DAXX mediates high phosphate-induced endothelial cell apoptosis in vitro through activating ERK signaling

Animal experiments

Male SD rats (aged 6–8 weeks) were randomly divided into two groups: the CKD group (n = 5) and sham group (n = 5), All animals were obtained from the Shanghai Model Organisms Center, Inc. (Shanghai, China). All animal experiments were approved by the Ethic Committee of Shanghai Tenth People’s Hospital, School of Medicine, Tongji University (2016-33; Shanghai, China). In brief, rats were housed under constant environmental conditions with a 12-h light–dark cycle. Standard rat chow and water were provided ad libitum before and after surgery (Hsu et al., 2015). A two-step 5/6 nephrectomy was carried out to induce CKD as previously described (Hanifa et al., 2019; Uchiyama et al., 2016). Rats were anesthetized by peritoneal injection of pentobarbital (30–50 mg/kg) (Merck KGaA, Darmstadt, Germany) and right kidney was then exposed through a flank incision and ∼2/3 of the right kidney were removed. One week later, the left kidney was exposed and the upper and lower poles were resected through a flank incision. For sham rats, each flank of the kidney underwent sham surgery by incising the flanks, exposing the kidney and suturing each flank. Eight weeks after the second step of nephrectomy, rats were sacrificed and blood samples were obtained by cardiac puncture and urine was collected in individual metabolic cages.

Biochemical measurements

The concentration of serum Pi and creatinine was assessed using an automated Olympus AU2700 chemistry analyzer (Tokyo, Japan). Urea in the urine was measured using Urea Assay Kit (MAK006, Merck KGaA) according to the manufacturer’s instructions.

Cell culture and treatment

HUVECs were obtained from Lonza Group, Ltd. (Basel, Switzerland) and cultured with Endothelial Cell Growth Medium (ScienCell Research Laboratories Carlsbad, CA, USA) in a 37 °C incubator with 5% CO₂. An appropriate amounts of Na2HPO4/NaH2PO4 buffer (1 M; pH7.4) was added into the cell culture medium to achieve different phosphorus concentrations (1.0 and 3.0 mM) with or without an inhibitor of Ca-Pi crystal formation, phosphonoformic acid (PFA, 1.0mM; Merck KGaA), as previously described (Di Marco et al., 2008; Peng et al., 2011; Zhou et al., 2016). Cells were treated with different Pi concentrations in the presence or absence of PFA for 24 h. ERK inhibitor U0126 (100 ng/mL, Thermo Fisher Scientific Inc, Waltham, MA, USA) was used to treat cells for 24 h when necessary, and DMSO was used as a vehicle control.

Overexpression and Small Interfering RNAs (siRNAs)

The plasmids of pLEGFP-DAXX which encode the full-length DAXX sequence were constructed in our laboratory to overexpress DAXX in HUVECs. DAXX siRNA and negative control siRNA (NC-siRNA) were purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China). The HUVECs in the logarithmic growth phase were inoculated into culture dishes before transfection, and the cells reached 60–80% confluence on the second day. Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) was used to prepare the mixture of DAXX-siRNA, NC-siRNA, or pLEGFP-DAXX-lipofectamine 3000 according to the instructions. The mixture was then dropped evenly into each of the six well plates that already contained the cells and the culture medium. The transfection method was carried out in strict accordance with the instructions of the transfection kit. Pi or U0126 was used to treat the cells 48 h after transfection. The DAXX-siRNA sequence was 5′-GGAGTTGGATCTCTCAGAA-3′ and the NC-siRNA 5′-GUACCGCACGUCAUUCGUAUC-3′. The siRNA-treated cells were collected 48 h post-transfection for subsequent experiments.

