TFEB SUMOylation in macrophages accelerates atherosclerosis by promoting the formation of foam cells through inhibiting lysosomal activity

Abstract

Atherosclerosis (AS) is a serious cardiovascular disease. One of its hallmarks is hyperlipidemia. Inhibiting the formation of macrophage foam cells is critical for alleviating AS. Transcription factor EB (TFEB) can limit the formation of macrophage foam cells by upregulating lysosomal activity. We examined whether TFEB SUMOylation is involved in this progress during AS. In this study, we investigated the role of TFEB SUMOylation in macrophages in AS using TFEB SUMOylation deficiency Ldlr^−/− (TFEB-KR: Ldlr^−/−) transgenic mice and TFEB-KR bone marrow-derived macrophages. We observed that TFEB-KR: Ldlr^−/− atherosclerotic mice had thinner plaques and macrophages with higher lysosomal activity when compared to WT: Ldlr^−/− mice. TFEB SUMOylation in macrophages decreased after oxidized low-density lipoprotein (OxLDL) treatment in vitro. Compared with wild type macrophages, TFEB-KR macrophages exhibited less lipid deposition after OxLDL treatment. Our study demonstrated that in AS, deSUMOylation of TFEB could inhibit the formation of macrophage foam cells through enhancing lysosomal biogenesis and autophagy, further reducing the accumulation of lipids in macrophages, and ultimately alleviating the development of AS. Thus, TFEB SUMOylation can be a switch to modulate macrophage foam cells formation and used as a potential target for AS therapy.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-023-04981-8.

Introduction

Atherosclerosis (AS) is the most common cardiovascular disease in clinic. Hypercholesterolemia is a major risk factor for AS [1–4]. With the continuous improvement in living conditions, the incidence of AS has increased. Therefore, it is very important to study the pathogenesis of AS and explore potential therapeutic targets.

Macrophages play an important role in the early stage of AS. Recruited and differentiated macrophages can remove lipids deposited under vascular endothelium via lysosomal system, which is an important process to maintain the homeostasis of blood lipids [5, 6]. If macrophages take up too much lipids, it results in foam-like lipid formation in cytoplasm. These cells are called macrophage foam cells [7, 8]. We can slow down the formation of macrophage foam cells by enhancing their ability to process lipids and ultimately achieve the inhibition of AS.

Small ubiquitin-related modifiers (SUMOs) are a member of ubiquitin-like proteins [9]. Through a series of enzymatic reactions, SUMOs covalently bind to the substrate proteins and subsequently alter their functions. This post-translational modification is known as SUMOylation [10–12]. Many proteins can undergo SUMOylation. SUMOylation plays an important role in many aspects, such as regulating transcriptional activity, intracellular signal transduction, ensuring normal cell division, and maintaining the stability and integrity of genome [13].

As a transcription factor, transcription factor EB (TFEB) plays an important role in regulating lysosomal biogenesis and autophagy [14–17]. Several studies have considered TFEB as a valuable target for AS therapy [18–20]. According to recent reports, macrophages can metabolize cholesterol in cells using lysosomal autophagy system and finally reduce the content of excess cholesterol in cells [21, 22]. In the AS model, the accumulation of atherosclerotic plaque in mice with TFEB conditionally overexpressed in macrophages was less and the content of proinflammatory factor interleukin-1β (IL-1β) in serum was also lower than that in wild type mice [5]. Therefore, TFEB is considered as a protective factor in the occurrence and development of atherosclerotic diseases. Although TFEB has been reported to be a SUMOylated protein [23], the function of TFEB SUMOylation in the development of AS remains unclear. In this study, we identified that TFEB SUMOylation was decreased after the stimulation of oxidized low-density lipoprotein (OxLDL) in macrophages, and TFEB-KR: Ldlr^−/− mice exhibited milder atherosclerotic lesions with high cholesterol diet. Our study demonstrated that TFEB SUMOylation promoted the formation of macrophage foam cells by inhibiting lysosomal biogenesis and autophagy and eventually aggravated the development of AS. This indicated that targeting TFEB SUMOylation is a potential therapeutic strategy for AS.

Materials and methods

Plasmids and antibodies

GFP-TFEB, Flag-TFEB, HA-SSRP1, His-SUPT16H, SUMO1 were generated using standard cloning procedures (Vazyme Biotech Co., Ltd). GFP-TFEB-K361R and Flag-TFEB-K361R were generated using site-directed mutagenesis (Strategene). Flag-SUMO1, HA-SUMO1 were gifts from Cheng laboratory. Antibodies against FLAG-M2, HA, and LC3 were purchased from Sigma, TFEB from Protein Tech, SUMO1 and LAMP1 from Abcam, ACTIN and His from Cell Signaling Technology.

Animal models

Animal studies were approved by the Animal Care and Use Committee from Xinhua Hospital, Affiliated to Medicine School of Shanghai Jiaotong University. Both TFEB-KR and Ldlr^−/− mice were C57BL/6 background mice. Ldlr^−/− mice were gifted from Professor Cheng's laboratory and TFEB-KR mice were generated by CRISPR/Cas9 gene targeting technology at Shanghai Bangyao Biotechnology Co. We used littermate derived, sex, age, and genetic background-matched mice in our experiments. TFEB-KR; Ldlr^−/− mice were created by crossing mating TFEB-KR with Ldlr^−/− mice to yield a double homozygote. Eight-weeks-old male littermates were used in the subsequent phenotype study. Atherosclerosis was induced by feeding 8-weeks-old WT: Ldlr^−/− and TFEB-KR: Ldlr^−/− littermates with a Western diet (Research diet) for 12 weeks.

