Vascular Endothelial Growth Factor Downregulates Apolipoprotein M Expression by Inhibiting Foxa2 in a Nur77-Dependent Manner

Abstract

Objective:

We aimed to investigate whether vascular endothelial growth factor (VEGF) influences apolipoprotein M (ApoM) expression and pre-β-high-density lipoprotin (HDL) formation, and whether forkhead box A2 (Foxa2) and Nur77 are involved in this process.

Methods and Results:

We analyzed the serum VEGF concentrations of 264 adults who underwent a medical checkup and found that VEGF concentration was positively correlated with serum triglyceride, total cholesterol, LDL cholesterol (LDL-C), very-low-density lipoprotein cholesterol (VLDL-C), and ApoB concentrations, but was negatively correlated with serum high-density lipoprotein cholesterol (HDL-C) and ApoM concentrations. We further investigated the effects of VEGF on ApoM expression and pre-β-HDL formation, and the mechanisms responsible, in HepG2 cells and mouse primary hepatocytes. VEGF markedly downregulated ApoM expression and pre-β-HDL formation. At the same time, expression of Foxa2 was also inhibited, whereas expression of Nur77 was increased by treatment with VEGF. Furthermore, small interfering (si) RNA knockdown of Foxa2 made the downregulation of VEGF on ApoM expression and pre-β-HDL formation even more obvious. In addition, siRNA knockdown of Nur77 significantly compensated for the inhibitory effect of VEGF on Foxa2 expression, whereas the Nur77 agonist cytosporone B led to the downregulation of Foxa2 expression more significantly than VEGF. Moreover, overexpression of a Nur77 transgene in C57BL/6 mice resulted in decreased serum ApoM and pre-β-HDL levels, whereas si-Nur77–treated mice displayed upregulated serum ApoM and pre-β-HDL levels.

Conclusion:

These results provide evidence that VEGF may first downregulate expression of Foxa2 by enhancing Nur77 activity and then decrease expression of ApoM and pre-β-HDL formation. Therefore, our study may be useful in understanding the critical effect of VEGF in the pathogenesis of atherosclerosis.

Introduction

A polipoprotein M (ApoM) is a recently characterized human apolipoprotein that is mainly associated with high-density lipoprotein (HDL) in human plasma, with a small proportion being present in triglyceride-rich lipoproteins and low-density lipoproteins (LDL).¹ It is mainly expressed in hepatocytes and in the tubular epithelial cells in kidney, although small amounts are also detected in fetal liver and kidney.^2,3 It has been demonstrated that ApoM is important for pre-β-HDL formation and thus influences the HDL cholesterol (HDL-C) concentration in plasma.^4,5 ApoM expression by hepatocytes is highly regulated at both the transcriptional and posttranscriptional levels. It is directly regulated by transcription factors, including forkhead box A2 (Foxa2), hepatic nuclear factor-1α (HNF-1α), liver receptor homolog-1 (LRH-1), and liver X receptor (LXR), all of which contribute to hepatic lipid and glucose metabolism. In addition, cytokines, especially proinflammatory factors, have been shown to exert pleiotropic and antagonistic effects on ApoM expression.⁶

Foxa2 is a transcription factor involved in pancreatic and hepatic development via the regulation of glucose homeostasis in liver and pancreatic β-cells.^7,8 Recent studies have revealed that Foxa2 regulates APOM transcription, leading to increased pre-β-HDL formation and plasma HDL levels. Treatment of wild-type mice and ob/ob mice with an adenovirus containing phosphorylation-defective Foxa2 not only improved glucose and lipid homeostasis but also increased hepatic ApoM mRNA expression and plasma levels of the protein. In contrast, haplo-insufficient Foxa2 ^+/− mice exhibited decreases in hepatic ApoM expression and in plasma pre-β-HDL and HDL levels. A binding site for Foxa2 in the APOM promoter was identified at position −474.⁹

Nur77 is an orphan member of the steroid/thyroid receptor superfamily of transcription factors that positively and negatively regulates gene expression. It is composed of an amino-terminal transactivation domain, a DNA-binding domain, and a carboxy-terminal ligand-binding domain.¹⁰ Nur77 binds to the NGFI-B (NGF-induced B factor)-response element (NBRE) and the Nur-response element (NurRE) sequences in DNA and is able to stimulate transcription from NBRE- and NurRE-dependent reporter genes.¹¹ Nur77 regulates distinct cellular processes in a tissue-specific manner. For example, Nur77 is expressed in human adult liver and has been shown to regulate gluconeogenesis and hepatic lipid metabolism.^12,13 Interestingly, Liu et al. showed that vascular endothelial growth factor (VEGF) could increase Nur77 protein expression and decrease Nur77 phosphorylation at the negative regulatory site serine 351.¹⁴

VEGF in cells is a homodimeric glycoprotein that is best known for its capacity to induce angiogenesis, increase vascular permeability, and stimulate production of the thrombogenic protein tissue factor.¹⁵ The role of VEGF in the molecular mechanism of atherosclerotic lesion formation remains controversial. Some studies have indicated that VEGF inhibits thickening of the media by promoting regeneration of vascular endothelial cells and by improving endothelial function. Others have reported that VEGF can promote atherosclerosis via promotion of monocyte chemotaxis and plaque neovascularization.¹⁶ Recently, Kimura et al. found that the serum VEGF concentration showed a negative correlation with HDL-C concentration and suggested that the serum VEGF concentration might be closely related to atherosclerosis accelerating factor, especially in men.¹⁷

