RNF90 promotes hepatic steatosis by degrading CPT1α to suppress fatty acid oxidation

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  Figure 1. KLF5 mediates RNF90 expression upregulation in the liver of hepatic steatosis. A, representative H&E and Oil red O staining images of liver tissues from mice fed the indicated diets (n = 5). B–C, western blot (B) and quantitative analysis (C) of RNF90 and KLF5 protein expression in liver tissues from the mice described in (A) (n = 4). D, RT-qPCR analysis of RNF90 mRNA expression in mouse liver tissues (12 W NCD, HFD: n = 8; ob/ob: n = 6; 19 W NCD, HFHC: n = 10). E–H, RT-qPCR (E, G; n = 4) and western blot (F, H; n = 3) analyses of the indicated genes’ expressions in POA-treated HepG2 and AML12 cells at different time points. I, schematic of RNF90 gene promoter regions analysis via NCBI’s in silico tools. J, predicted conserved KLF5 binding sites via the JASPAR database. K–L, analysis of RNF90 mRNA (K; n = 4) and protein (L; n = 3) expression in HEK293T cells with KLF5 overexpression (leftpanel) and KLF5 knockdown (rightpanel). M, dual-luciferase reporter assay analysis of RNF90 promoter activity in KLF5-overexpressed (leftpanel) or KLF5-knockdown (rightpanel) HEK293T cells (n = 4). N, dual luciferase reporter assay for analyzing the effect of KLF5 on truncated RNF90 promoter…

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  Figure 2. RNF90 aggravates hepatic steatosis in vitro. A, RT-qPCR (n = 4) and western blot (n = 3) analyses of RNF90 mRNA and protein expression in indicated HepG2 stable cell lines. B–C, Oil red O staining (B) and TG content measurement (C) in BSA- or POA-treated RNF90-modified HepG2 stable cell lines (n = 4). D–E, validation of RNF90 knockdown efficiency at the mRNA (D, n = 4) and protein levels (E, n = 3) in AML12 cells stably expressing shRNF90 or control shRNA. F–G, Oil red O staining (F) and TG content measurement (G) in BSA- or POA-treated AML12 stable cell lines with stable RNF90 knockdown (n = 4). H–I, RT-qPCR (H, n = 4) and western blot (I, n = 3) analyses of RNF90 mRNA and protein expression in HepG2 and AML12 cells stably overexpressing RNF90 wildtype or its E3 ligase-deficient mutant (RNF90-DM). J–K, Oil red O staining (J) and TG content detection (K) in BSA- or POA-treated HepG2/AML12 cells stably overexpressing RNF90 wildtype or RNF90-DM cells (n = 4). Data are presented as the mean ± SD, and statistical significance was analyzed by one-way ANOVA followed by Tukey’s post hoc test (A, C, D, G, H, and K). The exact p values are indicated in each graph. BSA, bovine serum…

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  Figure 3. RNF90 interacts with CPT1α and promotes its K48-linked ubiquitination. A, mass spectral information of RNF90 and its interacting protein CPT1α from the RNF90-IP/MS assay. B, representative immunofluorescence staining images showing the subcellular localization of RNF90 and CPT1α protein in L02 cells. C, quantitative analysis of the colocalization between RNF90 and CPT1α in the indicated regions from panel B. D–E, co-IP analysis to detect the interaction between RNF90 and CPT1α proteins using exogenous-specific antibodies (D) and endogenous-specific antibodies (E) in HEK293T cells. F–G, measurement of CPT1α ubiquitination levels in HEK293T cells cotransfected with Myc-Ub, HA-CPT1α, and either Flag-tagged RNF90 wildtype or its E3 ligase deficiency mutant plasmids (F) or with Myc-Ub, HA-CPT1α, and RNF90 knockdown plasmids (G). H–I, analysis of the ubiquitination type of CPT1α catalyzed by RNF90 in HEK293T cells cotransfected with HA- CPT1α, Flag-RNF90, and the indicated Myc-Ub-K48 mutants (H) or Myc-Ub-K63 mutants (I). J, detection of the ubiquitination sites of CPT1α in HEK293T cells cotransfected with Myc-Ub, Flag-RNF90 and the indicated HA-CPT1α mutant plasmids. co-IP,…

