MicroRNA-322 inhibition of calcification of arterial smooth muscle cells by regulation of galactosyltransferase 1-associating protein UBE2Q1 and Runx2

Abstract

Background and purpose: 

Vascular smooth muscle cell (SMC) calcification represents a prominent phenotypic alteration in atherosclerosis. MicroRNA-322 (miR-322) is crucially involved in myogenic stem cell growth and differentiation. The galactosyltransferase 1-associated protein (GTAP) is a ubiquitin-conjugating enzyme E2Q1 (UBE2Q1) that serves as a critical mediator of post-translational regulation of certain cellular enzymes and transcription factors. Runt-related transcription factor 2 (Runx2) plays a critical role in arterial calcification. However, the interplay between miR-322, UBE2Q1, and Runx2 during cardiovascular calcification remain largely unknown. Therefore, the purpose of this study is to delineate the molecular mechanisms by which miR-322 regulates vascular calcification.

Methods: 

Here we examined miR-322 expression in murine SMC, and determined whether miR-322 regulates SMC calcification via modulating expression of UBE2Q1 and calcifying proteins. Murine SMC cultures or aortic segments were exposed to inorganic phosphate (Pi) for induction of calcification. Expressions of calcification-related genes in SMC with lentivirus-mediated knockdown of UBE2Q1 were determined with Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Luciferase reporter assay was performed to validate miR-322 target binding and SMC were transfected with anti-miR-322 oligonucleotides to inhibit miR-322 function.

Results: 

Aortic rings derived from UBE2Q1^−/− mice exhibited much higher calcium content compared to aortic rings from wildtype (WT) animals, following calcification induction. Knockdown of UBE2Q1 by lentiviral short hairpin RNA (shRNA) significantly enhanced the calcium deposition and expression of osteogenic gene Runx2 in SMC. Enhanced UBE2Q1 expression dramatically reduced calcification while promoting expression of contractile proteins SM22α and α-SMA. Treatment with anti-miR-322 diminished the luciferase activity in SMC transfected with the reporter gene driven by the 3′-untranslated region of UBE2Q1 mRNA. Anti-miR-322 treatment also inhibited calcification significantly.

Conclusions: 

Our study identified miR-322 regulates vascular calcification by targeting UBE2Q1. The miR-322–dependent regulation of UBE2Q1 and calcification represents a novel regulatory mechanism that controls vascular SMC function during the pathogenesis of vascular calcification.

INTRODUCTION

Calcification of the artery is an age-dependent degenerative process that involves complicated interactions between environmental factors and genetic factors and dysregulation of calcium metabolism or imbalance between promotion and inhibition of calcification^[1]. Highly calcified arteries become stiff and rigid, and could lead to hypertension^[2], myocardial ischemia, and congestive heart failure^[3].

Vascular smooth muscle cells (SMC) are the primary type of cells in the arterial wall that play a key role in the development of vascular calcification^[4,5]. Under the influence of various proatherogenic and pro-calcific factors, SMC undergo a phenotypical transformation from myogenesis to osteogenesis^[6,7].

Vascular calcification frequently happens in patients with chronic kidney disease (CKD)^[8,9], primarily due to elevated plasma phosphate levels^[10]. Calcification of cultured SMC upon exposure to inorganic phosphate (Pi) is dependent on the sodium-phosphate cotransporter Pit-1^[11]. When calcification happens, SMC transdifferentiate into osteoblast-like cells and lose contractility^[5]. This process is characterized by decreased expression of smooth muscle lineage markers, for example, smooth muscle α-actin (α-SMA) and smooth muscle 22α (SM22α) as well as increased expression of Runt-related transcription factor 2 (Runx2), a master transcription factor involved in calcification^[12].

Ubiquitin is expressed in almost all tissues of eukaryotic organisms^[13,14]. Ubiquitination of proteins controls their degradation, translocation and biologic activity^[15]. The ubiquitin-proteasome system (UPS) consists of three major enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3)^[16]. Dysfunction of the UPS is closely related to vascular calcification^[17,18].