However, whether VEGF influences ApoM expression and pre-β-HDL formation is unclear, and whether Foxa2 and Nur77 are involved in this process has not yet been explored. In the present study, we investigated the novel effects and their mechanisms of VEGF on ApoM expression and pre-β-HDL formation in HepG2 cells and mouse primary hepatocytes. A series of assays were conducted and revealed that VEGF downregulated ApoM expression and pre-β-HDL formation. At the same time, expression of Foxa2 was also inhibited, whereas expression of Nur77 was increased, which could be compensated by Nur77-small interfering (si) RNA. Furthermore, Foxa2-siRNA blocked the effects of VEGF on ApoM expression and pre-β-HDL formation. Nur77-siRNA significantly compensated the inhibitory effect of VEGF on Foxa2 expression, whereas the Nur77 agonist cytosporone B (Csn-B) led to the downregulation of Foxa2 expression more significantly than VEGF. Moreover, overexpression of a Nur77 transgene in mice resulted in decreased serum ApoM and pre-β-HDL levels, whereas si-Nur77-treated mice displayed increased serum ApoM and pre-β-HDL levels. Taken together, our data suggest that VEGF might decrease the expression of ApoM and pre-β-HDL formation by downregulating the expression of Foxa2, in a process involving Nur77.

Materials and Methods

Materials

Recombinant VEGF and Csn-B were purchased from Sigma Chemical Company (St. Louis, MO). SU5614, GF109203X, LY2940024, -amino- 5- (4 - chlorophenyl) –7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2), and [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) were from Calbiochem Inc (San Diego, CA). The phosphoserine-351–Nur77 antibody was purchased from Santa Cruz Biotechnology Inc. (USA), and the BCA Protein Assay Kit was from Pierce Chemical (Rockford, IL). The RevertAidTM First Strand cDNA Synthesis Kit (#k1622) (Fermentas, 830 Harrington Court, Burlington, Ontario, Canada), DyNAmoTM SYBR^® Green qPCR Kit (Finnzymes, Espoo, Finland), and Immobilon-P Transfer Membranes (Millipore, USA) were obtained as indicated. All other chemicals were of the best grade available from commercial sources.

Subjects

The subjects of this study were 264 consecutive outpatients who attended Nanfang Hospital. They underwent a health check for the prevention of lifestyle-related diseases from the period of March, 2010, to May, 2010. The study population consisted of 177 males and 87 females (average age, 38.12±10.87 and 36.05±11.30 years, respectively, p=0.156). The age range was from 12 to 76 years. Four female and 83 male subjects were smokers. Fourteen people had diabetes mellitus (9 of whom received medication), 18 people had hyperlipidemia (6 of whom received medication), and 31 people were diagnosed with hypertension (26 of whom received medication). Participants provided informed written consent, and the study was approved by the Ethics Committee of Nanfang Hospital.

Measurement of serum VEGF, ApoM, and other biochemical parameters

The serum VEGF concentration was measured in duplicate using a commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). The assay system used is sensitive to 9 pg/mL (0.2 pM) VEGF and does not cross-react with platelet-derived growth factor or other homologous cytokines. The optical density at 450 nm was measured using a microtiter plate reader (Titertek Multiskan MC, Sterling, VA), and the VEGF concentration was determined by linear regression from a standard curve obtained using the VEGF supplied with the kit as the standard. The serum ApoM concentration was measured in duplicate using a commercial ELISA kit (USCN Life Science Inc., China). The optical density at 450 nm was measured on a microtiter plate reader (Titertek Multiskan MC, Sterling, VA), and the ApoM concentration was determined by linear regression from a standard curve using the ApoM supplied with the kit to prepare the standards. The T-Cho, triglyceride (TG), LDL-C, HDL-C, VLDL-C, ApoA1, ApoB, ApoA1/ApoB, and lipoprotein(a) (Lp[a]) concentrations were determined enzymatically using an automated analyzer.

Cell culture

HepG2 cells were cultured in 25-cm² vented flasks containing RPMI-1640 with 10% fetal calf serum (FCS) in the presence of benzylpenicillin (100 U/mL) and streptomycin (100 μg/mL) under standard culture conditions (5% CO₂, 37°C). For experiments, cells were seeded in six-well cell culture clusters, and were grown to 50%–70% confluence. Prior to the experiment, cells were washed twice with phosphate-buffered saline (PBS), and once with serum-free RPMI-1640 without antibiotic. Experimental media contained RPMI-1640 with 0.5% human serum albumin and one or more additives, i.e., VEGF, Csn-B, SU5614, GF109203X, LY2940024, PP2, BAPTA-AM, etc., at the concentrations described in the figure legends.

Mouse primary hepatocyte isolation

Mouse primary hepatocytes were harvested from 8-week-old C57BL/6 mice and isolated using a Krebs–Henseleit solution/collagenase perfusion protocol according to Amaxa Biosystems (Amaxa, Gaithersburg, MD). The viable hepatocyte population was further purified by Percoll gradient centrifugation.¹⁸ Hepatocytes were plated in collagen-coated plates at 3×10⁵ cells per well in a six-well plate in William E medium supplemented with 5% fetal bovine serum (FBS) and antibiotics. Cells were allowed to attach for 2 hr before switching to fresh medium and adding all treatments.