Serum lipids analyses

The retro-orbital plexus method was used to obtain blood samples. Serum levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were analyzed with both closed and open channel chemistry reagents on a LABOSPECT 008AS platform (Hitachi High-Tech Co., Tokyo, Japan).

Bone marrow transplantation

Eight-weeks-old male recipient mice were lethally irradiated with 900 rads irradiation on the day of transplantation. Bone marrow cells from the donor Ldlr^−/− mice or Ldlr^−/− mice with the whole body TFEB SUMOylation deficiency (TFEB-KR; Ldlr^−/−) were isolated and injected into each recipient mouse intravenously. Four weeks later, the mice were fed with a Western diet for 12 weeks and subjected for atherosclerotic lesion analysis.

Histology

To determine lesion size, cryosections (8 μm) of the aortic root were stained with Oil Red O (Sigma). Corresponding sections on separate slides were also stained for monocyte/ macrophage content using a monoclonal rat MAC-3 antibody (BD), and lysosome using a rabbit polyclonal LAMP1 antibody (Abcam).

Cell culture

BMDMs were differentiated with M-CSF for 7 days. BMDMs, 293T and RAW264.7 cells were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum (Invitrogen) and 1% antibiotics (penicillin/streptomycin) (Invitrogen).

Immunoprecipitation and immunoblotting

BMDMs or transfected cells were lysed in radioimmune precipitation assay buffer (50 mM Tris–HCl (pH 7.4), 400 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate, and a mixture of protease inhibitors) and cleared by centrifugation. Cleared cell lysates were incubated with 10 μl of anti-FLAG M2-agarose affinity gel (Sigma) or 2 μl of anti-SUMO1 (Abcam) with 10 μl of protein A/G (Sigma) for 2 h. After extensive washing, beads were boiled at 100 °C for 10 min. Proteins were resolved by SDS-PAGE and transferred onto PVDF membranes (Millipore), followed by immunoblotting using corresponding antibodies according to the instructions of the manufacturer. Immunoblots were analyzed using the LAS-4000 system (Fujifilm).

Bodipy labeling

Post OxLDL treatment, the cells were washed with PBS, and BODIPY (2 μM, Life Technologies, D3922) was added to the cells for 30 min at 37 °C. After washing with PBS, cells were mounted with Vectashield containing 40, 6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, MA, USA).

RNA isolation and qPCR

Total RNA was isolated from cells using Tripure isolation reagent (Roche). For mRNA analysis, an aliquot containing 2 μg of total RNA was reverse-transcribed using the cDNA synthesis kit (Takara). Real-time PCR was performed using SYBR Green PCR master mix (Applied Biosystems) and detected by the ABI Prism 7500 sequence detection system (Applied Biosystems). The primers for related genes are as follows:

• CLCN7:

  • Forward Primer-CGCCAGTCTCATTCTGCACT,
  • Reverse Primer-GAGGATCGACTTCCGGGTC;
• LAMP1:

  • Forward Primer-CAGCACTCTTTGAGGTGAAAAAC,
  • Reverse Primer-CCATTCGCAGTCTCGTAGGTG;
• CTSB:

  • Forward Primer-CAGGCTGGACGCAACTTCTAC,
  • Reverse Primer-TCACCGAACGCAAC CCTTC;
• CTSD:

  • Forward Primer-GCTTCCGGTCTTTGACAACCT,
  • Reverse Primer-CACCAAGCATTAGTTCTCCTCC;
• ATG4b:

  • Forward Primer-CGGCACTTAGGTCGAGATTGG,
  • Reverse Primer-ACTCCCATTTGCGCTATCTGA;
• ATG4d:

  • Forward Primer-AGGGGACAAACCCGTATCC,
  • Reverse Primer-CCATACTTGACGTTGTTCCAGG;
• ATG5:

  • Forward Primer-TGTGCTTCGAGATGTGTGGTT,
  • Reverse Primer-ACCAACGTCAAATAGCTGACTC;
• ATG9a:

  • Forward Primer-CCGAGGGGAGCAAATCACC,
  • Reverse Primer-TAGTCCACACAGCTAACCAGG;
• CD36:

  • Forward Primer-ATGGGCTGTGATCGGAACTG,
  • Reverse Primer-TTTGCCACGTCATCTGGGTTT;
• SCARB1:

  • Forward Primer-TTTGGAGTGGTAGTAAAAAGGGC,
  • Reverse Primer-TGACATCAGGGACTCAGAGTAG;
• MSR1:

  • Forward Primer-TGGAGGAGAGAATCGAAAGCA,
  • Reverse Primer-CTGGACTGACGAAATCAAGGAA;
• ABCG1:

  • Forward Primer-GTGGATGAGGTTGAGACAGACC,
  • Reverse Primer-CCTCGGGTACAGAGTAGGAAAG.

