![Nrf2 regulates L2hgdh expression to modulate the production of glutamine-derived L2HG. (A) Control or NRF2 silenced EPs from patients with SCD were cultured for 12 days in glutamine-free Dulbecco’s modified Eagle medium media supplemented with 15% dialyzed fetal bovine serum, 15% dialyzed human AB serum and 2 IU/mL erythropoietin under hypoxia and oxidative stress (1% O2/200 μM H2O2), and traced with 2mM U-13C5 glutamine for 12 hours. The ion intensity of m + 0, m + 1, m + 2, m + 3, m + 4, m + 5, and m + 6 and the relative ion intensity of selected trichloroacetic acid cycle intermediates (α-ketoglutarate [2OG], succinate, fumarate, malate, and citrate) and 2HG are shown. (B) Metabolites from (A) were treated with TSPC and determined the derivatized TSPC-2HG (total), TSPC-D2HG, and TSPC-L2HG. (C) Quantitative real-time PCR showing the relative messenger RNA levels of L2HG/D2HG generation/utilization enzyme genes Mdh1, Mdh2, lactate dehydrogenase A (Ldha), L2hgdh, and D2hgdh in spleen Ter119+ cells of SS/Nrf2+/+ and SS/Nrf2–/– mice. (D) Mouse L2hgdh gene promoter activities were measured via GFP reporter expression in the presence of DMF or after NRF2 silencing (shNRF2) in KU812 cells. (E) Transcriptional activities of the mouse L2hgdh promoter and ARE motif mutants (motifs I-IV, located −980, −810, −800, and −452 base pairs upstream of the TSS were measured via GFP reporter expression. The putative ARE motifs are underlined. Data from 3 different wells (A-B), 6 mice per group (C), or 3 biological replicates (C-E) are presented as the mean ± SD. ∗P < .05; ∗∗P < .01. GFP, green fluorescent protein; TSPC, N-(p-toluenesulfonyl)-l-phenylalanyl chloride.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/142/4/10.1182_blood.2022018159/2/blood_bld-2022-018159-gr3.jpeg?Expires=1752992809&Signature=J9pFRD9aD4nFnY0ulvBKLNxhk-vAFamRMw-S11dwv6h81Q-2zhswF8sgxHXMG1oXcw5e6VyRHJCAwf2XIyQx0EWa5CY2VtZ7YtaDnqUYpt1nDsS1j5BaAboQkPJtmbonUSU3BdLE9iwM959GeG9ZhZeTL6bBC7FtvLMoYiXB84fEa9v0U6LemuFXg0ekNn4AOXhmtrfdG37Xb0N8YVa6IvWoV-CpC-J7zNBoZhHGNQ0uJ6A4rasqXIsg1xPB9uTHFTrti9P3mGw~CsayOjK6PaoRezTolLFcEPlvqRkMpy~h7lx6C9F-3-a~eNfUuyq5j~NEbpru8gHNLuxWs4C7VA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Nrf2 regulates L2hgdh expression to modulate the production of glutamine-derived L2HG. (A) Control or NRF2 silenced EPs from patients with SCD were cultured for 12 days in glutamine-free Dulbecco’s modified Eagle medium media supplemented with 15% dialyzed fetal bovine serum, 15% dialyzed human AB serum and 2 IU/mL erythropoietin under hypoxia and oxidative stress (1% O2/200 μM H2O2), and traced with 2mM U-13C5 glutamine for 12 hours. The ion intensity of m + 0, m + 1, m + 2, m + 3, m + 4, m + 5, and m + 6 and the relative ion intensity of selected trichloroacetic acid cycle intermediates (α-ketoglutarate [2OG], succinate, fumarate, malate, and citrate) and 2HG are shown. (B) Metabolites from (A) were treated with TSPC and determined the derivatized TSPC-2HG (total), TSPC-D2HG, and TSPC-L2HG. (C) Quantitative real-time PCR showing the relative messenger RNA levels of L2HG/D2HG generation/utilization enzyme genes Mdh1, Mdh2, lactate dehydrogenase A (Ldha), L2hgdh, and D2hgdh in spleen Ter119+ cells of SS/Nrf2+/+ and SS/Nrf2–/– mice. (D) Mouse L2hgdh gene promoter activities were measured via GFP reporter expression in the presence of DMF or after NRF2 silencing (shNRF2) in KU812 cells. (E) Transcriptional activities of the mouse L2hgdh promoter and ARE motif mutants (motifs I-IV, located −980, −810, −800, and −452 base pairs upstream of the TSS were measured via GFP reporter expression. The putative ARE motifs are underlined. Data from 3 different wells (A-B), 6 mice per group (C), or 3 biological replicates (C-E) are presented as the mean ± SD. ∗P < .05; ∗∗P < .01. GFP, green fluorescent protein; TSPC, N-(p-toluenesulfonyl)-l-phenylalanyl chloride.