Lentivirus production and tail vein injection

All studies were done in accordance with protocols approved by the Animal Use and Care Committee at Nanfang Hospital. Lentiviral constructs for mouse Nur77 and VEGF were made as described previously.^19

–22 Viral multiplicity of infection for liver infection was estimated based on in vitro primary hepatocyte transduction efficiency: 0.5 mL of undiluted viral stocks supplemented with Polybrene (7.5 μg/mL) was added to 10⁵ primary hepatocytes cultured in 12-well plates, and green fluorescent protein (GFP)-positive cells were counted 96 hr after transduction. Anesthetized male C57BL/6 mice, aged 8 weeks, were injected with 150 μL of undiluted viral stocks supplemented with Polybrene (5 μg/mL) into the tail vein. Serum analysis or tissue harvesting was performed 7 days after injection.

Cholesterol efflux assays

J774 cells obtained from the American Type Culture Collecion (ATCC) were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS. Then they were labeled with [³H]cholesterol (2 μCi/ml) for 48 hr. On the one hand, labeled cells were washed and then incubated in serum-free medium containing 1% bovine serum albumin (BSA) for 24 hr of equilibration. The cells were then washed and subjected to different treatments: (1) 2.5% serum from control lentivirus treated mice was added to cells; (2) 2.5% serum from lentivirus encoding VEGF mice was added to cells. On the other hand, labeled cells were washed and then incubated in serum-free medium containing 1% BSA for 24 hr of equilibration in either the presence or absence of VEGF (100 ng/mL) in serum-free medium. For the cholesterol efflux, medium containing 25 μg/ml HDL was added to cells. Cholesterol efflux was determined by liquid scintillation counting, and the percentage of radiolabeled cholesterol released (percent cholesterol efflux) was calculated as (cpm in medium/[cpm in the cell+ medium])×100.^23,24

RNA Isolation and real-time quantitative PCR analysis

Total RNA from cells was extracted using TRIzol reagent in accordance with the manufacture's instructions. Real-time quantitative PCR, using SYBR Green detection chemistry, was performed on the Roche Light Cycler Run 5.32 Real-Time PCR System. Melt curve analyses of all real-time PCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value was considered. Quantitative measurements were determined using the ΔΔCt method, and expression of β-actin was used as the internal control.

Western blot analyses

Cells were harvested and protein extracts prepared as previously described.²⁵ These were then subjected to western blot analyses (10% sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE]; 30 μg protein per lane) using rabbit anti-ApoM (BD Biosciences, San Jose, CA), anti-Nur77 (Santa Cruz Biotech, Santa Cruz, CA), anti-VEGF (Abcam, Cambridge, MA), and goat anti-Foxa2 (Santa Cruz Biotech, Santa Cruz, CA) and β-actin (Santa, Cruz, Santa Cruz, CA) antibodies. The proteins were visualized using a chemiluminescence method (ECL Plus Western Blotting Detection System; Amersham Biosciences, Foster City, CA).

Transfection with siRNA

The siRNAs against Nur77 and Foxa2 and an irrelevant 21-nucleotide control siRNA were purchased from Biology Engineering Corporation in Shanghai, China. Cells (2×10⁶ cells/well) were transfected with the siRNA targeting Nur77, Foxa2, or the control in the absence or presence of appropriate plasmids using Lipofectamine 2000 (Invitrogen). Real-time RT-PCR and western blots were performed 48 hr after transfection.

Native agarose gel electrophoresis

Native agarose gel electrophoresis was used to determine pre-β-HDL and α-HDL levels, as described.²⁶ Proteins were applied to 0.7% agarose gels. After electrophoresis for 2.5 hr in 60 mM sodium barbital buffer (pH 8.6; Sigma-Aldrich) at 4°C, proteins were transferred to a nitrocellulose membrane in deionized water by capillary blotting for 16 hr. The membrane was probed with rabbit anti-ApoA1 antibody (Biodesign, Saco, ME), followed by incubation with anti-rabbit immunoglobuin G (IgG) conjugated with horseradish peroxidase (HRP; Amersham Biosciences, Piscataway, NJ). ApoA1 proteins were visualized by ECL Detection Reagents and quantified by densitometry, as described.²⁷

Statistical analysis

Data are expressed as mean±standard deviation (SD). Results were analyzed by one-way analysis of variance (ANOVA) and the Student t-test, using SPSS 13.0 software. Statistical significance was obtained when p values were less than 0.05.

Results

The serum VEGF concentration showed a negative correlation with ApoM concentration and HDL-C concentration

VEGF has been noted in the pathogenesis of atherosclerosis.²⁸ We first examined associations between serum VEGF concentrations and serum lipid and lipoprotein concentrations in 264 subjects. Table 1 shows the clinical and biochemical characteristics of these subjects. The average serum VEGF concentration of all subjects was 320.06±183.61 pg/mL (range, 90.91–975.90 pg/mL). The average serum HDL-C of all subjects was 1.61±0.36 mmol/L (range, 0.87–2.77 mmol/L). The average serum ApoM concentration of all subjects was 17.93±5.03 mg/L (range, 7.63–28.17 mg/L). Table 2 shows the univariate associations between serum VEGF concentration and age, serum lipid and lipoprotein concentrations. The average serum VEGF concentration of men was significantly higher than that of women (p=0.001). The serum VEGF concentration was negatively correlated with serum HDL-C and ApoM concentrations and the ApoA1/ApoB ratio in all subjects (p<0.05). Moreover, the serum VEGF concentration was positively correlated with serum TG, total cholesterol, LDL-C, VLDL-C, and ApoB concentrations in all subjects (p<0.05). In addition, the serum VEGF concentration was positively correlated with the serum Lp(a) concentration in subjects with diabetes mellitus and subjects with hyperlipidemia (p<0.05). There were no associations between serum VEGF concentration and age or ApoA1 concentrations.