ChIP-qPCR assay

WT and TFEB-KR BMDMs were stimulated with OxLDL for 16 h before crosslinking with 1% formaldehyde, and sonicated. Solubilized chromatin was immunoprecipitated with anti-TFEB antibody (Proteintech), washed, and then eluted. After crosslink reversal and proteinase K treatment, immunoprecipitated DNA was extracted with phenol–chloroform, ethanol precipitated. The DNA fragments were further analyzed by qPCR. The specific primers used to amplify Mcoln1 promoter region was as followed:

• Forward primer-AGGGGCTCTGGGCTACC;

• Reverse primer-GCCCGCCGCTGTC ACTG.

Flow cytometry

Cells were resuspended in 200 ml FACS buffer (PBS with 5% bovine calf serum) and placed on ice for 15 min to block. Cells were incubated with fluorescently labeled antibodies on ice in the dark for 20 min and then washed with PBS, centrifuged at 500×g for 5 min, resuspended in 200 ml FACS buffer, and evaluated on a FACSCanto (Becton Dickinson) flow cytometer. Data were further analyzed using FlowJo software (Tree Star).

Protein digestion

100 μg of protein per condition was transferred into a new Eppendorf tube and the final volume was adjusted to 100 μl with 8 M urea. 2 μl of 0.5 M TCEP was added and the sample was incubated at 37 °C for 1 h, and then 4 μl of 1 M iodoacetamide was added to the sample and the incubation was last for 40 min protected from light at room temperature. After that, five volumes of − 20 °C pre-chilled acetone was added to precipitate the proteins overnight at − 20 °C. The precipitates were washed by 1 ml pre-chilled 90% acetone aqueous solution for twice and then re-dissolved in 100 μl 100 mM TEAB. Sequence grade modified trypsin (Promega, Madison, WI) was added at the ratio of 1:50 (enzyme: protein, weight: weight) to digest the proteins at 37 °C overnight. The peptide mixture was desalted by C18 ZipTip and then lyophilized by SpeedVac.

Nano-HPLC–MS/MS analysis

The sample was analyzed by online nanospray LC–MS/MS on Orbitrap Fusion™ mass spectrometer (Thermo Fisher Scientific, MA, USA) coupled to an EASY-nanoLC 1000 system (Thermo Fisher Scientific, MA, USA). 1 μl peptide was loaded (analytical column: Acclaim PepMap C18, 75 μm × 25 cm) and separated with a 60 min gradient. The column flow rate was maintained at 600 nL/min with the column temperature of 40 °C. The electrospray voltage of 2 kV versus the inlet of the mass spectrometer was used. The chromatographic gradient is shown below (A: 0.1% formic acid in water; 0.1% formic acid in CAN):

Time	Duration	Flow (nl/min)	Mixture (%B)
00:00	00:00	600	5
03:00	03:00	600	8
37:00	34:00	600	20
51:00	14:00	600	30
52:00	01:00	600	90
60:00	08:00	600	90

The mass spectrometer was run under data dependent acquisition mode, and automatically switched between MS and MS/MS mode. The parameters was: (1) MS: scan range (m/z) = 350–1550; resolution = 60,000; AGC target = 4e5; maximum injection time = 50 ms; include charge states = 2–6 (2) HCD-MS/MS: resolution = 15,000; isolation window = 1.6; AGC target = 5e4; maximum injection time = 120 ms; collision energy = 35.

Luciferase assay

RAW264.7 cells in a 24-well plate were transiently transfected with indicated plasmids through FuGENE 6 transfection reagent (FuGENE). The cells were incubated for 24 h and then OxLDL (50 μg/ml) was used to stimulate the cells for 8 h. Finally, Dual-Luciferase Reporter Assay System was used to assess the activity of the corresponding promoter (Promega). Lamp1-Luc, a LAMP1 promoter-luciferase construct, was performed as described previously [24].

Statistical analysis

Statistical analyses were performed with a 2-tailed unpaired Student’s t test. All data shown represent the results obtained from triplicated independent experiments with SEM (mean ± SD). The values of P < 0.05 were considered statistically significant.

Results

OxLDL stimulation reduced TFEB SUMOylation

TFEB has been reported to undergo SUMOylation and this modification has a key regulatory role in its transcriptional activity [23]. We confirmed this finding by co-transfecting TFEB and SUMO1 plasmids in 293T cells and observed that TFEB is indeed a SUMOylated protein (Fig. 1A). Simultaneously, we identified that the SUMOylation site of human TFEB was lysine 361, since TFEB SUMOylation band was completely disappeared when lysine 361 was mutated to arginine (K361R) (Fig. 1A). Although SUMOylation has proved a very important regulatory effect on the transcriptional activity of TFEB, the specific molecular mechanism under different stimulation conditions is not completely clear, including during macrophage foam cells formation.

Fig. 1.

