Inhibition of IGF2BP1 attenuates renal injury and inflammation by alleviating m6A modifications and E2F1/MIF pathway

Abstract

Septic acute kidney injury (AKI) is characterized by inflammation. Pyroptosis often occurs during AKI and is associated with the development of septic AKI. This study found that induction of insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) to a higher level can induce pyroptosis in renal tubular cells. Meanwhile, macrophage migration inhibitory factor (MIF), a subunit of NLRP3 inflammasomes, was essential for IGF2BP1-induced pyroptosis. A putative m6A recognition site was identified at the 3'-UTR region of E2F transcription factor 1 (E2F1) mRNA via bioinformatics analyses and validated using mutation and luciferase experiments. Further actinomycin D (Act D) chase experiments showed that IGF2BP1 stabilized E2F1 mRNA dependent on m6A. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) indicated that E2F1 acted as a transcription factor to promote MIF expression. Thus, IGF2BP1 upregulated MIF through directly upregulating E2F1 expression via m6A modification. Experiments on mice with cecum ligation puncture (CLP) surgery verified the relationships between IGF2BP1, E2F1, and MIF and demonstrated the significance of IGF2BP1 in MIF-associated pyroptosis in vivo. In conclusion, IGF2BP1 was a potent pyroptosis inducer in septic AKI through targeting the MIF component of NLRP3 inflammasomes. Inhibiting IGF2BP1 could be an alternate pyroptosis-based treatment for septic AKI.

Keywords: IGF2BP1; Transcriptional regulation; m6A RNA methylation; pyroptosis; septic acute kidney injury.

Figure 1

IGF2BP1 is a pyroptosis inducer in septic AKI. (A) Total m6A levels in CLP and Sham mice, as measured by dot blot. (B-D) IGF2BP1 mRNA and protein levels in CLP and Sham mice, as measured by qPCR (B), WB (C), and IHC (D; Scale bar, 100 μm). (E) Location of IGF2BP1 protein in the kidney tissue from mice, as measured by immunofluorescence. Immunofluorescent staining of IGF2BP1 (red) and nuclear staining with DAPI (blue) are shown. Scale bar, 150 μm. (F-H) Induction of IGF2BP1 by the indicated concentration of LPS for 12 h in HK2 cells, as measured by qPCR (F), WB (G), and immunofluorescence (H; Scale bar, 50 μm). Immunofluorescent staining of IGF2BP1 (red) and nuclear staining with DAPI (blue) are shown. (I) IGF2BP1 protein expression in sh-IGF2BP1/sh-NC-expressing HK2 cells following treatment with the indicated concentration of LPS for 12 h, as measured by WB. (J) Total m6A level in sh-IGF2BP1/sh-NC-expressing HK2 cells following induction with or without LPS for 12 h, as measured by dot blot. (K-N) Pyroptosis levels in sh-IGF2BP1/sh-NC-expressing HK2 cells following induction with or without LPS for 12 h. Cell viability (K), pyroptosis-related proteins (L), LDH release (M), secretory Caspase-1, IL-1β, and IL-18 levels (N) were measured. Statistical analysis was performed using a t-test (B, K, M, N) and one-way ANOVA (F). *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 2

IGF2BP1 induces pyroptosis via targeting MIF. (A & B) MIF interacted with NLRP3. The co-localization of MIF protein and NLRP3 protein in HK2 cells was measured by immunofluorescence (A). Immunofluorescent staining of NLRP3 (red), MIF (green), and nuclear staining with DAPI (blue) are shown. Scale bar, 50 μm. The combination of MIF protein and NLRP3 protein in HK2 cells with or without LPS stimulation was measured by co-IP (B). (C-E) MIF protein levels in the CLP and Sham mice, as measured by IHC (C; Scale bar, 100 μm), WB(D), and ELISA (E). (F & G) MIF protein levels in HK2 cells treated with the indicated concentration of LPS for 12 h, as measured by immunofluorescence (F; Scale bar, 50 μm) and WB (G). Immunofluorescent staining of MIF (green) and nuclear staining with DAPI (blue) are shown. (H) MIF protein levels in sh-MIF/sh-NC-expressing HK2 cells treated with the indicated concentration of LPS for 12 h, as measured by WB. (I-K) Pyroptosis levels in sh-MIF/sh-NC-expressing HK2 cells following the induction with or without the indicated concentration of LPS for 12 h. Pyroptosis-related proteins (I), LDH release (J), secretory IL-1β, and IL-18 levels (K) were measured. (L-N) Serum MIF (L), creatinine (M), and IL-1β (N) levels in CLP mice treated with or without ISO-1, as measured by ELISA. (O-Q) Pyroptosis level in sh-IGF2BP1-expressing HK2 cells with MIF over-expression following the induction with the indicated concentration of LPS for 12 h. Pyroptosis-related proteins (O), secretory IL-1β level (P), and secretory IL-18 level (Q) were measured. Statistical analysis was performed using a t-test (E, K-N, P-Q). *P < 0.05, **P < 0.01 and ns, non-significance.