Table 1.

Clinical and Biochemical Characteristics of Studied Subjects (n=264)

	Mean	SD	Minimum	Maximum
Age (years)	37.44	11.04	12.00	76.00
Triglyeride (mmol/L)	1.87	1.21	0.49	8.61
Total cholesterol (mmol/L)	5.18	1.00	2.99	7.80
HDL-C (mmol/L)	1.61	0.36	0.87	2.77
LDL-C (mmol/L)	2.77	0.79	1.02	4.69
VLDL-C (mmol/L)	0.79	0.41	0.10	2.78
ApoA-I (g/L)	1.38	0.13	1.06	1.77
ApoB (g/L)	1.04	0.26	0.40	1.64
ApoA-I/ ApoB	1.42	0.45	0.70	3.40
LP(a) (g/L)	0.21	0.15	0.05	0.80
ApoM (mg/L)	17.93	5.03	7.63	28.17
VEGF (pg/ml)	320.06	183.61	90.91	975.90

HDL-C, High-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; VLDL-C, very-low-density lipoprotein cholesterol; Apo, apolipoprotein; LP(a), lipoprotein (a); VEGF, vascular endothelial growth factor.

Table 2.

Associations (Pearson Correlation Coefficients) of Serum VEGF Concentration with Clinical and Biochemical Characteristics of Subjects Studied (n=264)

VEGF
					Hypertension				Hyperlipidemia				Diabetes mellitus
	Healthy		Smokers		Untreated		Received medication		Untreated		Received medication		Untreated		Received medication
	r	p	r	p	r	p	r	p	r	p	r	p	r	p	r	p
Age (years)	0.014	0.816	0.017	0.825	0.032	0.766	0.021	0.823	0.019	0.622	0.041	0.718	0.047	0.661	0.323	0.723
BMI (kg/m²⁾	0.193	0.735	0.231	0.770	0.191	0.789	0.331	0.634	0.219	0.775	0.167	0.576	0.115	0.637	0.324	0.813
TG (mmol/L)	0.351	<0.001	0.383	<0.001	0.353	<0.001	0.431	<0.001	0.513	<0.001	0.312	<0.001	0.219	<0.001	0.332	<0.001
CHO (mmol/L)	0.219	<0.001	0.221	<0.001	0.251	<0.001	0.195	<0.001	0.339	<0.001	0.325	<0.001	0.431	<0.001	0.367	<0.001
HDL-C (mmol/L)	−0.242	<0.001	−0.235	<0.001	−0.237	<0.001	−0.436	<0.001	−0.237	<0.001	−0.135	<0.001	−0.312	<0.001	−0.239	<0.001
LDL-C (mmol/L)	0.175	0.005	0.196	0.002	0.171	0.006	0.218	0.004	0.353	0.008	0.219	0.001	0.115	0.005	0.169	0.007
VLDL (mmol/L)	0.385	<0.001	0.385	<0.001	0.385	<0.001	0.385	<0.001	0.385	<0.001	0.385	<0.001	0.385	<0.001	0.385	<0.001
ApoA1 (g/L)	−0.041	0.551	−0.037	0.768	−0.051	0.116	−0.076	0.619	−0.040	0.143	−0.039	0.146	−0.131	0.221	−0.096	0.137
ApoB (g/L)	0.274	<0.001	0.271	<0.001	0.273	<0.001	0.312	<0.001	0.331	<0.001	0.196	<0.001	0.412	<0.001	0.513	<0.001
ApoA1/ ApoB	−0.248	<0.001	−0.275	<0.001	−0.566	<0.001	−0.553	<0.001	−0.713	<0.001	−0.685	<0.001	−0.191	<0.001	−0.212	<0.001
LP(a) (g/L)	0.057	0.379	0.098	0.515	0.045	0.396	0.051	0.779	0.315	0.026	0.219	0.037	0.069	0.041	0.089	0.039
ApoM (mg/L)	−0.129	0.039	−0.217	0.002	−0.106	0.003	−0.419	0.028	−0.513	0.027	−0.313	0.040	−0.173	0.031	−0.197	0.041

BMI, Body mass index; TG, triglyceride; CHO, cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; VLDL, very-low-density lipoprotein; Apo, apolipoprotein.

VEGF downregulates ApoM expression and pre-β-HDL formation

Because the serum VEGF concentration was negatively associated with the plasma ApoM concentration, we next examined the effect of VEGF on ApoM expression in HepG2 cells and mouse primary hepatocytes by real-time quantitative PCR and western blot assays. As shown (Fig. 1A,B), VEGF decreased ApoM expression at both the transcriptional and translational levels in a dose-dependent manner. The inhibitory effects of VEGF on ApoM expression were increased with cell culture time. The strongest effects were seen after 24 hr in culture (Fig. 1C,D).

FIG. 1.

Dose-dependent and time-dependent effects of vascular endothelial growth factor (VEGF) on apolipoprotein M (ApoM) expression and pre-β-high-density lipoprotein (HDL) formation. HepG2 cells and mouse primary hepatocytes were treated with VEGF at 0 ng/mL, 1 ng/mL, 10 ng/mL, and 100 ng/mL for 24 hr. Cells were also treated with 100 ng/mL VEGF for 0 hr, 6 hr, 12 hr, and 24 hr. (A and C) APOM transcript levels were measured by real-time quantitative PCR. (B and D) ApoM protein expression was assessed by western blotting. (E and F) HDL subfractions of media from VEGF-treated cells were analyzed by native agarose gel electrophoresis. All the results are expressed as mean±standard deviation (SD) of three independent experiments, each performed in triplicate. (*) p<0.05 versus vehicle.