OxLDL stimulation reduced TFEB SUMOylation. A Human TFEB was SUMOylated at K361. Cell lysates from 293T cells transfected with the indicated plasmids were immunoprecipitated with anti-Flag beads. The precipitated proteins or lysates were immunoblotted with anti-Flag or anti-TFEB antibodies. B Endogenous TFEB could be SUMOylated in bone marrow–derived macrophages. BMDMs were immunoprecipitated with anti-SUMO1 antibody. The precipitated proteins or lysates were immunoblotted with anti-SUMO1 or anti-TFEB antibodies. C TFEB SUMOylation was decreased after OxLDL treatment. BMDMs were treated with OxLDL (50 μg/ml) for 24 h. The alteration of TFEB SUMOylation was detected using immunoprecipitation; n = 3. D Using the BLAST function from the PubMed database, compare the DNA sequences of TFEB and identify the SUMOylation modification sites. E Construction of genetic mouse model with the whole body TFEB SUMOylation deficiency (TFEB-KR, K346R) and the results were confirmed through PCR sequencing

To detect the SUMOylation of endogenous TFEB, we performed immune co-precipitation assay in bone marrow-derived macrophages (BMDMs). A band at approximately 100 kDa reflecting endogenous TFEB SUMOylation was detected (Fig. 1B). WT and TFEB-KR BMDMs were confirmed by F4/80 and CD11b co-staining [25] (Fig. S1). To clarify the role of TFEB SUMOylation in AS, BMDMs were treated with OxLDL (50 μg/ml) for 24 h. The SUMOylation band of TFEB was significantly weakened after OxLDL stimulation (Fig. 1C). These results suggested that TFEB SUMOylation was involved in the process of AS. Through protein sequence alignment, we found that TFEB SUMOylation site is highly conserved across various species (Fig. S2A). To further study the role of TFEB SUMOylation in the development of AS in vivo, we mapped the TFEB SUMOylation site in mice (Fig. 1D) and constructed the TFEB SUMOylation deficiency mice (K346R) using CRISPR/Cas9 base editing technology. We named it TFEB-KR mice (Fig. 1E).

TFEB-KR: Ldlr^−/− mice exhibited milder atherosclerotic lesions with high cholesterol diet

Since TFEB-KR mice had whole body TFEB SUMOylation deficiency, we performed bone marrow transplantation experiments in these mice prior to feeding them with a high cholesterol diet for 12 weeks (Fig. S3). The results revealed that TFEB-KR: Ldlr^−/− mice exhibited less atherosclerotic plaques in the aorta when compared with the control mice (Fig. 2A). The ratio of atherosclerotic plaques area to total aortic area in WT: Ldlr^−/− mice was 20.1%, 19.3%, 26.0%, and 19.2% and that in TFEB-KR: Ldlr^−/− mice was 12.3%, 16.3%, 16.2%, and 14.1%. In addition, we observed that the average aorta lesion in TFEB-KR: Ldlr^−/− mice was approximately 30.3% less (Fig. 2B) than that in the control mice. Furthermore, TFEB-KR: Ldlr^−/− mice had thinner aortic sinus plaques and smaller atherosclerotic plaques (Fig. 2C). MAC-3 is a glycoprotein that is expressed on the surface of monocytes and macrophages [26, 27]. Lysosomal associated membrane protein 1 (LAMP1) is a type 1 transmembrane protein that exists on the lysosomal membrane and can indirectly reflect the activity of lysosomes [28, 29]. TFEB-KR: Ldlr^−/− mice exhibited stronger LAMP1 (red) but not MAC-3 (green) expression when compared with the control mice (Fig. 2D/E). These results demonstrated that the number of macrophages infiltrating into atherosclerotic plaques was comparable between the two groups. However, macrophages in atherosclerotic plaques from TFEB-KR: Ldlr^−/− mice exhibited higher lysosomal activity. Further, we measured the levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the serum collected from the two groups. The content of TG, TC, and LDL-C but not of HDL-C was slightly higher in TFEB-KR: Ldlr^−/− mice (Fig. 2H). This may be due to the higher lysosomal activity of macrophages in the plaques from TFEB-KR: Ldlr^−/− mice, which can decompose and metabolize more lipids accumulated under the vascular endothelium. Further, we monitored the body weight between the two groups. No significant difference existed in body weight between the two groups even in the presence of western diet (Fig. 2F). Moreover, the content of IL-1β, interleukin-4 (IL-4), and interleukin-6 (IL-6) was evaluated in the two groups from the AS model mice. TFEB-KR: Ldlr^−/− mice exhibited lower IL-1β and higher IL-4 levels even though without significant difference. However, no difference was observed in terms of IL-6 secretion level (Fig. 2G). Collectively, we identified that TFEB SUMOylation plays an important role in the process of AS, and TFEB-KR: Ldlr^−/− mice had stronger ability to resist AS.

Fig. 2.