Figure 3

MIF is transcriptionally promoted by IGF2BP1. (A) MIF mRNA levels in sh-IGF2BP1/sh-NC-expressing HK2 cells following treatment with or without LPS for 12 h, as measured by qPCR (A). (B) MIF protein levels in sh-IGF2BP1/sh-NC-expressing HK2 cells following treatment with or without LPS for 12 h, as measured by WB. The quantification of the relative MIF protein levels was shown in the right panel. (C) Actinomycin D (Act D) chase experiments for MIF mRNA expression in sh-IGF2BP1/sh-NC-expressing HK2 cells following treatment with or without the indicated concentration of LPS for 12 h. (D) MIF promoter activities in HK2 cells following the treatment with or without LPS for 12 h. (E) MIF promoter activities in HK2 cells in the absence or presence of α-Amanitin, as measured by luciferase assays. (F & G) IGF2BP1 did not affect the protein stability of MIF. HK2 cells were treated with CHX (100 μM) during administration with or without LPS for 12 h. The ratio between MIF and GAPDH was graphed in the middle panel, and the relative MIF level with or without CHX was also calculated in HK2 cells treated with or without LPS (right panel). (H) MIF mRNA levels in AKI patients (n=38) and control samples (n=12), as measured in qPCR. (I) IGF2BP1 mRNA levels in AKI patients (n=38) and control samples (n=12), as measured in qPCR. (J) Correlation between IGF2BP1 mRNA and MIF mRNA in AKI patients (n=38) and control samples (n=12), as analyzed by Pearson analysis. Statistical analysis was performed using one-way ANOVA (A, D, E), two-way ANOVA (B, C), t-test (G), Wilcoxon rank sum test (H, I), and Pearson analysis (J). **P < 0.01, ***P < 0.001, and ns, non-significance.

Figure 4

IGF2BP1 promotes MIF transcription via E2F1. (A) The specific binding site bases of transcription factor E2F1, downloaded from the JASPAR website (https://jaspar.genereg.net/). (B) The conserved E2F1 binding motif of the MIF promoter between humans and mice. (C) MIF promoter activities with or without intact E2F1 motif with E2F1 overexpressed or knocked down in HK2 cells, as measured by luciferase assay. (D & E) MIF mRNA and protein levels with E2F1 overexpressed or knocked down in HK2 cells, as measured by qPCR (D) and WB (E) in HK2 cells respectively. (F) The interaction between probes compassing the E2F1 motif and nuclear exacts (NE) from HK2 cells, as measured by EMSA. The proportion of wild-type (Wt) probes and mutant (mut) probes in each assay was different. (G) The interaction between probes compassing the E2F1 motif and nuclear exacts (NE) from HK2 cells, as measured by EMSA. The type or dose of antibodies was different for each assay. (H) ChIP-seq of E2F1 related to MIF in HeLa cells, as shown on the Cistrome Data Browser website (http://cistrome.org/db). (I & J) ChIP assays using anti-E2F1 antibody and control IgG antibody in HK2 cells with or without the indicated concentration of LPS for 12 h (I). QPCR was used to quantify immunoprecipitated chromatin fragments using primers specific to E2F1 binding sites (J). (K & L) ChIP assays using anti-E2F1 antibody and IgG antibody in HK2 cells with E2F1 knocked down (K). qPCR was used to quantify immunoprecipitated chromatin fragments (L). F1/R1 region compassed the E2F1 motif in the MIF promoter, while F2/R2 region represented an unrelated region in the MIF promoter. (M-O) IGF2BP1 promotes MIF promoter activities and expression via E2F1 in HK2 cells. MIF promoter activities, mRNA levels, and protein levels in sh-E2F1/sh-NC-expressing HK cells with or without inducible IGF2BP1 expression, as measured by luciferase assay (M), qPCR (N), and WB (O) respectively. Statistical analysis was performed using one-way ANOVA (C, D) and t-test (J, L-N). **P < 0.01, ***P < 0.001, and ns, non-significance.