The serum VEGF concentration was inversely associated with the HDL cholesterol concentration. Moreover, ApoM was required for pre-β-HDL formation and could be downregulated by VEGF. Therefore, we next examined the effect of VEGF on pre-β-HDL formation by agarose gel electrophoresis and immunoblotting (Fig. 1E,F). The expression of pre-β-HDL was decreased by treatment with VEGF, suggesting that ApoM expression can be downregulated by VEGF in both HepG2 cells and mouse primary hepatocytes.

Foxa2 is involved in VEGF-induced down-regulation of ApoM

APOM expression is directly regulated by transcription factors, including HNF-1β, LRH-1, Foxa2, and LXRα, all of which contribute to hepatic lipid and glucose metabolism.⁶ To confirm whether the expression of these transcription factors is affected by VEGF, real-time quantitative PCR and western blot analysis were performed. As shown (Fig. 2A–E), expression of Foxa2 mRNA and protein was decreased when cells were treated with VEGF, whereas expression of HNF-1α, LRH-1, and LXRα was not affected by VEGF treatment.

FIG. 2.

Foxa2 is involved in vascular endothelial growth gactor (VEGF)-induced downregulation of apolipoprotein M (ApoM) expression. (A) HepG2 cells were incubated with VEGF (100 ng/mL) for 24 hr. Total RNA was extracted and real-time quantitative PCR was performed to determine the mRNA expression levels of hepatocyte nuclear factor-1α (HNF-1α), liver receptor homolog-1 (LRH-1), forkhead box A2 (Foxa2), and liver X receptor α (LXRα. (B) Mouse primary hepatocytes were incubated with VEGF (100 ng/mL) for 24 hr. Total RNA was extracted and real-time quantitative PCR was performed to determine the mRNA expression levels of HNF-1α, LRH-1, Foxa2, and LXRα. (C) HepG2 cells and mouse primary hepatocytes were incubated with VEGF (100 ng/mL) for 24 hr. Western blot assays using antibodies against HNF-1α, LRH-1, Foxa2, LXRα, and β-actin were conducted. (D and E) VEGF reduced the expression of Foxa2 at both mRNA and protein levels. Cells were treated with 100 ng/mL VEGF for 0 hr, 6 hr, 12 hr, and 24 hr. (D) Total RNA was extracted and real-time quantitative PCR was performed to determine the transcript levels of Foxa2 mRNA. (E) Western blot assays using antibodies against human Foxa2 and β-actin were conducted. (F–I) The cells were transfected with control or Foxa2 siRNA, and then incubated with VEGF (100 ng/mL) for 24 hr. (F) Protein samples were immunoblotted with anti-Foxa2 or anti-β-actin antibodies. (G and H) mRNA and protein expression of ApoM were determined by real-time quantitative PCR and western blotting. (I) High-density lipoprotein (HDL) subfractions of media from VEGF-treated cells were analyzed by native agarose gel electrophoresis as shown above. (J–M) The cells were transfected with control or pcDNA-Foxa2, and then incubated with VEGF (100 ng/mL) for 24 hr. (J) Protein samples were immunoblotted with anti-Foxa2 or anti-β-actin antibodies. (K and L) mRNA and protein expression of ApoM were determined by real-time quantitative PCR and western blotting. (M) HDL subfractions of media from VEGF-treated cells were analyzed by native agarose gel electrophoresis as shown above. Similar results were obtained in three independent experiments. Data are mean±standard deviation (SD). (*) p<0.05 versus baseline. (^#) p<0.01 versus control and VEGF group.

Foxa2 has been shown to regulate APOM transcription, leading to increased pre-β-HDL formation and plasma HDL levels. Therefore, we examined the effects of Foxa2 siRNA on the downregulation of ApoM induced by VEGF. Treatment with siRNA targeting Foxa2 downregulated Foxa2 protein expression by 75% and 81% in HepG2 cells and mouse primary hepatocytes, respectively (Fig. 2F), and made the downregulation of VEGF on ApoM expression even more obvious (Fig. 2G,H). At the same time, pre-β-HDL formation in cells treated with a combination of Foxa2 siRNA and VEGF was significantly decreased compared to those treated with VEGF alone (Fig. 2I). Next, we examined the effect of recombinant plasmid overexpressing Foxa2 (pcDNA-Foxa2) on the downregulation of ApoM and pre-β-HDL formation induced by VEGF. As shown (Fig. 2J, 2K, 2L, and 2M), the suppression effect on ApoM expression and pre-β-HDL formation by VEGF was markedly compensated by treatment with pcDNA-Foxa2. These data suggest VEGF downregulates ApoM expression by inhibiting the activity of Foxa2.

A recent study showed that ApoA1-deficient mice had dramatically decreased ApoM levels, suggesting a connection between ApoM and ApoAI metabolism.²⁹ Thus, we addressed the possibility that VEGF could affect ApoM expression through an ApoA1 pathway. However, both mRNA levels and protein expression were unaffected by treatment with VEGF (data not shown), suggesting that expression of ApoA1 is not involved in VEGF-induced downregulation of ApoM expression.