TFEB-KR: Ldlr^−/− mice exhibited milder atherosclerotic lesions with high cholesterol diet. A Representative images of en face oil red O staining of aortas from WT: Ldlr^−/− and TFEB-KR: Ldlr^−/− littermates; n = 4. B Quantitative analysis of the ratio of lesion areas to total aortic areas in each mouse; n = 4. C Representative images of cross-sections of the aortic sinus stained with oil red O; n = 4. D The staining of Lamp1 in Mac-3-positive macrophages in lesions of WT: Ldlr^−/− and TFEB-KR: Ldlr^−/− mice; n = 4 (ZOOM 0.8). E The staining of Lamp1 in Mac-3-positive macrophages in lesions of WT: Ldlr^−/− and TFEB-KR: Ldlr^−/− mice; n = 4 (ZOOM 3). F Body weight of WT: Ldlr^−/− and TFEB-KR: Ldlr^−/− mice on a normal chow diet or a western diet; n = 8. G Detection of inflammatory factors amounts in WT: Ldlr^−/− and TFEB-KR: Ldlr^−/− mice after feeding western diet; n = 8. H Cholesterol levels, and lipoprotein profiles of WT: Ldlr^−/− and TFEB-KR: Ldlr^−/− mice on a normal chow diet or a western diet; n = 8

TFEB SUMOylation inhibited lysosomal biogenesis and activity

Lyso-Tracker is a fluorescent probe labeled with weak alkalinity; therefore, it can selectively stain acidic lysosomes [30, 31]. Upon OxLDL stimulation, TFEB-KR BMDMs exhibited stronger Lyso-Tracker and LAMP1 staining signals and formed larger vacuoles when compared with WT BMDMs (Fig. 3A/B). Further, quantitative analysis revealed higher MFI of Lyso-Tracker in TFEB-KR BMDMs after OxLDL treatment (Fig. 3C/D). Considering the impact of OxLDL on the acidity and alkalinity of the cellular environment, we also used other methods to detect the activity of lysosomes after OxLDL stimulation. Real-time quantitative PCR detection revealed that genes associated with lysosomal biogenesis (Clcn7, Lamp1, Ctsb, and Ctsd) and autophagy (Atg4b, Atg4d, Atg5 and Atg9a) were significantly upregulated in TFEB-KR BMDMs after OxLDL stimulation [32–36] (Fig. 3E). The activity of cathepsin B (CTSB) can reflect the function of lysosome [37]. We observed that CTSB activity was approximately 26.1% higher in TFEB-KR BMDMs after OxLDL treatment (Fig. 3G). LC3 is a class of microtubule associated protein, which plays a very important role in autophagy [38, 39]. We observed that the expression of LAMP1 and LC3 II was drastically increased in TFEB-KR BMDMs after OxLDL treatment (Fig. 3F, Fig. S2B). These results indicated that TFEB-KR macrophages had higher lysosomal activity than wild type macrophages after OxLDL treatment.

Fig. 3.

TFEB SUMOylation inhibited lysosomal biogenesis and activity. A Under OxLDL stimulation, Lyso-Tracker Red staining was more pronounced in TFEB-KR BMDMs. WT and TFEB-KR BMDMs were treated with OxLDL (50 μg/ml) for 24 h, followed by Lyso-Tracker Red staining; n = 3. B Under OxLDL stimulation, LAMP1 staining was more pronounced in TFEB-KR BMDMs. WT and TFEB-KR BMDMs were treated with OxLDL (50 μg/ml) for 24 h, followed by LAMP1 staining; n = 3. C Under OxLDL stimulation, Lyso-Tracker Red staining was more pronounced in TFEB-KR BMDMs. WT and TFEB-KR BMDMs were treated with OxLDL (50 μg/ml) for 24 h, followed by Lyso-Tracker Red staining, which was quantified using flow cytometry; n = 3. D Quantitative analysis of the Lyso-Tracker MFI after OxLDL treatment in WT and TFEB-KR BMDMs; n = 3. E Under OxLDL stimulation, the expression of TFEB target genes were increased more significantly in TFEB-KR BMDMs. WT and TFEB-KR BMDMs were treated with OxLDL (50 μg/ml) for 16 h and then relative mRNA levels of TFEB target genes were assessed by Real-time quantitative PCR; n = 3. F Under OxLDL stimulation, the protein levels of LAMP1 and LC3 were more significantly in TFEB-KR BMDMs. WT and TFEB-KR BMDMs were treated with OxLDL (50 μg/ml) for 24 h and then the cells lysates were immunoblotted with anti-LAMP1 / anti-LC3 or anti-Actin antibodies; n = 3. G Under OxLDL stimulation, the activity of cathepsin B was higher in TFEB-KR BMDMs. WT and TFEB-KR BMDMs were treated with OxLDL (50 μg/ml) for 24 h and then the activity of cathepsin B was measured; n = 3

TFEB-KR macrophages exhibited less lipid deposition upon OxLDL stimulation

BODIPY is used as a dye for natural oils and fats and a tracer for oils and other non-polar oils [40]. Oil red O and BODIPY staining revealed that lipid deposition was more pronounced in the wild type BMDMs (Fig. 4A/B). We quantitatively analyzed the results of BODIPY staining using flow cytometry. The results revealed that the lipid content was approximately 31.6% less in TFEB-KR BMDMs (Fig. 4C/D) than that in the WT group. Further, we measured the intracellular cholesterol content by relevant kit (BioVision, K603-100, CA, USA) and observed that TFEB-KR BMDMs had less cholesterol (Fig. 4E) than the WT group. These results suggested that TFEB-KR BMDMs had less lipid deposition after OxLDL stimulation.

Fig. 4.

