HIF-independent regulation of thioredoxin reductase 1 contributes to the high levels of reactive oxygen species induced by hypoxia

Abstract

Cellular adaptation to hypoxic conditions mainly involves transcriptional changes in which hypoxia inducible factors (HIFs) play a critical role. Under hypoxic conditions, HIF protein is stabilized due to inhibition of the activity of prolyl hydroxylases (EGLNs). Because the reaction carried out by these enzymes uses oxygen as a co-substrate it is generally accepted that the hypoxic inhibition of EGLNs is due to the reduction in oxygen levels. However, several studies have reported that hypoxic generation of mitochondrial reactive oxygen species (ROS) is required for HIF stabilization. Here, we show that hypoxia downregulates thioredoxin reductase 1 (TR1) mRNA and protein levels. This hypoxic TR1 regulation is HIF independent, as HIF stabilization by EGLNs inhibitors does not affect TR1 expression and HIF deficiency does not block TR1 hypoxic-regulation, and it has an effect on TR1 function, as hypoxic conditions also reduce TR1 activity. We found that, when cultured under hypoxic conditions, TR1 deficient cells showed a larger accumulation of ROS compared to control cells, whereas TR1 over-expression was able to block the hypoxic generation of ROS. Furthermore, the changes in ROS levels observed in TR1 deficient or TR1 over-expressing cells did not affect HIF stabilization or function. These results indicate that hypoxic TR1 down-regulation is important in maintaining high levels of ROS under hypoxic conditions and that HIF stabilization and activity do not require hypoxic generation of ROS.

Figure 1. Effect of hypoxic conditions on TR1 mRNA and protein levels.

(A) EMT6 cells were labeled with ⁷⁵Se and incubated under normoxic (Nx) or hypoxic (Hx) conditions for 12 h. Cells were lysed and selenoprotein levels analyzed by autoradiography (left panel). Protein standards are shown in lane 1 and protein extracts of EMT6 cells cultured in (lane 2) normoxic or (lane3) hypoxic conditions (right panel). (B) TR1 protein levels were analyzed by western blotting in EMT6 and DT cell lines grown under normoxic (Nx) or hypoxic (Hx) conditions for 12 h. (C) EMT6 and (D) DT cell lines were grown under normoxic (Nx) or hypoxic (Hx) conditions and the mRNA levels of TR1, VEGF and ADM determined by quantitative RT-PCR and normalized to the content of β-actin mRNA in the same sample. The data represent the fold induction of each mRNA over the mean from the normoxic values. The mean from 3 different experiments ± SE is shown.

Figure 2. Effect of HIF-1α stabilization by EGLN inhibitors on TR1 expression.

(A) EMT6 and DT cells were treated with 150 µM of deferoxamine (DFX), 100 µM of L-mimosine (LM) or left untreated (Ctrl) for 12 h and the levels of HIF-1α and TR1 protein detected by western blotting. The mRNA levels of TR1, VEGF and ADM from (B) EMT6 cells and (C) DT cells, treated with deferoxamine (DFX) or left untreated (Ctrl) for 8 h, were measured by quantitative RT-PCR. The data represent the fold-change relative to untreated cells after normalization with the β-actin content. The mean and ± SE of 3 independent experiments is shown.

Figure 3. Hypoxic regulation of TR1 expression in HIF-1α deficient cells.

TR1 and HIF-1α protein levels, from (A) EMT6 cells and (B) DT cells infected with retrovirus encoding a scramble sequence (SCR) or a HIF-1α shRNA (si HIF-1α) and cultured under normoxic (Nx) or hypoxic (Hx) conditions for 12 h, were determined by western blotting. (C) EMT6 cells or (D) DT cells infected with the indicated retroviruses were cultivated for 12 h under normoxic (Nx) or hypoxic conditions (Hx) and TR1, VEGF and ADM mRNA levels measured by quantitative RT-PCR. The fold-change relative to the normoxic values and normalized with the β-actin is shown. Data represent the mean and ± SE of 3 independent experiments.

Figure 4. Effect of hypoxic conditions, EGLN inhibitors or HIF-1α deficiency on TR1 activity.

(A) TR1 activity was measured as indicated in Methods in EMT6 and DT cells grown under normoxic (Nx) or hypoxic (Hx) conditions for 12 h. (B) EMT6 and DT cells were treated with 150 µM deferoxamine (DFX) or left untreated (Ctrl) for 12 h and TR1 activity measured. (C) EMT6 and DT cells infected with retrovirus encoding a scramble sequence (SCR) or a HIF-1α shRNA (si HIF-1α) were cultured under normoxic (Nx) or hypoxic (Hx) conditions for 12 h and TR1 activity measured. Data represent the means of 3 independent experiments ± SE.

Figure 5. Effect of TR1 deficiency on ROS levels and HIF activity in cells cultured under hypoxic conditions.

(A) EMT6 and (B) DT cells infected with retroviruses encoding a scramble sequence (SCR) or a TR1 shRNA (siTR1) were grown under normoxic (Nx) or hypoxic (Hx) conditions for 12 h and ROS levels determined as described in Methods. As a positive control for ROS generation, cells were treated with 100 µM of H₂O₂ where indicated. Data represent the fluorescence value of each sample after subtracting the fluorescence value from unstained control cells. The mean ± SE of 3–4 independent experiments is shown. HIF-1α, TR1 and β-actin protein levels were determined by western blotting in (C) EMT6 and (D) DT cells infected with retroviruses encoding a scramble sequence (SCR) or a TR1 shRNA (siTR1) and cultured under normoxic (Nx) or hypoxic (Hx) conditions for 12 h. mRNA levels of the HIF target genes, VEGF and ADM, were analyzed by quantitative RT-PCR in (E) EMT6 or (F) DT cell lines infected with retroviruses encoding a scramble sequence (SCR) or a TR1 shRNA (siTR1) and cultured under normoxic (Nx) or hypoxic (Hx) conditions for 12 h. Data show the means of 3 independent experiments ± SE.

Figure 6. ROS levels and HIF stabilization and activity in hypoxic TR1 over-expressing cells.

ROS levels were determined in (A) EMT6 and (B) DT cells infected with retroviruses encoding the TR1 gene (TR1) or empty retroviruses (Ctrl) and cultured under normoxic (Nx) or hypoxic (Hx) conditions for 12 h. Data represent the fluorescence value of each sample after subtracting the fluorescence value from unstained control cells. The mean ± SE of 4–7 independent experiments is shown. Protein levels of HIF-1α and TR1 were determined by western blotting in (C) EMT6 and (D) DT cells infected with empty retroviruses (Ctrl) or retroviruses encoding the TR1 gene (TR1) and cultured for 12 h under normoxic (Nx) or hypoxic (Hx) conditions. VEGF and ADM mRNA levels were analyzed by quantitative RT-PCR in EMT6 (E) or DT (F) cell lines with retroviruses encoding the TR1 gene (TR1) or with empty retroviruses (Ctrl) and cultured under normoxic (Nx) or hypoxic (Hx) conditions for 12 h. Data show the means of 3 independent experiments ± SE.