Downregulation of Foxa2 expression induced by VEGF is mediated by the Nur77 pathway

VEGF has been shown to induce expression of Nur77 and concomitantly decrease Nur77 phosphorylation at serine-351, a negative regulatory site that inhibits Nur77 transcriptional activity.^14,30 To test whether VEGF inhibits Foxa2 expression by stimulating expression of Nur77, the effects of VEGF on the expression of Nur77 in HepG2 cells and mouse primary hepatocytes were evaluated. Results showed that expression of Nur77 mRNA and protein were markedly increased when cells were treated with VEGF (Fig. 3A,B).

FIG. 3.

Nur77 is involved in vascular endothelial growth factor (VEGF)-induced downregulation of forkhead box A2 (Foxa2) expression. (A) HepG2 cells and mouse primary hepatocytes were treated with 100 ng/mL VEGF for 0 min, 15 min, 30 min, and 45 min. Total RNA was extracted and real-time quantitative PCR was performed to determine the Nur77 mRNA transcript levels. (B) HepG2 cells and mouse primary hepatocytes were treated with 100 ng/mL of VEGF for 0 hr, 1 hr, 2 hr, and 3 hr, respectively. Nur77 protein expression was measured by western blot assays. (C–E) HepG2 cells and mouse primary hepatocytes were transfected with control or Nur77 siRNA, and then incubated with VEGF (100 ng/mL) for 24 hr. (C) Protein samples were immunoblotted with anti-Nur77 or anti-β-actin antibodies. (D and E) mRNA and protein expression of Foxa2 was determined using real-time quantitative PCR and western blot assays. (F and G) HepG2 cells and mouse primary hepatocytes were untreated or incubated for 24 hr with, 100 ng/mL VEGF, 5 μg/mL Csn-B, and 100 ng/mL VEGF+5 μg/mL Csn-B. Then mRNA and protein expression of Foxa2 was determined using real-time quantitative PCR and western blot assays. (H) Western blot of serum apoliporpotein M (ApoM) and isolated high-density lipoprotein (HDL) from C57BL/6 mice (WT), and WT mice treated with Ad-Nur77 or si-Nur77. Each lane represents an individual animal. Values are means±standard deviation (SD) of three independent experiments. (I) Western blot analysis of Nur77, Foxa2, and ApoM protein levels in liver of C57BL/6 mice (WT), and WT mice treated with Ad-Nur77 or si-Nur77. (J) Western blot analysis of VEGF, Nur77, Foxa2, and ApoM protein levels in liver of C57BL/6 mice (WT), and WT mice treated with Ad-VEGF. Each lane represents an individual animal. Values are means±SD of three independent experiments. (K) HepG2 cells were treated with 100 ng/mL VEGF for the times indicated. Total cell lysates were prepared and equal amounts of protein were immunoblotted with antibodies to total Nur77 and Nur77 phosphorylated at serine-351. All the results are expressed as mean±SD of three independent experiments, each performed in triplicate. (*) p<0.05 versus vehicle. (^#) p<0.01 versus cells incubated with cytosporone B (Csn-B) alone.

Next, we examined the effect of Foxa2 siRNA on the VEGF-induced upregulation of Nur77. However, in both HepG2 cells and mouse primary hepatocytes, treatment with siRNA for Foxa2 had no effect on VEGF-induced Nur77 expression (data not shown). We then investigated the effect of Nur77 siRNA on Foxa2 expression in VEGF-stimulated cells. Treatment with siRNA for Nur77 downregulated Nur77 protein expression by 79% and 83% in HepG2 cells and mouse primary hepatocytes, respectively (Fig. 3C), and the reduction in Foxa2 expression by VEGF was significantly compensated by treatment with siRNA targeting Nur77 (Fig. 3D,E). We further examined the effects of the Nur77 agonist Csn-B on the downregulation of Foxa2 expression when cells were treated with VEGF. As demonstrated (Fig. 3F,G), treatment with both Csn-B and VEGF led to a greater reduction in Foxa2 expression than by treatment with VEGF alone. These observations suggest that the modulation of Foxa2 expression by VEGF is dependent on enhanced expression of Nur77.

We subsequently investigated the role of Nur77 on ApoM expression through a Foxa2 pathway in vivo by using a lentivirus expressing an siRNA for Nur77 (si-Nur77) and by overexpression of a Nur77 transgene (Ad-Nur77) in 8-week-old C57BL/6 mice. As shown in Fig. 3H, silencing of Nur77 in C57BL/6 mice by si-Nur77 led to an approximately 22% increase in plasma ApoM levels and a 15.5% increase in plasma pre-β-HDL (as judged from western blots) and an 8.9% increase in plasma HDL-C. Overexpression of Nur77 in C57BL/6 mice led to a 31% decrease in plasma ApoM levels and a 21% decrease in plasma pre-β-HDL (as judged from western blots) and a 13.5% reduction in plasma HDL-C. However, the plasma ApoA1 level was found to be similar between the two groups. Furthermore, Foxa2 and ApoM expression was upregulated in si-Nur77–treated mice whereas adenoviral overexpression of Nur77 in mice resulted in downregulated Foxa2 and ApoM expression in the liver (Fig. 3I).

Next, we investigated the causal role of VEGF on suppressing ApoM expression and pre-β-HDL formation through increasing Nur77 expression and decreasing Foxa2 expression in vivo by overexpression of a VEGF transgene (Ad-VEGF) in 8-week-old C57BL/6 mice. Overexpression of VEGF in C57BL/6 mice led to a 22.6% decrease in plasma ApoM levels and a 15.8% decrease in plasma pre-β-HDL (as judged from western blots) and a 7.5% reduction in plasma HDL-C. In addition, as shown in Fig. 3J, adenoviral overexpression of VEGF in mice could significantly upregulate Nur77 expression while downregulating Foxa2 and ApoM expression in the liver. Moreover, we examined the effects of VEGF on cholesterol efflux in J774 cells by liquid scintillation counting assays. However, treatment with both 2.5% serum from Ad-VEGF treated mice and VEGF (100 ng/mL) had no effect on cholesterol efflux in J774 cells.