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  Figure 4. RNF90 regulates CPT1α stability in an E3 ligase activity-dependent manner. A, immunoblotting analysis of CPT1α protein expression in HEK293T cells cotransfected with HA-CPT1α and increasing concentrations of Flag-tagged RNF90 wildtype (RNF90-WT) or its E3 ligase-deficient mutant (RNF90-DM) plasmids. B–D, immunoblotting (B, C) and densitometric quantification (D) analyses showing the effect of RNF90-WT or RNF90-DM on CPT1α protein half-life by CHX chase assay. E, immunoblotting analysis of CPT1α protein expression in HEK293T cells cotransfected with increasing amounts of RNF90 plasmid and either HA-tagged CPT1α-WT or HA-tagged ubiquitination-deficient CPT1α mutant (CPT1α-K195R) plasmids. F–G, immunoblotting (F) and quantification (G) analyses of CPT1α-WT or CPT1α-K195R protein half-life as regulated by RNF90 in HEK293T cells. H–I, immunoblotting (H) and quantification (I) analyses of RNF90 knockdown on CPT1α protein half-life in CHX-treated HEK293T cells. J, immunoblotting analysis to delineate the degradation pathway of CPT1α protein in HEK293T cells transfected with the indicated plasmids, and subsequently treated with CHX alone or in combination with MG132, CQ, or NH4Cl prior…

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  Figure 5. Hepatic RNF90 deficiency alleviates diet-induced hepatic steatosis in mice by enhancing FAO. A–C, body weight (A), liver weight (B), liver weight-to-body weight ratio (C) in RNF90-HKO and RNF90-Flox mice at the indicated time points after feeding with NCD or HFD (n = 8). D–E, representative images of H&E staining and Oil red O staining (D), and quantitative analysis of Oil red O positive area (E) in liver tissues from RNF90-HKO and RNF90-Flox mice fed a NCD or HFD for 14 weeks (n = 5). F, measurement of hepatic TG, TC contents in RNF90-HKO and RNF90-Flox mice after 14 weeks of HFD feeding (n = 8). G–H, representative images of H&E and Oil red O staining (G), and quantitative analysis of Oil red O-stained positive area (H) in liver tissues from RNF90-HKO and RNF90-Flox mice fed a NCD or HFHC for 19 weeks (n = 5). I–J, detection of hepatic TG (I) and TC (J) contents in RNF90-HKO and RNF90-Flox mice after 19 weeks of HFHC feeding (n = 8). K, immunoblotting analysis of CPT1α protein expression in liver tissues from RNF90-HKO and RNF90-Flox mice fed a HFD for 14 weeks or HFHC for 19 weeks (n = 4). L, detection of FAO activity in liver tissues from RNF90-HKO and RNF90-Flox mice fed a…

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  Figure 6. Hepatic RNF90 overexpression suppresses FAO and exacerbates hepatic lipid accumulation. A, representative EGFP fluorescence images of freshly isolated liver tissues from mice injected with AAV8 using a small animal imaging device. B–C, immunoblotting (B) and densitometric quantification (C) of RNF90, CPT1α protein expression in liver tissues from HFD- or HFHC-fed mice injected with AAV8. β-actin was used as control, and relative protein levels were quantified by normalizing target band intensities to β-actin using Image J software (n = 4). D–E, representative images of H&E staining and Oil red O staining (D), and quantitative analysis of Oil red O-stained positive area (E) in liver tissues from AAV8-injected mice fed a HFD (14 weeks) or HFHC (19 weeks) (n = 5). F–G, measurements of liver TG, TC contents (F), and FAO activity (G) in AAV8-injected mice fed a HFD (14 weeks) or HFHC (19 weeks) (n = 8). H, RT-qPCR analysis of HMGCS2 mRNA expression in liver tissues from HFD- or HFHC-fed mice injected with AAV8. I–J, measurement of liver β-HB (I) and ATP (J) contents in AAV8-injected mice fed a HFD (14 weeks) or HFHC (19 weeks) (n = 8). Statistical significance was analyzed by one-way…

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  Figure 7. CPT1α is essential for RNF90-mediated regulation of FAO and steatosis in hepatocytes. A, HepG2 cells stably overexpressing Flag-tagged RNF90 were transfected with either HA-tagged CPT1α plasmid or empty control plasmid. At 12 h post transfection, cells were treated with POA for 24 h, then harvested for immunoblotting analysis of Flag-tagged RNF90 and HA-tagged CPT1α protein expression, with β-actin as the loading control. B, measurement of FAO activity in HepG2 cells treated as described in (A) (n = 4). C–D, representative Oil red O staining images, quantitative analysis of Oil red O-positive area (C), and TG content quantification (D) in HepG2 cells treated as described in (A) (n = 4). E, HepG2-shRNF90-3# cells were transfected with shCPT1α plasmid or empty control plasmid. After 12 h of transfection, cells were treated with POA for 24 h, then harvested for immunoblotting analysis of RNF90 and CPT1α protein expression, with β-actin as the loading control. F, measurement of FAO activity in HepG2 cells treated as described in (E) (n = 4). G–H, representative Oil red O staining images, quantitative analysis of Oil red O-positive area (G) and TG content quantification (H) in HepG2…