Regulation of MDM2 E3 ligase-dependent vascular calcification by MSX1/2

Vascular calcification increases morbidity and mortality in patients with cardiovascular and renal diseases. Previously, we reported that histone deacetylase 1 prevents vascular calcification, whereas its E3 ligase, mouse double minute 2 homolog (MDM2), induces vascular calcification. In the present study, we identified the upstream regulator of MDM2. By utilizing cellular models and transgenic mice, we confirmed that E3 ligase activity is required for vascular calcification. By promoter analysis, we found that both msh homeobox 1 (Msx1) and msh homeobox 2 (Msx2) bound to the MDM2 promoter region, which resulted in transcriptional activation of MDM2. The expression levels of both Msx1 and Msx2 were increased in mouse models of vascular calcification and in calcified human coronary arteries. Msx1 and Msx2 potentiated vascular calcification in cellular and mouse models in an MDM2-dependent manner. Our results establish a novel role for MSX1/MSX2 in the transcriptional activation of MDM2 and the resultant increase in MDM2 E3 ligase activity during vascular calcification.

MSX binding element (MSXE)-disrupted MDM2 promoter luciferase was constructed by Bionics (Bionics). All plasmids were checked before use by direct sequencing.
RVSMCs were used at passages 2 to 7. A10 cells derived from embryonic rat aorta were purchased from American Type Culture Collection (ATCC, CRL-1476, Manassas, VA, USA) and were maintained in 10% FBS in DMEM with antibiotics. Human embryonic kidney 293T cells were obtained from the Seoul Korean Cell Line Bank (KCLB, 21573, Seoul, Korea) and were maintained in 10% FBS in DMEM with antibiotics. All cells were incubated in a humidified atmosphere with 5% CO2.

Induction of vascular calcification in vitro
For the induction of vascular calcification in VSMCs, the cells cultured in growth medium were changed with calcification medium containing 2 mM or 4 mM inorganic phosphate (pH 7.4) for up to 3 days or 6 days. The medium was changed every 2 days. Calcium deposition in VSMCs was determined after washing with 1x PBS twice.

Quantification of calcium deposition
Cells and tissues were decalcified with 0.6N HCl at 4°C for 24 hours. The calcium content of the HCl supernatants was determined colorimetrically using the QuantiChrom calcium assay kit (QuantiChromTM Calcium Assay Kit, BioAssay Systems, Hayward, USA) according to the manufacturer's instructions. Briefly, 5 μL of the samples was transferred to a 96-well plate and 200 μl working reagent was added (1:1, solution A and B). Mixed samples were briefly incubated and absorbance was measured at 570 nm by using an ELx808 absorbance reader (BTELX808, BioTek Instruments, Winooski, VT, USA). After decalcification, cells were washed 3 times with 1xPBS and lysed with 0.1N NaOH/0/1% SDS to extract proteins.
The calcium content of cells was then normalized to the total protein amount, whereas that of the tissues was normalized to tissue dry weight.

Alizarin Red S staining
The cells were washed with 1xPBS and fixed with 10% formalin for 30 min at RT. After being washed three time with distilled water, the cells were stained with 40 mM alizarin red S solution (pH 4.2, Sigma-Aldrich) for 1 hour at room temperature and washed with 1xPBS to remove nonspecific staining.
To determine arterial calcification, the aorta was collected and stored in 70% ethanol. The arterial tissue sample was placed in 10 mL of alizarin working solution containing 0.8% Alizarin red S in 0.5% KOH for 24 h with rotation and then switched to 10 ml of 0.05% KOH for 24 h to remove nonspecific stain from the tissues.

Immunoprecipitation and Western blot analysis
Cells and tissues were lysed with 0.5% NP-40 solution including 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1 μg/mL each of leupeptin, pepstatin, and aprotinin. One milligram of proteins was then immunoprecipitated overnight at 4 °C with the indicated antibody. After extensive washing with lysis buffer, the immunocomplexes were analyzed by western blotting assay. The lysates were separated by SDS-PAGE and transferred overnight at 120 mA onto a PVDF (Millipore, Bedford, MA, USA) and blocked with 5% skim milk in 1x TBST. Membranes were incubated with the specific primary antibodies overnight at 4°C. After 3 washes in 1x TBST, membranes were incubated with horseradish peroxidase-linked secondary antibodies for 1 hour at room temperature. Membranes were again washed 3 times in 1x TBST, and protein bands were visualized by enhanced chemiluminescence using Western Blotting Luminol Reagent (Santa Cruz) and a Fusion FX7 imaging system (Vilber, Marne-la-vallée, France).

Promoter luciferase reporter assay and MTT assay
For the luciferase assay, A10 cells were plated in 24-well plates and transiently transfected with plasmids containing pCMV-beta-galactosidase using Lipofectamine (ThermoFisher) and Lipofectamine RNAiMAX (ThermoFisher) according to the manufacturers' protocols. The cells were treated with 4 mM Pi for 3 days. The luciferase activity was measured by using the Luciferase Assay System (E1500, Promega) and normalized to -galactosidase activity. The data are representative of at least two to three independent experiments.

Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation assays were performed by using an EpiQuik chromatin immunoprecipitation kit (EpiGentek, Farmingdale, NY, USA) according to the manufacturer's instructions. Briefly, A10 cells were treated with 1% formaldehyde for 10 min for cross-linking between proteins and DNA and sonicated to shear the chromatin into fragments of < 500 bp. The chromatin complex was immunoprecipitated with indicated primary antibody, while the negative control was immunoprecipitated with nonimmunized IgG. Primers for qPCR of rat MDM2 promoter flanking the MSXE were forward, 5′-TGC