Feedback repression of PPARα signaling by Let-7 microRNA - PubMed (original) (raw)
Feedback repression of PPARα signaling by Let-7 microRNA
Tomoki Yagai et al. Cell Rep. 2021.
Abstract
Peroxisome proliferator-activated receptor α (PPARα) controls hepatic lipid homeostasis and is the target of lipid-lowering fibrate drugs. PPARα activation represses expression of let-7 microRNA (miRNA), but the function of let-7 in PPARα signaling and lipid metabolism is unknown. In the current study, a hepatocyte-specific let-7b/c2 knockout (let7b/c2ΔHep) mouse line is generated, and these mice are found to exhibit pronounced resistance to diet-induced obesity and fatty liver. Let-7 inhibition by hepatocyte-specific let-7 sponge expression shows similar phenotypes as let7b/c2ΔHep mice. RNA sequencing (RNA-seq) analysis reveals that hepatic PPARα signaling is repressed in let7b/c2ΔHep mice. Protein expression of the obligate PPARα heterodimer partner retinoid X receptor α (RXRα) is reduced in the livers of let7b/c2ΔHep mice. Ring finger protein 8 (Rnf8), which is a direct target of let-7, is elevated in let7b/c2ΔHep mouse liver and identified as a E3 ubiquitin ligase for RXRα. This study highlights a let-7-RNF8-RXRα regulatory axis that modulates hepatic lipid catabolism.
Keywords: PPARα; RNF8; RXRα; fatty liver disease; let-7; nuclear receptors; obesity; ubiquitin-proteasome system.
Published by Elsevier Inc.
Conflict of interest statement
Declaration of interests The authors declare no competing interests.
Figures
Figure 1.. Expression of let-7 miRNA family members is decreased in mouse liver in response to PPARα activation
(A–D) Fold change of let-7 miRNA precursors in PPARα-activated Ppara+/+ and _Ppara_ΔHep mouse livers measured by qRT-PCR. Individual let-7 miRNA precursor expression in livers treated with Wy-14,643 (A) or overnight fasting (C). Precursor abundance ratios in mice treated with Wy-14,643 (B) or overnight fasting (D). Data are normalized to Actb expression and presented as mean ± SEM (n = 4 mice per group; *p < 0.05, #p < 0.01, §p < 0.001) (E) Time course of mature let-7 miRNA abundance in wild-type mouse liver after Wy-14,643 administration measured by qRT-PCR. Data are normalized to U6 expression and presented as mean ± SEM (n = 4 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001)
Figure 2.. _let7b/c2_ΔHep and let-7 sponge AAV-transduced mice are resistant to HFD-induced obesity
(A) let7b/c2+/+ and _let7b/c2_ΔHep mice after HFD feeding for 8 weeks. (B) Body weight alterations of let7b/c2+/+ and _let7b/c2_ΔHep mice during HFD feeding. (C) Oil red O and H&E staining of let7b/c2+/+ and _let7b/c2_ΔHep liver sections after HFD feeding. Bars, 100 μM. (D–F) Tissue weight ratio of lean (D), fat (E), and liver (F) to total body weight for let7b/c2+/+ and _let7b/c2_ΔHep mice after HFD feeding. (G) Biochemical analysis for serum triglycerides (TGs) in let7b/c2+/+ and _let7b/c2_ΔHep mice after HFD feeding. (H) EGFP fluorescence in mouse primary hepatocytes infected with either EGFP or let-7 sponge AAV vectors after 5 days in culture. Bars, 300 μm. (I) Body weight alterations in wild-type mice infected with EGFP or let-7 sponge expressing AAV and then placed on HFD for 8 weeks. (J–L) Tissue weight ratio of lean (J), fat (K), and liver (L) to total body weight for wild-type mice infected with EGFP or let-7 sponge expressing AAV then placed on HFD for 8 weeks. (M) Biochemical analyses for serum TG concentration in EGFP and let-7 sponge AAV-infected mice after HFD feeding. Data are presented as mean ± SEM (n = 4–5 mice per group; *p < 0.05, **p < 0.01)
Figure 3.. PPARα target gene expressions were repressed by RXRα protein reduction in _let7b/c2_ΔHep and let-7 sponge AAV-transduced mice
(A) Heatmap of PPARα target genes identified by differential gene expression analysis of RNA-seq data from let7b/c2+/+ and _let7b/c2_ΔHep livers after HFD feeding. (B and C) mRNA analysis by qRT-PCR of PPARα target genes involved in fatty acid oxidation and cell proliferation (B) and lipid accumulation and glucose metabolism (C) in HFD-fed let7b/c2+/+ and _let7b/c2_ΔHep livers. (D) Western blot analysis for PPARα target genes in HFD-fed let7b/c2+/+ and _let7b/c2_ΔHep liver lysates. (E) Western blot analysis of PPARα and RXRα protein expression in nuclear fractions isolated from let7b/c2+/+ and _let7b/c2_ΔHep hepatocytes. (F–H) Western blot analysis of RXRα and the densiometric quantification in whole-liver lysates from _let7b/c2_ΔHep- mice (F), let-7 sponge expressing AAVinflected mice (G), and _pre-let-7c-1-_-AAVinfected mice (H). Data are presented as mean ± SEM (n = 4–5 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001
Figure 4.. RNF8 is decayed by let-7 miRNA, and RXRα protein is ubiquitinated by RNF8 E3 ubiquitin ligase
(A) Predicted let-7 miRNA binding sites in Rnf8 mRNA. (B–F) Western blot analysis and densitometric quantification of RNF8 protein (B, D, and F) and qRT-PCR of Rnf8 mRNA (C and E) in let7b/c2+/+ and _let7b/c2_ΔHep (B and C); EGFP and let-7 sponge AAV-transduced (D and E); EGFP and pre-let-7c-1 AAV-transduced (F) livers treated with HFD feeding. (G) 3′ UTR reporter assays in HepG2 cells transfected with Rnf8 wild-type or mutant 3′ UTR reporter constructs and a let-7c mimic expression vector. (H and I) Western blot analysis (H) and densitometric quantification (I) of RXRα expression in _Rxra_- and _Rnf8_-transfected Hepa-1 cells. (J and K) Fold change of Rnf8 (J) and Rxra (K) mRNA by qRT-PCR analysis in _Rxra_- and _Rnf8_-transfected Hepa-1 cells. (L) Western blot analysis and the densitometric quantification of RXRα in _Rxra_- and _Rnf8-_transfected Hepa-1 cells treated with the proteasome inhibitor MG-132. (M) Ubiquitination assays for _Rxra_- and _Rnf8_-transfected and MG-132-treated Hepa-1 cells. RXRα was immunoprecipitated and polyubiquitin detected by anti-ubiquitin antibody. RXRα expression was confirmed in whole-cell lysate as input. (N) Scheme of 3-step inhibition for PPARα/RXRα pathway that the current study demonstrates.
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