferation, migration, and survival of human endothelial cells

Abstract

Glutaminase-1 (GLS1) is a mitochondrial enzyme found in endothelial cells (ECs) that metabolizes glutamine to glutamate and ammonia. Although glutaminolysis modulates the function of human umbilical vein ECs, it is not known whether these findings extend to human ECs beyond the fetal circulation. Furthermore, the molecular mechanism by which GLS1 regulates EC function is not defined. In this study, we show that the absence of glutamine in the culture media or the inhibition of GLS1 activity or expression blocked the proliferation and migration of ECs derived from the human umbilical vein, the human aorta, and the human microvasculature. GLS1 inhibition arrested ECs in the G₀/G₁ phase of the cell cycle and this was associated with a significant decline in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer improved the proliferative, but not the migratory, response of GLS1-inhibited ECs. Glutamine deprivation or GLS1 inhibition also stimulated the production of reactive oxygen species and this was associated with a marked decline in heme oxygenase-1 (HO-1) expression. GLS1 inhibition also sensitized ECs to the cytotoxic effect of hydrogen peroxide and this was prevented by the overexpression of HO-1. In conclusion, the metabolism of glutamine by GLS1 promotes human EC proliferation, migration, and survival irrespective of the vascular source. While cyclin A contributes to the proliferative action of GLS1, HO-1 mediates its pro-survival effect. These results identify GLS1 as a promising therapeutic target in treating diseases associated with aberrant EC proliferation, migration, and viability.

Keywords: 6-Diazo-5-oxo-L-norleucine (DON, PubChem CID: 5359375); CB-839 (PubChem CID: 71577426); Endothelial cells; Glutaminase; Migration; Proliferation; bis-2-(5-Phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES, PubChem CID: 3372016).

1.

Glutamine (Gln) stimulated the proliferation and migration of human endothelial cells. (A) Gln stimulated the proliferation of HUVECs in a time-dependent manner. (B) Gln stimulated the proliferation of HUVECs in a concentration-dependent manner. HUVECs were incubated in the absence and presence of different concentrations of Gln for three days. (C) Gln deprivation for three days inhibited DNA synthesis in HUVECs, HAECs, and HMECs. (D) Gln deprivation for three days inhibited the migration of HUVECs, HAECs, and HMECs. Results are means ± SD (n=4–6). *Statistically significant effect of Gln addition or deprivation.

2.

GLS1 inhibition blocked glutamine (Gln)-mediated increases in intracellular glutamate and proliferation of HUVECs. (A) Gln-mediated increases in intracellular glutamate were inhibited by GLS1 inhibitors. HUVECs were incubated with Gln in the presence or absence of DON (20μM), BPTES (20μM), or CB-839 (20μM) for 24 hours. (B) DON inhibited the proliferation of HUVECs in a concentration-dependent manner. (C) BPTES inhibited the proliferation of HUVECs in a concentration-dependent manner. (D) CB-839 inhibited the proliferation of HUVECs in a concentration-dependent manner. For the proliferation experiments, HUVEC were incubated in the presence or absence of GLS1 inhibitors for three days. Results are means ± SD (n=6). *Statistically significant effect of GLS1 inhibitors. ⁺Statistically significant effect of Gln

3.

GLS1 inhibition blocked DNA synthesis in human endothelial cells. (A-C) GLS1 inhibitors blocked DNA synthesis in HUVECs, HAECs, and HMECs. (D) GLS1 inhibition had no effect on endothelial cell viability. HUVECs were treated with DON (20μM), BPTES (20μM), or CB-839 (20μM) for 24 hours prior to DNA synthesis or viability measurements. Results are means ± SD (n=4–6). *Statistically significant effect of GLS1 inhibitors.

4.

GLS1 inhibition blocked the migration of human endothelial cells. (A) DON inhibited the migration of HUVECs. (B) BPTES inhibited the migration of HUVECs. (C) CB-839 inhibited the migration of HUVECs. (D) DON (20μM) or BPTES (20μM) inhibited the migration of HAECs. (E) DON (20μM) or BPTES (20μM) inhibited the migration of HMECs. Cells were treated with the GLS1 inhibitors for 24 hours prior to the migration assay. Results are means ± SD (n=4–6). *Statistically significant effect of GLS1 inhibitors.

5.