Figure 5

IGF2BP1 m6A-dependently stabilizes E2F1 mRNA. (A & B) E2F1 protein expression in the CLP and Sham mice, as measured by IHC (A; Scale bar, 100 μm) and WB (B). (C) Act D chase experiments for E2F1 mRNA in HK2 cells with or without knockdown of IGF2BP1 following treatment with or without LPS for 12 h. (D) Act D chase experiments for E2F1 mRNA in HK2 cells with or without knockdown of METTL3 following treatment with or without LPS for 12 h. (E & F) The mRNA and protein levels of E2F1 and MIF were measured using qPCR (F) and WB (G) in HK2 cells with IGF2BP1 knocked down, treated with or without DAA for 12 h. (G) Cell viabilities of sh-METTL3/sh-NC-expressing HK2 cells with the treatment of LPS in the absence or presence of VX765, as measured by CCK-8 assay. (H-J) Pyroptosis level in sh-METTL3/sh-NC-expressing HK2 cells with or without the indicated concentration of LPS in the absence or presence of VX765. Pyroptosis-related proteins (H), LDH release (I), secretory IL-1β and IL-18 levels (J) were measured. Statistical analysis was performed using two-way ANOVA (C-E) and one-way ANOVA (G, I, J). *P < 0.05, **P < 0.01, ***P < 0.001, and ns, non-significance.

Figure 6

E2F1 mRNA is m6A methylated and stabilized by IGF2BP1. (A) DART-seq data from the GSE54365 set analyzed on the RMVar website (https://rmvar.renlab.org/), which was based on the A549 cells with or without knockdown of METTL3 and revealed an m6A peak within the 3′-UTR region of E2F1 mRNA. (B & C) The enrichment of E2F1 mRNA in RIP experiments in HK2 cells with or without METTL3 knockdown (B) and with or without METTL3 overexpression (C). F1/R1 region encompassed the potential m6A site in the 3′-UTR region of E2F1 mRNA, while F2/R2 region represented an unrelated region nearby. (D) The construction for the E2F1 3′-UTR region containing pmir-GLO luciferase reporters. (E) Luciferase activities of pmir-GLO reporters in sh-IGF2BP1/sh-NC-expressing HK2 cells with or without the treatment of LPS for 12 h, as measured by luciferase assay. (F) Schematic presentation of the mutation of the m6A region in E2F1 3′-UTR. (G) Luciferase activities of pmir-GLO reporters that contained wild-type (WT) or m6A site-mutated (Mut) E2F1 3′-UTR region in HK2 cells following treatment with LPS for 12 h, as measured by luciferase assay. (H) Act D chase assays for the WT or mutated-F-Luc-E2F1 fusion mRNA levels in HK2 cells with or without the knockdown of IGF2BP1 following treatment with LPS for 12 h. Statistical analysis was performed using two-way ANOVA (B, C, H) and one-way ANOVA (E, G). **P < 0.01, ***P < 0.001, and ns, non-significance.

Figure 7

Inhibiting IGF2BP1 is a potential strategy to protect against renal septic injury in vivo. (A) Representative images of H&E and PAS staining of kidney tissues from CLP mice with or without the knockdown of IGF2BP1. Scale bar, 50 μm. (B) Expression of IGF2BP1 in the CLP kidney injected with Lv-IGF2BP1 or Lv-NC, as measured by WB. (C) Renal function serum creatinine (Scr) level in mice receiving CLP surgery after injection of Lv-IGF2BP1 or Lv-NC. (D-G) The expression level of E2F1 mRNA (D), MIF mRNA (E), IL-6 mRNA (F), and TNF-α mRNA (G) in kidney tissues from mice injected with Lv-IGF2BP1 or Lv-NC, as measured by qPCR. (H) Serum IL-1β levels in mice injected with Lv-IGF2BP1 or Lv-NC, as measured by ELISA. Group comparisons were performed by t-test (C-H). N = 6/group, *P<0.05, **P < 0.01.