Because it has been shown that phosphorylation plays an important role in Nur77 activity,³¹ we then tested the effect of VEGF on Nur77 phosphorylation. Western blotting with antibodies to Nur77 phosphorylated at serine-351 showed that concomitantly with increased Nur77 expression and reduced Foxa2 expression, Nur77 serine-351 phosphorylation was decreased in response to VEGF (Fig. 3K). These results indicate that reduction of Nur77 serine phosphorylation by VEGF might be involved in VEGF-induced enhancement of Nur77 activity and inhibition of Foxa2 activity.

KDR and KDR-mediated signaling mechanisms are required for induction of Nur77 expression by VEGF

Because the major positive signal of VEGF is generated by kinase insert domain receptor (KDR; VEGFR2),³² we used the specific KDR inhibitor SU5614 to explore whether KDR is required for induction of Nur77 expression by VEGF. HepG2 cells were pretreated with dimethyl sulfoxide or with SU5614 for 30 min and then incubated with or without VEGF, and the expression of Nur77 and phosphorylation of Nur77 serine 351 were measured. The results showed that the VEGF-induced upregulation of Nur77 expression and reduction of Nur77 serine 351 phosphorylation were notably abolished by SU5614 (Fig. 4A,B), revealing that VEGF must bind to KDR before changes in Nur77 expression and phosphorylation are observed.

FIG. 4.

Signaling pathways mediating vascular endothelial growth factor (VEGF)-induced Nur77 expression. (A) HepG2 cells were pretreated with or without 5 μmol/L kinase insert domain receptor (KDR) inhibitor SU5614 for 30 min and then treated for a further 45 min or 3 hr in the presence or absence of 100 ng/mL VEGF. Total cell lysates were prepared and equal amounts of protein were immunoblotted with antibodies to total Nur77 or Nur77 phosphorylated at serine 351. (B) HepG2 cells were pretreated for 30 min with dimethyl sulfoxide (control group and VEGF group), 5 μmol/L KDR inhibitor SU5614 (SU+V), 3 μmol/L PKC inhibitor GF109203X (GF+V), 20 μg/mL cell-permeable Ca²⁺ chelator BAPTA-AM (BP+V), 10 μmol/L phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY+V), or 3 μmol/L Src kinase inhibitor PP2 (PP+V) and then treated for 45 min with nothing (control group), 100 ng/mL VEGF alone (VEGF group), or 100 ng/mL VEGF in the presence of inhibitors. Total RNA was extracted and real-time quantitative PCR was performed to determine the expression of Nur77 mRNA. All results are expressed as mean±standard deviation (SD) of three independent experiments, each performed in triplicate.

Biologically relevant VEGF/KDR signaling mainly occurs via four pathways, including the phospholipase C-γ (PLC-γ)/protein kinase C (PKC)/mitogen-activated protein (MAP) kinase pathway, the PLC-γ/IP₃/Ca²⁺ pathway, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the Src kinase/focal adhesion kinase (Src/FAK) pathway.³³ Thus, the PKC inhibitor GF109203X, the cell-permeable Ca²⁺ chelator BAPTA-AM, the PI3K inhibitor LY294002, and the Src kinase inhibitor PP2 were used to determine which pathway is involved in induction of Nur77 expression by treatment with VEGF (Fig. 4B). GF109203X, BAPTA-AM, and LY294002 significantly suppressed VEGF-induced expression of Nur77 by 83%, 95%, and 43%, respectively. In contrast, PP2 caused a significant enhancement of VEGF-induced Nur77 expression by 2.3-fold.

Discussion

ApoM is a novel apolipoprotein that is predominantly associated with HDL. Its expression is reduced in some patients with diabetes and in animal models of diabetes. Overexpression of either the human or mouse APOM gene in LDL-receptor knockout mice could protect against the development of early atherosclerotic lesions. In humans, APOM is mainly expressed in the liver and kidney, where it is highly regulated by transcription factors, hormones, and cytokines. Cytokines, especially proinflammatory factors, have been shown to exert pleiotropic and antagonistic effects on ApoM expression.⁶ In the present study, we first demonstrated that VEGF significantly downregulated ApoM expression and pre-β-HDL formation in HepG2 cells and mouse primary hepatocytes. Moreover, we found that the modulation of ApoM expression by VEGF is mediated by inhibiting Foxa2 expression in a Nur77-dependent manner.

VEGF is a potent growth factor for endothelial cells and an inducer of angiogenesis that is important for endothelial integrity and thus for vascular function.^34,35 In addition, VEGF may have an effect on atherosclerotic plaque growth and instability through angiogenesis-independent processes.^36,37 In the present study, we found that the serum VEGF concentration was positively correlated with serum triglyceride, total cholesterol, LDL-C, , VLDL-C, and ApoB concentrations, which are classical risk factors for coronary atherosclerosis.³⁸ In addition, we observed that the serum VEGF concentration was negatively associated with serum ApoM concentration and HDL-C levels, which might be explained by our result that VEGF could downregulate ApoM expression and pre-β-HDL formation in HepG2 cells and mouse primary hepatocytes. Our observations that the serum VEGF concentration has a close relationship with these atherosclerosis-related factors suggest that serum VEGF concentration will be a candidate marker for development of atherosclerosis.