Flow cytometry

An annexin V-FITC/PI Apoptosis Detection Kit (Beyotime, Shanghai, China) was used to detect the apoptotic rate of cells according to the manufacturer’s instructions. In brief, HUVECs treated with 1.0 mM or 3.0 mM Pi and NC-siRNA or DAXX-siRNA for 24 h were washed with precooled culture medium twice, centrifuged at 2,000 rpm for 5 min to collect cells (3 ×10⁵), and then suspended with 500 µl binding buffer (culture medium with magnesium, calcium, 0.5% NaN3 and 1% FBS). Thereupon, Annexin V (5 µl) and PI (10 µl) were added to the cells and incubated for 10 min at room temperature in the dark. A flow cytometer (Attune NxT; Thermo Fisher Scientific, Inc) and ModFitLT software were used to detect and calculate the percentage of apoptotic cells.

Quantitative PCR (qPCR)

Total RNA was extracted from HUVECs by Trizol reagent (Takara, Japan) and cDNA was synthesized using AMV reverse transcriptase (Merck Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. qPCR was carried out by SYBR Green PCR Master Mix (Takara) on an Applied 7300 Real-Time PCR System (Thermo Fisher Scientific, Inc.). Relative mRNA levels were determined by normalization to the endogenous GAPDH mRNA. The sequence-specific primer pairs for DAXX was 5′-AGAAGCAAACAGGATCAGGG-3′ forward and 5′-GCTGGGCAGGGTACATATC-3′ reverse.

Western blot analysis

The protein extracted from HUVECs lysate was detected using the BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.), and an equal amount of protein was loaded and separated on 12% SDS-PAGE for each experiment. Following electrophoresis, the protein was transferred to PVDF membranes. The primary antibodies used included cleaved caspase-3 (1 µg/ml; ab2302), DAXX (1:5000, ab32140), ERK (1:1000, ab17942) and phospho-ERK(1:1000, ab201015) (all from Abcam, Cambridge, MA, USA). The membranes were incubated for 1 h with blocking buffer plus an horseradish peroxidase-conjugated goat anti-rabbit IgG H&L secondary antibody (1:10000; ab205718, Abcam). The band intensities were analyzed using Scion Image (WinB403).

Immunohistochemistry

Frozen aorta tissues from a CKD rat model or sham rats embedded in optimal cutting temperature compound was cut into 5- µm-thick sections and fixed in 4% formaldehyde (Merck KGaA). Following washing 3 times with cold PBS, the sections were incubated with anti-Von Willebrand Factor (1:100, ab6994), and anti-DAXX (1:100, ab239806) primary antibodies (both from Abcam) overnight at 4 °C. Next, the sections were washed 3 times with PBS and incubated with Alexa Fluor 488 or Alexa Fluor 594-labeled secondary antibody for 1 h at room temperature in the dark. Three digital images were obtained from every aortic section using a confocal microscope and photographs were taken using Olympus confocal software (Olympus Corporation, Tokyo, Japan). The integrated optical density was assessed using Image-pro plus 4.5 software (Media Cybernetics, Inc., Rockville, MD, USA).

Statistical analyses

Data are represented as the mean ± SEM of at least three independent experiments. The differences between two groups were compared using one–way ANOVA or Student’s t-test using GraphPad Prism statistical software (GraphPad Software, Inc., La Jolla, CA, USA). P < 0.05 was considered to indicate a statistically significant difference.

Hyperphosphatemia was correlated with upregulated DAXX expression in endothelial cells

