EP300-mediated H3 acetylation elevates MTHFD2 expression to reduce mitochondrial dysfunction in lipopolysaccharide-induced tubular epithelial cells

Abstract

Sepsis represents an organ dysfunction resulting from the host's maladjusted response to infection, and can give rise to acute kidney injury (AKI), which significantly increase the morbidity and mortality of septic patients. This study strived for identifying a novel therapeutic strategy for patients with sepsis-induced AKI (SI-AKI). Rat tubular epithelial NRK-52E cells were subjected to lipopolysaccharide (LPS) exposure for induction of in-vitro SI-AKI. The expressions of E1A binding protein p300 (EP300) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in NRK-52E cells were assessed by western blot and qRT-PCR, and their interaction was explored by chromatin immunoprecipitation performed with antibody for H3K27 acetylation (H3K27ac). The effect of them on SI-AKI-associated mitochondrial dysfunction of tubular epithelial cells was investigated using transfection, MTT assay, TUNEL staining, 2',7'-Dichlorodihydrofluorescein diacetate probe assay, Mitosox assay, and JC-1 staining. MTHFD2 and EP300 were upregulated by LPS exposure in NRK-52E cells. LPS increased the acetylation of H3 histone in the MTHFD2 promoter region, and EP300 suppressed the effect of LPS. EP300 ablation inhibited the expression of MTHFD2. MTHFD2 overexpression antagonized LPS-induced viability reduction, apoptosis promotion, reactive oxygen species overproduction, and mitochondrial membrane potential collapse of NRK-52E cells. By contrast, MTHFD2 knockdown and EP300 ablation brought about opposite consequences. Furthermore, MTHFD2 overexpress and EP300 ablation counteracted each other's effect in LPS-exposed NRK-52E cells. EP300-mediated H3 acetylation elevates MTHFD2 expression to reduce mitochondrial dysfunction of tubular epithelial cells in SI-AKI.

Keywords: E1A binding protein p300; methylenetetrahydrofolate dehydrogenase 2; mitochondrial dysfunction; sepsis-induced acute kidney injury; tubular epithelial cells.

Figure 1.

MTHFD2 was upregulated by LPS exposure and its overexpression antagonized LPS-induced apoptosis of NRK-52E cells. (A and B) The expression of MTHFD2 in NRK-52E cells that had received a 24-h exposure to 0, 0.1, 0.5 and 1 μg/mL LPS was analyzed by qRT-PCR and Western blot, with GAPDH serving as the normalizer. (C and D) The expression of MTHFD2 in NRK-52E cells transfected with shMTHFD2/shNC or MTHFD2 overexpression plasmids/NC was assessed by qRT-PCR and Western blot, with GAPDH serving as the normalizer. (E/F/G/H). NRK-52E cells were transfected with shMTHFD2/shNC or MTHFD2 overexpression plasmids/NC and exposed to 1 μg/mL LPS for 24 h. (E and F) The expression of MTHFD2 was assessed by qRT-PCR and Western blot, with GAPDH serving as the normalizer. (G). The viability of NRK-52E cells was measured by MTT assay. (H). The apoptosis of NRK-52E cells was checked using TUNEL staining (magnification, × 200; scale bar, 100 µm).*p < .05; **p < .01; ***p < .001; *compares the values located at both ends of the line segment. LPS: lipopolysaccharide; MTHFD2: methylenetetrahydrofolate dehydrogenase 2; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Figure 2.

