DNAJB4 Protects Against Atherosclerosis by Stabilizing the HSP70-LXRα Axis in Hepatic Lipid Metabolism

Figures

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  Figure 1. Distribution of DNAJB4 in the liver of wild-type (WT) mice. Five-month-old male wild-type (WT) C57BL/6 mice fed with a regular chow diet were sacrificed. Immunohistochemistry of DNAJB4 (green) and (A) albumin (red), (B) vWF (red), and (C) F4/80 (red) in the liver of WT mice. The nucleus was stained with DAPI. The arrows indicated the expression of DNAJB4 in the nucleus.

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  Figure 2. DNAJB4 protein expression is decreased in the liver of apoe-/- mice. Livers from five-month-old male wild-type (WT) C57BL/6 mice and apoe-/- mice fed with a regular chow diet were extracted, lysed, or harvested for histological analysis. (A) Western blot analysis of DNAJB4 and β-actin in livers. (B) Immunohistochemistry of DNAJB4 (green) in the liver sections of five-month-old apoe-/- and WT mice. The nucleus was stained with DAPI. Data are expressed as mean ± SEM from five mice (n=5). *p < 0.05 vs. WT mice.

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  Figure 3. Genetic deletion of dnajb4 increases hepatic lipid accumulation in apoe-/- mice. The apoe-/-dnajb4-/- mice were generated by cross-breeding apoe-/- and dnajb4-/- mice. Livers from five-month-old male apoe-/- mice and apoe-/-dnajb4-/- mice fed with a regular chow diet were extracted for hepatic lipid analysis or harvested for histological analysis. (A and B) Representative histological images were obtained using (A) hematoxylin and eosin (H&E) staining and (B) oil red O staining. (C) The hepatic levels of triglyceride, fatty acid, glycerol, total cholesterol, free cholesterol, cholesterol ester, and bile acid. Data are expressed as mean ± SEM from five mice (n=5). *p < 0.05 vs. the apoe-/- mice.

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  Figure 4. Genetic deletion of dnajb4 alters the protein expression in the livers of apoe-/- mice. The apoe-/-dnajb4-/- mice were generated by cross-breeding apoe-/- and dnajb4-/- mice. The livers were extracted from five-month-old male apoe-/- and apoe-/-dnajb4-/- mice fed a regular chow diet and then lysed. (A) Proteomic analysis using LC-MS/MS identified altered proteins in the livers of apoe-/-dnajb4-/- mice compared to apoe-/- mice. (B) The ten selected canonical pathways related to lipid metabolism were identified using Ingenuity Pathway Analysis (IPA, 76765844M). Pathway significance was determined using -log(p-value), and pathways with -log(p-value) > 1.3 were considered statistically significant. (C) The protein-protein interaction (PPI) network was analyzed using the STRING database.

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  Figure 5. Genetic deletion of dnajb4 impairs hepatic cholesterol metabolism in apoe-/- mice. The apoe-/-dnajb4-/- mice were generated by cross-breeding apoe-/- and dnajb4-/- mice. The livers were extracted from five-month-old male apoe-/- and apoe-/-dnajb4-/- mice fed a regular chow diet and then lysed. (A and B) Western blot analysis of LRP1, SR-B1, LDLR, LAL, ACAT2, nCEH, CYP7A1, ABCG5, ABCG8, ABCA1, ABCG1, LXRα, and β-actin in livers of apoe-/- mice and apoe-/-dnajb4-/- mice. Data are expressed as the mean ± SEM from five mice (n=5). *p < 0.05 vs. the apoe-/- mice.

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  Figure 6. The genetic deletion of dnajb4 decreases LXRα gene expression and promotes LXRα protein degradation. Livers and primary hepatocytes from five-month-old male wild-type (WT) C57BL/6 mice, apoe-/- mice, and apoe-/-dnajb4-/- mice fed a regular chow diet were isolated. (A) Representative confocal microscopy images of a proximity ligation assay (PLA) were obtained using anti-LXRα and anti-DNAJB4 antibodies. The nucleus was stained with DAPI, and PLA signals (green) were observed. (B) Co-immunoprecipitation assay of the interaction among HSP70, DNAJB4, and LXRα in the liver of WT mice. (C) Western blot analysis of LXRα and DNAJB4 in the nuclear and cytoplasmic fractions of Huh7 cells after transfection with scramble or DNAJB4 siRNA. (D) The mRNA expression of LXRα in the liver of WT and dnajb4-/- mice. (E) Western blot analysis of LXRα in primary hepatocytes isolated from WT and dnajb4-/- mice was performed after treatment with cycloheximide (20 μg/ml) at the indicated times (0, 2, 4, 6, and 8 h). (F) Western blot analysis of HSP70, LXRα, and DNAJB4 in Huh7 cells transfected with scramble or DNAJB4 siRNA, followed by treatment with or without proteasome inhibitor MG-132 (1 μM) for 12 h.…