Previous studies have identified a number of differentially expressed genes using microarray analysis in high Pi-treated HUVECs (Qin et al., 2015). Qin et al. (2015) identified BMP4, ARPC1A, OR2D3, MYLK, GDF6, HPRT1, CDC45, ACSL1, TUBB2C, and DAXX as the top 10 differentially expressed genes in simulated hyperphosphatemia (Table S1). Given the important role of DAXX in regulating https://www.ncbi.nlm.nih.gov/pubmed/23771797 survival (Sakaue et al., 2017; Zeng et al., 2013), we attempted to investigate whether DAXX mediated high Pi-induced HUVECs apoptosis and identify its underlying mechanisms. The rat model of CKD was firstly established by 5/6 nephrectomy. Figures 1A and 1B show that CKD rats exhibited severe abnormalities in phosphorus metabolism and reveals a significant enhancement of serum creatinine, as compared with the sham-operated group. The urea level was increased in CKD rats as compared with Sham-operated rats (0.62 ± 0.11 vs 0.38 ± 0.09, P < 0.05). To assess whether DAXX is correlated with endothelial dysfunction in CKD rats, the DAXX expression in endothelial cells was examined using immunohistofluorescence. Figures 1C–1I shows that the staining intensity of DAXX protein in endothelial cells was obviously enhanced in CKD rats, compared with sham-operated group.

High Pi resulted in a significant increase in DAXX expression

In vitro, HUVECs were treated with normal or high Pi and the expression level of DAXX was then assessed using qPCR and western blot analysis. As shown in Fig. 2A, high Pi resulted in an increased mRNA expression of DAXX in HUVECs as compared with normal Pi. PFA (1 mM), an inhibitor of Ca-Pi crystal formation, reversed the high Pi-induced DAXX upregulation. Western blot analysis further verified that high Pi increased the protein level of DAXX in HUVECs as compared with normal Pi, and additional treatment of PFA reversed the high Pi-induced DAXX expression (Figs. 2B and 2C). These results demonstrated that high Pi resulted in a significant increase in DAXX expression.

DAXX mediated high phosphate-induced endothelial cell apoptosis

The frequent increase of DAXX in simulated hyperphosphatemia implied that DAXX may plays a regulatory role in disease progression. Therefore, the biological effect of DAXX in regulating HUVECs apoptosis was then examined. The results from qPCR and western blot analysis showed that DAXX level was significantly upregulated in HUVECs following DAXX overexpression (Figs. 3A, 3C and 3E). As expected, DAXX was markedly downregulated in HUVECs treated with DAXX-specific siRNAs (Figs. 3B, 3D and 3F). Functionally, the results from flow cytometry showed that high Pi resulted in a significant increase of HUVECs apoptosis, whereas DAXX knockdown repressed high Pi-induced cell apoptosis (Figs. 3G–3J).

In HUVECs treated with high Pi, a significant activation of cleaved caspase-3 was observed as compared with that in HUVECs treated with normal Pi, whereas knockdown of DAXX markedly inhibited high Pi-induced cleaved caspase-3 activation, further indicating the role of DAXX in regulating high Pi-induced HUVECs apoptosis (Figs. 4A and 4B).

DAXX mediated high Pi-induced HUVECs apoptosis through activating ERK signaling

Previous studies have reported that MAPK signaling is the most prominently aberrant pathway in high Pi-treated HUVECs, and ERK activation contributes to endothelial cell apoptosis. The present study investigated the association between DAXX and ERK activation. The western blot analysis results showed that DAXX overexpression upregulated the phosphorylated ERK (p-ERK) activity in the presence of normal Pi (Figs. 4C and 4D). Furthermore, high Pi resulted in a significant increase in p-ERK activity in HUVECs as compared with normal Pi, whereas DAXX knockdown markedly repressed the high Pi-induced activation of ERK signaling (Figs. 4C and 4D), demonstrating the regulatory role of DAXX in the activation of ERK signaling. Functional studies further showed that DAXX overexpression in HUVECs promoted cell apoptosis in the presence of normal Pi, whereas additional treatment with U0126, a specific inhibitor of ERK activation, for 24 h inhibited that effect (Figs. 5A–5D). Western blot analysis also demonstrated that DAXX overexpression in HUVECs resulted in a significant activation of cleaved caspase-3 in the presence of normal Pi, whereas additional treatment with U0126 markedly inhibited, the DAXX-induced activation of cleaved caspase-3 (Figs. 5E and 5F). These results confirmed that DAXX mediated high Pi-induced endothelial cell apoptosis by activating ERK signaling.