GLS1 knockdown inhibited the proliferation and migration of HUVEC. (A) GLS1 siRNA inhibited HUVEC GLS1 protein expression. (B) GLS1 siRNA inhibited HUVEC DNA synthesis. (C) GLS1 siRNA inhibited HUVEC migration. Cells were transfected with GLS1 siRNA (0.1μM) or non-targeting (NT) siRNA (0.1μM) for 48 hours prior to the proliferation or migration assay. Results are means ± SD (n=4). *Statistically significant effect of GLS1 silencing.

6.

Effect of eNOS on the proliferation, migration, and activity of GLS activity in HUVEC. (A) Effect of L-NAME on endothelial cell proliferation. Cells were incubated in the presence or absence of L-NAME (0–5mM) for three days. (B) Effect of L-NAME on endothelial cell migration. Cells were incubated in the presence or absence of L-NAME (0–5mM) for 24 hours. (C) Effect of BPTES and DEA-NO on GLS Activity. Cells were incubated in the presence or absence of BPTES (20μM) or DEA-NO (20μM) for 24 hours. Results are means ± SD (n=4–6). *Statistically significant effect of L-NAME or BPTES.

7.

Effect of glutamine (Gln) metabolites on the proliferation and migration of Gln-deprived HUVECs. A. α-Ketoglutarate (α-KG) and asparagine (Asp) are required for endothelial cell proliferation in Gln-depleted conditions. B. α-KG and Asp are required for endothelial cell migration in Gln-depleted conditions. Cells were grown in Gln (2mM)-replete or -deprived conditions with or without supplementation of glutamate (Glu;2Mm), ammonium chloride (NH₄Cl;2mM), dimethyl-α-KG (DMKG;2mM), aspartate (Asp;100μM), and/or asparagine (Asn;100μM) for three days prior to performing the proliferation or migration assay. Results are means ± SD (n=5–6). *Statistically significant effect of Gln deprivation. ⁺Statistically significant effect of DMKG and/or Asn supplementation.

8.

GLS1 inhibition blocked cell cycle progression and cyclin A expression by HUVEC. (A) Effect of DON (20μM) or BPTES (20μM) exposure for 24 hours on the distribution of HUVECs in the cell cycle. (B) Effect of DON (20μM) or BPTES (20μM) exposure for 24 hours on cell cycle protein expression and phosphorylation determined by western blotting. (C) Effect of DON (20μM) or BPTES (20μM) exposure for 24 hours on cyclin A mRNA expression. (D) Effect of DON (20μM) or BPTES (20μM) exposure for 24 hours on cyclin A promoter activity. Results are means ± SD (n=4). *Statistically significant effect of GLS1 inhibitors.

9.

Cyclin A contributed to the anti-proliferative effect of GLS1 inhibition in HUVEC. (A) AdCyclin A increased cyclin A protein levels in GLS1-inhibited HUVEC. (B) AdCyclin A increased DNA synthesis in GLS1-inhibited HUVEC. (C) AdCyclin A had no effect on migration in GLS1-inhibited HUVEC. Cells were infected with AdCyclin A (50 MOI) or AdGFP (50 MOI) for 24 hours and then incubated in the presence or absence of BPTES (10μM) for another 24 hours prior to performing the proliferation or migration assay. Results are means ± SD (n=3–4). *Statistically significant effect of AdCyclin A. ⁺Statistically significant effect of BPTES.

10.

GLS1 preserved oxidative balance and viability of human endothelial cells. (A) Glutamine (Gln) deprivation for 24 hours increased ROS production in HUVECs, HAECs, and HMECs. (B) GLS1 inhibition increased ROS production in HUVECs. Cells were treated with DON (20μM), BPTES (20μM), or CB-839 (20μM) for 24 hours. (C) BPTES (20μM) exposure for 24 hours decreased HO-1 protein levels in HUVECs. (D) AdHO-1 increased survival of GLS1-inhibited HUVECs exposed to ROS. Cells were infected with AdCyclin A (50 MOI) or AdGFP (50 MOI) for 24 hours, incubated in the presence or absence of BPTES (20μM) for another 24 hours, and then exposed to hydrogen peroxide (250μM) for 4 hours. Results are means ± SD (n=4). *Statistically significant effect of Gln deprivation or GLS1-inhibition. ⁺Statistically significant effect of AdHO-1.