Tissue specific role of ABCA1 in lung cholesterol homeostasis under high-cholesterol diet

Abstract

Background: ATP-binding cassette subfamily A1 (ABCA1) and sterol 27-hydroxylase (CYP27A1) are essential regulators of cholesterol metabolism. However, their tissue-specific roles, particularly in the lung, under high-cholesterol diet (HCD) conditions remain unclear.

Objective: Using the liver as a reference, this study aimed to investigate the tissue-specific regulation of ABCA1 in the lung under HCD or CYP27A1 knockout (KO) conditions, and to explore its potential regulatory mechanism.

Methods: CYP27A1 KO and wild-type (WT) mice on a C57BL/6J background were fed either a normal diet (ND) or HCD for 12 weeks. Transcriptome sequencing (RNA-seq) was conducted on lung tissue samples.

Results: HCD feeding in WT mice caused significant hepatic lipid accumulation, while no notable lipid deposition was observed in lung tissue. ABCA1 and CYP27A1 expression were downregulated in the liver but upregulated in the lung. In CYP27A1 ^(-/-) mice, hepatic lipid accumulation was more severe with further suppression of ABCA1, whereas ABCA1 expression in the lung remained elevated. Transcriptome analysis revealed that upregulated genes in lung tissue were significantly enriched in the inflammation-related nuclear factor kappa-B (NF-κB) signaling pathway. Furthermore, experiments confirmed that the expression of NF-κB pathway was consistent with the upregulation of ABCA1.

Conclusion: ABCA1 exhibits marked tissue specificity under HCD feeding or CYP27A1 KO conditions. In the liver, ABCA1 downregulation may exacerbate cholesterol metabolic imbalance, while its upregulation in the lung may play an important role in maintaining cholesterol homeostasis. Moreover, the increase in pulmonary ABCA1 expression in CYP27A1 KO mice may be associated with activation of the NF-κB signaling pathway.

Keywords: ABCA1; cholesterol homeostasis; high-cholesterol diet; lung metabolism; tissue specificity.

Figure 1

Tissue specific expression of CYP27A1 and ABCA1 under HCD. (A) Modeling schematic diagram of CYP27A1^(−/−) and WT mice with C57BL/6J background receiving ND and HCD for 12 weeks, respectively. (B) ORO staining of liver and lung tissues in WT mice fed with ND or HCD, respectively. (C) Protein levels of ABCA1, LDLR, HMGCR, and CYP27A1 in liver tissues. (D) Relative mRNA levels of ABCA1, LDLR, HMGCR, and CYP27A1 in liver tissues. (E) Protein levels of ABCA1, LDLR, HMGCR, and CYP27A1 in lung tissues. (F) Relative mRNA levels of ABCA1, LDLR, HMGCR, and CYP27A1 in lung tissues. n = 3–5 mice/group. Data are presented as mean ± SEM. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 2

Effects of HCD and CYP27A1 KO on body weight, organ ratio, and blood lipids in C57BL/6J mice. (A) Body weight growth curves of mice fed with ND and HCD. And body weights of mice measured once a week. (B) Organ ratio of mice in four groups. (C) Levels of total TC, TG, LDL, and HDL in serum. n = 7–10 mice/group. Data are presented as mean ± SEM. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 3

Specific effect of HCD and CYP27A1 KO on lipid accumulation in liver and lung tissues. (A) Levels of total TC, TG in liver tissues. (B) Levels of total TC, TG in lung tissues. (C) HE staining of liver tissues of CYP27A1^(−/−) and WT mice receiving ND and HCD, respectively. (D) HE staining of lung tissues of CYP27A1^(−/−) and WT mice receiving ND and HCD, respectively. (E) ORO staining of liver tissue in CYP27A1^(−/−) and WT mice receiving ND and HCD, respectively. (F) ORO staining of lung tissue in CYP27A1^(−/−) and WT mice receiving ND and HCD, respectively. Scale bars = 20 μm. n = 3–5mice/group. Data are presented as mean ± SEM. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 4

Differential expression of cholesterol metabolism-related proteins and ABCA1 in liver and lung tissues. (A) Protein levels of ABCA1, LDLR, HMGCR, and CYP27A1 in liver tissues. (B) Protein levels of ABCA1, LDLR, HMGCR, and CYP27A1 in lung tissues. (C) Relative mRNA levels of ABCA1, LDLR, HMGCR, and CYP27A1 in liver tissues. (D) Relative mRNA levels of ABCA1, LDLR, HMGCR, and CYP27A1 in lung tissues. (E) Immunohistochemical staining of ABCA1 in liver tissue with CYP27A1^(−/−) and WT mice receiving ND and HCD, respectively. (F) Immunohistochemical staining of CYP27A1 in liver tissue with CYP27A1^(−/−) and WT mice receiving ND and HCD, respectively. (G) Immunohistochemical staining of ABCA1 in lung tissue with CYP27A1^(−/−) and WT mice receiving ND and HCD, respectively. (H) Immunohistochemical staining of CYP27A1 in lung tissue with CYP27A1^(−/−) and WT mice receiving ND and HCD, respectively. Scale bars = 20 μm. n = 3–5 mice/group. Data are presented as mean ± SEM. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 5

Subcellular localization of ABCA1 in lung tissue. (A) Co-staining of ABCA1 (red) and SFTPC (green, alveolar type II marker); nuclei stained with DAPI (blue). (B) Co-staining of ABCA1 (red) and CD68 (green, macrophage marker) to determine cell-type-specific expression. n = 3–5 mice/group.

Figure 6

Preliminary exploration of the mechanism of ABCA1 upregulation in lung tissue. (A) The number of differentially expressed genes compared between two groups. (B,E) The differential genes between WTND vs. WTHCD, KOND vs. KOHCD were analyzed by Venn analysis, resulting in 288 common differential genes and gene heatmaps genes. (C,D) Volcanic diagram of differentially expressed genes in the lungs of WTND vs. WTHCD, KOND vs. KOHCD mice. The color represents p < 0.05 and the fold change >1 (red), p < 0.05, the fold change is <1 (green) and not significant (blue). (F,G) The top 25 signaling pathways in KEGG enrichment analysis. (H) Protein levels of p-NFκB, NFκB in lung tissues. (I) Relative mRNA levels of TNF-α, IL-1β, and IL-10 in lung tissues. n = 3–5 mice/group. Data are presented as mean ± SEM. ^*p < 0.05 and ^**p < 0.01.