MTHFD2 was upregulated by LPS exposure and its overexpression antagonized mitochondrial dysfunction of NRK-52E cells. (A–F) NRK-52E cells were transfected with shMTHFD2/shNC or MTHFD2 overexpression plasmids/NC and exposed to 1 μg/mL LPS for 24 h. (A and C) The level of ROS in NRK-52E cells was detected using mitosox assay (magnification, × 200; scale bar, 100 µm). (B and D) the mitochondrial membrane potential of NRK-52E cells was detected using JC-1 staining (magnification, × 200; scale bar, 100 µm). (E and F) the expresison of TOM20 was detected by Western blot. *p < .05; **p < .01; ***p < .001; *compares the values located at both ends of the line segment. LPS: lipopolysaccharide; MTHFD2: methylenetetrahydrofolate dehydrogenase 2; ROS: reactive oxygen species; DCFH-DA: 2′,7′-dichlorodihydrofluorescein diacetate; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; NC: negative control; shNC: short hairpin RNA against NC; shMTHFD2: short hairpin RNA against MTHFD2.

Figure 3.

EP300-mediated H3K27ac at the MTHFD2 promoter was essential to the antagonistic impact of MTHFD2. (A and B) The expression of EP300 in NRK-52E cells that had received a 24-h exposure to 1 μg/mL LPS was analyzed by qRT-PCR and Western blot, with GAPDH serving as the normalizer. (C and D) The expression of EP300 in NRK-52E cells transfected with shEP300/shNC was assessed by qRT-PCR and Western blot, with GAPDH serving as the normalizer. (E and F) In NRK-52E cells that had received a 24-h exposure to 1 μg/mL LPS following transfection with EP300 overexpression plasmids/NC, the expression of MTHFD2 was detected by qRT-PCR and Western blot. (G). Whether EP300-mediated H3K27 acetylation occurs at the MTHFD2 promoter was determined using chromatin immunoprecipitation, in NRK-52E cells that had received a 24-h exposure to 1 μg/mL LPS following transfection with EP300 overexpression plasmids/NC or not. (H). NRK-52E cells were transfected with NC plus shNC, shEP300 plus NC, MTHFD2 overexpression plasmids plus shNC or shEP300 plus MTHFD2 overexpression plasmids and exposed to 1 μg/mL LPS for 24 h, the viability of NRK-52E cells was measured by MTT assay. **p < .01; ***p < .001; *compares the values located at both ends of the line segment. LPS: lipopolysaccharide; MTHFD2: methylenetetrahydrofolate dehydrogenase 2; EP300: E1A binding protein p300; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; NC: negative control; shNC: short hairpin RNA against NC; shEP300: short hairpin RNA against EP300).

Figure 4.

EP300-mediated H3K27ac at the MTHFD2 promoter was essential to the antagonistic impact of MTHFD2 on LPS-induced apoptosis and mitochondrial dysfunction of NRK-52E cells. (A–F). NRK-52E cells were transfected with NC plus shNC, shEP300 plus NC, MTHFD2 overexpression plasmids plus shNC or shEP300 plus MTHFD2 overexpression plasmids and exposed to 1 μg/mL LPS for 24 h. (A and D). The apoptosis of NRK-52E cells was checked using TUNEL staining (magnification, × 200; scale bar, 100 µm). (B and E). The level of ROS in NRK-52E cells was detected mitosox assay (magnification, × 200; scale bar, 100 µm). (C and F). The mitochondrial membrane potential of NRK-52E cells was detected using JC-1 staining (magnification, × 200; scale bar, 100 µm).*p < .05, **p < .01; ***p < .001; *compares the values located at both ends of the line segment. TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; ROS: reactive oxygen species; DCFH-DA: 2’,7’-dichlorodihydrofluorescein diacetate; H3K27ac: acetylation of histone H3 lysine 27; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; NC: negative control; shNC: short hairpin RNA against NC; shEP300: short hairpin RNA against EP300.

Figure 5.

MTHFD2 overexpression reversed the decreased TOM20 expression by EP300 silencing. NRK-52E cells were transfected with NC plus shNC, shEP300 plus NC, MTHFD2 overexpression plasmids plus shNC or shEP300 plus MTHFD2 overexpression plasmids and exposed to 1 μg/mL LPS for 24 h. (A and B) The expression of TOM20 was detected by Western blot. ***p < .001; *compares the values located at both ends of the line segment.