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  Figure 7. Genetic deletion of dnajb4 reduces nuclear HSP70 association and HSP70-regulated LXRα gene expression. The livers or primary hepatocytes from five-month-old male WT mice, dnajb4-/- mice, apoe-/- mice, and apoe-/-dnajb4-/- mice fed with a regular chow diet were extracted or isolated. (A) Western blot analysis of HSP70 in the livers of apoe-/- mice and apoe-/-dnajb4-/- mice. (B) Western blot analysis of HSP70 in primary hepatocytes isolated from WT mice and dnajb4-/- mice were treated with cycloheximide (20 µg/ml) at the indicated times (0, 2, 4, 6, and 8 h). (C) Representative confocal microscopy images and quantified fluorescence intensity of HSP70 in the livers of apoe-/- mice and apoe-/-dnajb4-/- mice. The nuclear and cytoplasmic fluorescence intensities were quantified using ImageJ, and the nuclear regions were defined by DAPI staining. Chromatin immunoprecipitation (ChIP) with anti-HSP70 antibody followed by qRT-PCR of the LXRα promoter in (D) Huh7 cell line transfected with scramble siRNA or DNAJB4 siRNA and in (E) primary hepatocytes from apoe-/- mice and apoe-/-dnajb4-/- mice. Data are expressed as the mean ± SEM from five mice (n=5). *p < 0.05 vs. the WT or apoe-/- mice.

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  Figure 8. DNAJB4 is required for the protective effect of curcumin in hepatic cholesterol metabolism in apoe-/- mice. Four-month-old male apoe-/- mice and apoe-/-dnajb4-/- mice fed with a regular chow diet were orally administered with curcumin (20 mg/kg) or vehicle (PBS) for four weeks. Mouse livers were extracted and lysed. (A and B) Western blot analysis of protein levels of DNAJB4, HSP70, LXRα, CYP7A1, ABCG5, ABCG8, ABCA1 and β-actin in the livers of apoe-/- mice and apoe-/-dnajb4-/- mice. Data are expressed as the mean ± SEM from five mice (n=5). * p < 0.05 vs. the vehicle group. # p < 0.05 vs. the curcumin group in apoe-/- mice.

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  Figure 9. DNAJB4 is required for the protective effect of andrographolide in hepatic cholesterol metabolism in apoe-/- mice. Four-month-old male apoe-/- mice and apoe-/-dnajb4-/- mice fed with a regular chow diet were orally administered with andrographolide (20 mg/kg) or vehicle (PBS) for four weeks. Mouse livers were extracted and lysed. (A and B) Western blot analysis of DNAJB4, HSP70, LXRα, CYP7A1, ABCG5, ABCG8, ABCA1 and β-actin in the livers of apoe-/- mice and apoe-/-dnajb4-/- mice. Data are expressed as the mean ± SEM from five mice (n=5). *p < 0.05 vs. the vehicle group. #p < 0.05 vs. the andrographolide group in apoe-/- mice.

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  Figure 10. The beneficial effects of curcumin or andrographolide in hepatic cholesterol accumulation in apoe-/- mice are partially dependent on DNAJB4. Four-month-old male apoe-/- mice and apoe-/-dnajb4-/- mice fed with a regular chow diet were orally administered with curcumin (20 mg/kg), andrographolide (20 mg/kg), or vehicle (PBS) for four weeks. Mouse livers were extracted and lysed. (A) Representative histological images, as shown by Oil Red O staining, of the liver tissues. (B) The hepatic levels of triglyceride, fatty acid, glycerol, total cholesterol, free cholesterol, cholesterol ester, and bile acid. Data are expressed as mean ± SEM from five mice (n=5). *p < 0.05 vs. the vehicle group in apoe-/- mice.

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  Figure 11. The genetic deletion of dnajb4 attenuated the protective effects of curcumin and andrographolide against atherosclerosis progression. Four-month-old male apoe-/- mice and apoe-/-dnajb4-/- mice fed with a regular chow diet were orally administered with curcumin (20 mg/kg), andrographolide (20 mg/kg), or vehicle (PBS) for four weeks. The hearts and plasma of the mice were collected. (A) Representative images and quantifications of atherosclerotic lesions at the aortic roots were obtained after H&E staining and analysis. (B) Plasma levels of cholesterol, high-density lipoprotein cholesterol (HDL-c), non-HDL-c, triglyceride, TNF-α, IL-1β, IL-6, MCP-1, and MIP-2. Data are expressed as mean ± SEM from four to five mice (n=4-5). * p < 0.05 vs. the vehicle group in apoe-/- mice. # p < 0.05 vs. the vehicle group in apoe-/-dnajb4-/- mice.

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  Figure 12. Schematic illustration of the proposed molecular mechanisms by which the genetic deletion of DNAJB4 disturbs hepatic lipid metabolism and exacerbates hyperlipidemia-induced atherosclerosis by impairing HSP70-LXRα signaling. In the context of dnajb4 deficiency, the sequence of events is illustrated as follows: (1) The disruption of the DNAJB4-HSP70 leads to decreased protein stability of HSP70 and LXRα. (2) This results in reduced nuclear HSP70 association and diminished binding of HSP70 to the LXRα promoter. (3) Consequently, the transcription of LXRα and its downstream target genes involved in cholesterol metabolism is significantly downregulated. (4) These transcriptional alterations disrupt hepatic cholesterol homeostasis, leading to altered lipoprotein receptor expression and impaired cholesterol clearance. (5) Ultimately, this cascade of molecular dysregulation results in the aggravation of hepatic lipid accumulation, hyperlipidemia, and the progression of atherosclerotic lesions.