TFEB-KR macrophages exhibited less lipid deposition upon OxLDL stimulation. A TFEB-KR BMDMs inhibited the formation of macrophage foam cells. WT and TFEB-KR BMDMs were incubated with OxLDL (50 μg/ml) for 24 h, fixed, and stained with oil red O; n = 3. B Under OxLDL stimulation, there was less lipid deposition in TFEB-KR BMDMs. WT and TFEB-KR BMDMs were incubated with OxLDL (50 μg/ml) for 24 h, LDs were stained using BODIPY and observed using immunofluorescence; n = 3. C Under OxLDL stimulation, there was less lipid deposition in TFEB-KR BMDMs. WT and TFEB-KR BMDMs were incubated with OxLDL (50 μg/ml) for 24 h, LDs were stained using BODIPY and quantified using flow cytometry; n = 3. D Quantitative analysis of the BODIPY MFI after OxLDL treatment in WT and TFEB-KR BMDMs; n = 3. E Under OxLDL stimulation, TFEB-KR BMDMs had lower total cholesterol levels. WT and TFEB-KR BMDMs were incubated with OxLDL (50 μg/ml) for 24 h and the content of total cholesterol in WT and TFEB-KR BMDMs was assessed before or after OxLDL treatment; n = 3. F TFEB SUMOylation did not significantly alter the expression of main scavenger receptors in macrophages after OxLDL treatment. WT and TFEB-KR BMDMs were treated with OxLDL (50 μg/ml) for 16 h and then relative mRNA levels of main scavenger receptors were assessed by Real-time quantitative PCR; n = 3. G TFEB SUMOylation did not affect LDL uptake in WT and TFEB-KR BMDMs; n = 3. H Under OxLDL stimulation, the cholesterol efflux was significantly increased in TFEB-KR BMDMs. Analysis of cholesterol efflux in WT and TFEB-KR BMDMs after OxLDL treatment through cholesterol efflux assay kit; n = 3

In fact, the formation of macrophage foam cells is a way of metabolizing cholesterol by macrophages. The lipid metabolism process is very complex. It can be roughly divided into three steps: uptake, catabolism, and efflux of cholesterol [41–43]. Cholesterol uptake and efflux are mediated by a series of receptors. The major receptors related to cholesterol uptake include cluster of differentiation 36 (CD36), scavenger receptor class B member 1 (Scarb1), and macrophages scavenger receptor 1 (Msr1) [44–46]. The main receptor of cholesterol efflux is ATP binding cassette subfamily G member 1 (Abcg1) [47]. Upon OxLDL stimulation at 16 h, the mRNA levels of the above receptors were elevated in both WT and TFEB-KR groups and there was no difference between the two groups (Fig. 4F). Therefore, TFEB SUMOylation did not affect the expression of receptors related to cholesterol uptake and efflux in macrophages. Further, the cholesterol uptake capacity was assessed between the WT and TFEB-KR BMDMs, which was observed to be comparable in the two groups (Fig. 4G). Finally, the cholesterol efflux was measured by relevant kit (BioVision, K582-100, CA, USA) and we observed that the cholesterol efflux of TFEB-KR BMDMs was significantly increased upon OxLDL stimulation at 6 h (Fig. 4H). These results suggested that because of stronger lysosomal function, less lipid deposition was present in TFEB-KR macrophages.

TFEB SUMOylation inhibited the transcriptional activity of TFEB by preventing its binding activity

Chloroquine (CQ) can be used as a lysosomal inhibitor. Alkaline CQ interacts with acidic lysosomes and inhibits the activity of lysosomes [48]. It has been reported that TFEB translocated into the cell nuclei with its transcriptional activity enhanced upon CQ stimulation [15]. We overexpressed WT and TFEB-KR plasmids in 293T cells. The cells were further stimulated with CQ for 15 h, and as a result, both WT and TFEB-KR proteins were able to translocate into the nuclei (Fig. 5A), and there was no difference in the ratio of TFEB translocation into the nuclei between the two groups (Fig. 5B). By analyzing the structure of TFEB, we observed that TFEB SUMOylation site was close to the DNA binding domain [49] (Fig. 5C). Therefore, we used chromatin immuneprecipitation to test whether the binding ability of TFEB to its target genes was different between the two groups. Mucolipin-1 (Mcoln-1), also known as transient receptor potential channel mucolipin-1 (Trpml1), is a classical target for TFEB and plays an important role in maintaining the biological function of lysosome [50]. We evaluated Mcoln-1 level in both groups with or without OxLDL stimulation. The binding ability of TFEB to Mcoln-1 promoter was significantly increased in TFEB-KR macrophages upon stimulation (Fig. 5D). These results indicated that TFEB SUMOylation inhibited its transcriptional activity by inhibiting its binding to its target genes.

Fig. 5.