Recently, Wolfrum et al. showed that Foxa2 could regulate APOM transcription, leading to increased pre-β-HDL formation and plasma HDL levels.⁹ In the present study, we showed that VEGF could decrease ApoM expression at both the transcriptional and translational levels in a dose-dependent manner, and decrease pre-β-HDL formation. The consistency between their results and our findings may further indicate that VEGF could downregulate ApoM expression and pre-β-HDL formation through inhibiting the activity of Foxa2. Numerous studies have demonstrated that Nur77 can be rapidly induced in response to various stimuli including growth factors, inflammatory stimuli, and mechanical stimuli, and regulate metabolic function in a cell- and tissue-specific manner.^39,40

The liver exhibits a key regulatory function in metabolism; it controls the regulation of both glucose and lipid homeostasis. Nur77 is expressed in human adult liver,⁴¹ and recently, it has been shown to regulate gluconeogenesis and hepatic lipid metabolism.^12,13 Moreover, Liu et al. showed that VEGF could markedly induce expression of Nur77 and decrease Nur77 phosphorylation at the negative regulatory site serine-351.¹⁴ This raises the question of whether the effects of VEGF on the expression of ApoM and pre-β-HDL formation is through a signaling pathway that influences Nur77 expression and thus affects Foxa2.

We showed that overexpression of a Nur77 transgene in C57BL/6 mice resulted in decreased serum ApoM and pre-β-HDL levels whereas si-Nur77-treated mice displayed upregulated serum ApoM and pre-β-HDL levels. Moreover, si-Nur77-treated mice upregulated Foxa2 and ApoM expression while adenoviral overexpression of Nur77 in mice resulted in downregulated Foxa2 and ApoM expression in the liver. Furthermore, we showed that the reduction of Foxa2 expression by VEGF was significantly compensated by treatment with siRNA targeting Nur77. In addition, simultaneous treatment with both the Nur77 agonist Csn-B and VEGF led to a more significant reduction of Foxa2 expression than treatment with VEGF alone. This suggests that VEGF downregulates ApoM expression by inhibiting Foxa2 activity in a Nur77-dependent manner. Remarkably, little is known about the direct downstream target genes of the nuclear receptor Nur77. Thus, whether Foxa2 can be directly regulated by Nur77 or whether another mechanism is involved needs to be explored further.

Most biologically relevant VEGF signaling is mediated via the KDR, which is activated through ligand-stimulated receptor dimerization and trans(auto)phosphorylation of tyrosine residues in the cytoplasmic domain.^33,42,43 VEGF upregulates Nur77 expression and reduces Nur77 serine-351 phosphorylation in HepG2 cells, changes that are notably abolished by the specific KDR inhibitor SU5614, revealing that VEGF must bind to KDR before Nur77 expression is increased and Nur77 serine phosphorylation is reduced. VEGF was able to induce strong PLC-γ tyrosine phosphorylation and activation, leading to generation of diacylglycerol and inositol 1,4,5-trisphosphate and subsequent activation of PKC and Ca²⁺ mobilization.⁴⁴

We showed that both a PKC inhibitor and a cell-permeable Ca²⁺ chelator significantly suppressed VEGF-induced expression of Nur77, suggesting that activation of PLC-γ and subsequent mobilization of intracellular Ca²⁺ and activation of PKC play key roles in VEGF-induced Nur77 expression. Moreover, the PI3K inhibitor also significantly suppressed VEGF-induced expression of Nur77. This result indicates that the PI3K/Akt pathway is also involved in VEGF-induced Nur77 expression and is supported by the report that PI3K-dependent Akt is capable of phosphorylating Nur77 at Ser-351 directly and the phosphorylation of this residue is critical for the transactivation activity of Nur77.^45,46

In conclusion, we have provided strong evidence that VEGF reduces ApoM expression and pre-β-HDL formation in HepG2 cells and mouse primary hepatocytes. Expression of Foxa2 is also inhibited while the activity of Nur77 is increased when cells are treated with VEGF. As shown in Fig. 5, all these findings suggest that VEGF may first downregulate the expression of Foxa2 through the Nur77 pathway and then decrease the expression of ApoM and pre-β-HDL formation. Therefore, our study may be useful in understanding the critical effect of VEGF in the pathogenesis of atherosclerosis.

FIG. 5.

Schematic representation of inhibitory effects of vascular endothelial growth factor (VEGF) on apoliporpotein M (ApoM) expression. The results of the present study revealed the following scheme for the possible mechanism by which VEGF downregulates ApoM expression. When HepG2 cells and mouse primary hepatocytes are treated with VEGF, biologically relevant VEGF/ kinase insert domain receptor (KDR) signaling is mediated mainly via four pathways, including the phospholipase C-γ/protein kinase C/mitogen-activated protein kinase (PLC-γ/PKC/MAPK) pathway, the PLC-γ/IP₃/Ca²⁺ pathway, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and the Src/focal adhesion kinase (FAK) pathway. Then, Nur77 expression is increased and Nur77 serine-351 phosphorylation is reduced, which in turn decreases the expression of forkhead box A2 (Foxa2), and finally inhibits ApoM expression and pre-β-high-density lipoprotein (HDL) formation.

Footnotes

Acknowledgments

The authors gratefully acknowledge the financial support from the National Natural Sciences Foundation of China (81071416), and Guangdong Provincial Natural Sciences Foundation of China (07300312).

Author Disclosure Statement

There are no conflicts of interest or potential conflicts of interest.

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