TFEB SUMOylation inhibited its transcriptional activity by preventing the binding activity. A TFEB SUMOylation did not affect its nuclear localization. Wild-type and TFEB-KR plasmids labeled with GFP were transfected into 293T cells respectively and then stimulated the cells with chloroquine for 15 h to observe the nuclear entry of TFEB; n = 3. B Quantitative analysis of the ratio of TFEB entering the nuclei in WT and TFEB-KR groups; n = 3. C The structure of TFEB. D Under OxLDL stimulation, TFEB SUMOylation inhibited its transcriptional activity. WT and TFEB-KR BMDMs were treated with OxLDL (50 μg/ml) for 16 h and then the binding ability of TFEB and its target gene MCOLN1 was measured by chromatin immunoprecipitation; n = 3

TFEB SUMOylation inhibited the binding between TFEB and FACT complex to attenuate TFEB transcriptional activity

To further explore how TFEB SUMOylation affecting its transcriptional activity, we used mass spectrometry to find the key regulators affected by TFEB SUMOylation in this process. We overexpressed wild-type and mutant Flag-TFEB in 293T cells, and then enriched TFEB protein by immunoprecipitation (Fig. 6A). Through mass spectrometry analysis, we found that TFEB SUMOylation affected its interaction with other proteins, among which the interaction with facilitates chromatin transcription (FACT) complex was the most affected (Fig. 6B). FACT complex, a heterodimeric histone chaperone composed of structure specific recognition protein 1 (SSRP1) and SPT16 homolog, facilitates chromatin remodeling subunit (SUPT16H), mediates the disassembly and assembly of nucleosomes, thus improving the efficiency of transcription. A recent study showed that TFEB can regulate its transcriptional activity by binding to FACT, but the molecular mechanism is not fully understood [51]. To verify the influence of TFEB SUMOylation on the interaction between TFEB and FACT complex observed in MS analysis, we used sodium arsenite (NaAsO₂) to stimulate 293T cells with overexpressed wild-type and mutant Flag-TFEB, respectively. NaAsO₂ is a kind of oxidative stress stimulator known to induce efficient translocation of TFEB from the cytosol to the nucleus. Our results showed that TFEB SUMOylation did inhibit the interaction between TFEB and FACT complex, and this inhibition effect was more obvious after TFEB was activated (Fig. 6C). Finally, we used luciferase reporter gene experiment to verify that TFEB SUMOylation affected its transcriptional activity through the FACT complex during the process of macrophage foam cells formation. As previously described, LAMP1 is a target gene for TFEB and then we constructed a related reporter gene plasmid (Fig. 6D). We used OxLDL to stimulate macrophages to imitate the process of foam cells formation in RAW264.7 cells. Our results showed that TFEB SUMOylation inhibited the activity of LAMP1 promoter during the process of macrophage foam cells formation, but this inhibition completely disappeared when the FACT complex and TFEB were co-transfected (Fig. 6E). The above results showed that TFEB SUMOylation attenuated its transcriptional activity by inhibiting its interaction with FACT complex during the process of macrophage foam cells formation.

Fig. 6.

TFEB SUMOylation inhibited the binding between TFEB and FACT complex to attenuate TFEB transcriptional activity. A Identification of TFEB related proteins enriched by immunoprecipitation (IP:Flag) using Coomassie brilliant blue staining. B Through mass spectrometry analysis, TFEB SUMOylation can affect its interaction with the FACT complex. C TFEB SUMOylation is critical for its interaction with FACT complex. 293T cells were transfected with the indicated plasmids, and after 24 h of transfection, NaAsO₂ (250 μM) was used to stimulate the cells for 2 h. Finally, the cells were immunoprecipitated with anti-Flag beads. The precipitated proteins or lysates were immunoblotted with anti-Flag/anti-His or anti-HA antibodies. D Schematic diagram of constructing a reporter gene plasmid containing the LAMP1 promoter sequence. E The FACT complex can rescue the inhibitory effect of TFEB SUMOylation on its transcriptional activity during OxLDL stimulation. RAW264.7 cells were transfected with indicated plasmids and after 24 h of transfection, OxLDL (50 μg/ml) was used to stimulate the cells for 8 h. Finally, the activity of the corresponding promoter was checked by report gene assay; n = 3

Discussion

Our results revealed that TFEB SUMOylation plays a very important role in the formation of macrophage foam cells and ultimately affects the development of AS. By analyzing AS animal models, we observed that macrophages with TFEB SUMOylation deficiency had higher lysosomal activity, which could deal with excessive lipid content and ultimately alleviate the development of AS. To identify the molecular mechanism underlying TFEB SUMOylation in the formation of macrophage foam cells, we used OxLDL to stimulate BMDMs to mimic AS conditions in vitro. We observed that after OxLDL treatment, TFEB SUMOylation was decreased and thus TFEB exhibited stronger binding ability to its target genes in TFEB-KR BMDMs. The higher transcriptional activity of TFEB led to TFEB-KR BMDMs presenting stronger lysosomal activity, which enabled BMDMs to process more cholesterol and ultimately alleviate lipid deposition. Therefore, the study herein demonstrated that under OxLDL stimulation, TFEB undergone deSUMOylation and deSUMOylated TFEB had stronger transcriptional activity and enhanced lipid degradation by enhancing lysosomal activity in macrophages, thereby inhibiting the formation of macrophage foam cells and ultimately alleviating the development of AS (Fig. 7).

Fig. 7.

Model depicting the role of TFEB SUMOylation in macrophage foam cells formation. Under OxLDL stimulation, TFEB would undergo deSUMOylation and deSUMOylation of TFEB has stronger transcriptional activity and promotes lipid degradation by enhancing lysosomal activity, thereby ultimately inhibiting the formation of macrophage foam cells

Macrophages play an important role in the early stage of AS, particularly in the formation of macrophage foam cells. Marcel et al. reported that under LDL stimulation, the autophagy-lysosome system in macrophages is activated, which plays important roles in lipid processing and metabolism [7]. TFEB is a transcription factor that has an essential role in regulating lysosomal biogenesis and autophagy. A recent study by Razani et al. reported that enhancing lysosomal activity by specifically overexpression of TFEB in macrophages can alleviate AS [5]. Therefore, TFEB is considered as a protective factor for AS.

In our study, we found that TFEB SUMOylation was significantly decreased after OxLDL stimulation. However, some questions remain to be addressed. First, OxLDL is a macromolecule complex that cannot directly act on TFEB, it is unclear which signal specifically reduces the SUMOylation of TFEB upon the stimulation. Secondly, the molecular mechanism of TFEB deSUMOylation in macrophages remains unclear. SUMOylation is a dynamic and reversible process. The process of SUMOylation involves a series of enzymatic reactions mediated by E1/E2/E3. However, deSUMOylation is regulated by SUMO-specific proteases (SENPs) [52–55]. The decrease in TFEB SUMOylation may be due to the inhibition of the activity of one or more enzymes in E1/E2/E3 or the upregulation of the activity or expression of the corresponding SENPs. There are six known SENPs, namely, SENP1, 2, 3, 5, 6, and 7 [55]. Which SENPs mediated the deSUMOylation of TFEB is yet to be investigated.

To the best of our knowledge, this is the first study on mice to report that TFEB-KR: Ldlr^−/− mice had milder atherosclerosis onset and macrophages in plaques had stronger lysosomal activity. This is consistent with the study by Yang et al., who reported that the high activity of TFEB in macrophages can make macrophages possess stronger lysosomal activity, which can alleviate the development of AS [56]. Finally, we observed that TFEB SUMOylation inhibited the transcriptional activity of TFEB by inhibiting its interaction with FACT complex. Since TFEB is a master regulator on lysosomal biogenesis and autophagy, the mechanisms for regulating TFEB activity should be sophisticated and diverse. Although we found that SUMOylation affected the interaction between TFEB and FACT complex, of course, it should affect its interaction with other proteins either. Therefore, there must be other mechanisms that affect the activity of TFEB through its SUMOylation. We speculated that there are several possible mechanisms: (1) TFEB SUMOylation may inhibit the binding ability of TFEB to its co-activators. (2) TFEB SUMOylation may enhance the binding ability of TFEB to its co-inhibitors. (3) TFEB SUMOylation may interact with other signaling pathways that can regulate the transcriptional activity of TFEB.

Collectively, our study demonstrated that TFEB SUMOylation is involved in the pathogenesis of AS by regulating the formation of macrophage foam cells. TFEB SUMOylation promoted the formation of macrophage foam cells by inhibiting lysosomal biogenesis and autophagy and eventually aggravated the development of AS. Therefore, TFEB SUMOylation in macrophages might be applied as a novel target for AS therapy. Drugs that can reduce TFEB SUMOylation in macrophage foam cells should be developed in the future, to enhance the ability of macrophages to process lipids, thereby alleviating or inhibiting the development of AS.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AS

Atherosclerosis

TFEB

Transcription factor EB

SUMOs

Small ubiquitin-like modifiers

SENPs

SUMO specific peptidases

OxLDL

Oxidized low-density lipoprotein

IL-1β

Interleukin-1β

LAMP1

Lysosomal associated membrane protein 1

CLCN7

Chloride channel, voltage-sensitive 7

TG

Triglyceride

TC

Total cholesterol

HDL-C

High-density lipoprotein cholesterol

LDL-C

Low-density lipoprotein cholesterol

IL-4

Interleukin-4

IL-6

Interleukin-6

CTSB

Cathepsin B

CTSD

Cathepsin D

CD36

Cluster of differentiation 36

Scarb1

Scavenger receptor class B member 1

Msr1

Macrophages scavenger receptor 1

Abcg1

ATP binding cassette subfamily G member 1

CQ

Chloroquine

Mcoln-1

Mucolipin-1

Trpml1

Transient receptor potential channel mucolipin-1

FACT

Facilitates chromatin transcription

SSRP1

Structure specific recognition protein 1

SUPT16H

SPT16 homolog, facilitates chromatin remodeling subunit

NaAsO₂

Sodium arsenite

Author contributions

RC and TT designed and conceived the project, provided the concept, contributed to the manuscript and supervised the research. KZW designed and performed experiments, analyzed and interpreted the data, and prepared the manuscript. RC and TT designed experiments and edited the manuscript. LFW, WZ and GLH assisted with BMDM preparation and performed flow cytometry. All authors analyzed and interpreted the data, revised for critical intellectual content and approved the final manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Natural Science Foundation of China (82002937, 82000904).

Data availability

All the data supporting the findings of this study are available within the paper. Data will be made available on reasonable request, not applicable for material.

Declarations

Conflict of interest

The funders had no conflicts of interest in the design of the study; in the collection, analysis or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Ethics approval

Animal studies were approved by the Animal Care and Use Committee from Xinhua Hospital, Affiliated to Medicine School of Shanghai Jiaotong University.

Consent to participate

Not applicable.

Consent for publication

All authors have been involved in writing the manuscript and consented to publication.