Ubiquitin-conjugating enzyme E2 Q1 (UBE2Q1), also known as galactosyltransferase 1-associated protein (GTAP), is a member of the class III ubiquitin-conjugating enzyme family^[19]. UBE2Q1 has been implicated in several types of cancers as well as reproductive capability^[20–22]. siRNA inhibition of UBE2Q1 could increase the expression of p53^[21]. Loss of p53 gene has been linked to increased osteogenesis in bone marrow stromal cells^[23] and mesenchymal stem cells^[24]. Also, with the applications of the yeast two-hybrid system database and membrane based human proteome arrays, an E3 ligase called STUB1 (CHIP) is predicted to interact with UBE2Q1^[25]. STUB1 recognizes and binds to Runx2, a master regulator in osteogenesis and calcification. In osteoblasts, STUB1 targets Runx2 for proteasome-mediated degradation, thus inhibiting osteogenic differentiation^[26]. In a previous study by Sun et al.^[27], SMC-specific Runx2 knockout inhibits vascular calcification, indicating an important role of Runx2 in osteogenic trans-differentiation of SMC during the pathogenesis of vascular calcification.

MicroRNAs (miRs) are a class of non-coding small RNA species that promote mRNA cleavage and/or translational repression through base-paring to the 3′ untranslated region (UTR) of target mRNA^[28]. Conflicting reports have been reported concerning the role of miRs as an inhibitor^[29,30] or stimulator^[31,32] of gene expression. Recent studies implicated miRs in cardiovascular cell functions^[33,34]. For example, MiR-322/424/503 have been reported to participate in myogenic stem cell growth and differentiation^[35,36]. However, it is unknown whether miR-322 affects vascular osteogenic gene expression and calcification in vascular smooth muscle cell (SMC) during the development of vascular calcification.

In the current study, we investigated whether miR-322 impacts SMC calcification by regulating UBE2Q1 expression. We hypothesized that miR-322 serves as a critical regulatory factor of UBE2Q1 expression in SMC as well as UBE2Q1-associated vascular calcification.

METHODS

Animals and tissue collection

Age and sex matched sv129 wild type controls [wild type (WT), UBE2Q1^+/+, n = 14], heterozygous UBE2Q1-deficient (UBE2Q1^+/−, n = 4), and homozygous UBE2Q1-knockout (UBE2Q1^−/−, n = 15) mice were used in in vivo and ex vivo studies. C57BL/6J (n = 4) mice were used for isolation of SMC from aortic tissues as described previously^[37]. Anesthesia was conducted using 3% isoflurane in 100% oxygen (1.0 L/min) for 10 to 15 minutes. Euthanasia was performed by cervical disarticulation under anesthesia. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at Houston.

Histopathology and immunohistochemistry of paraffin sections

The heart and aorta were harvested from 12-month-old male mice, fixed in 10% formaldehyde, and embedded in paraffin. Sections (5 μm) were prepared using a microtome and mounted on slides for H&E staining. Images were acquired under a microscope (Nikon Eclipse TE2000-U, Tokyo, Japan) and analyzed using the inForm cell image analysis software (PerkinElmer).

SMC isolation, culture, and growth analysis

SMC were isolated from the aorta of 6-week-old male C57BL/6J mice with collagenase digestion using a standard protocol, and cultured in DME medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS). Cells were identified as SMC by the “hill and valley” growth pattern and by immunofluorescence with anti-α-SMA antibody. The average of triplicates was presented. All the experiments were performed using SMC at less than 10 passages.

Production of lentiviral vector for UBE2Q1 overexpression

Plasmid pET100D that contains murine UBE2Q1 cDNA was constructed by Dr. Michael Wassler-Akesson from the University of Texas Health Science Center at Houston, and used as the template for UBE2Q1 cDNA subcloning. Primers for polymerase chain reaction (PCR) amplification are: 5′-GGAATTCGAGCGGAGCGGAGGATGCAG-3′ (sense) and 5′-CGGGATCCGCCATCTTCCTTTGGGGGTGTGTA-3′ (antisense). PCR products of predicted size were isolated and digested by restriction endonuclease EcoRI and BamHI (New England Biolabs, Ipswich, MA) in 37°C and ligated with p3XFLAG-CMV™-14 Expression Vector (Sigma-Aldrich, St. Louis, MO). Ligation was performed with DNA Ligation Kit (Takara, Shiga, Japan).

The digested plasmid and PCR product were ligated using Ligation Mix from the Ligation Kit. Ligated DNA was transformed into One Shot TOP10 chemically competent Escherichia coli cells. After transformation, E. coli cells were cultured overnight at 37°C. Selected colonies were amplified in LB prior to isolation of plasmid DNA. Clones with verified UBE2Q1 gene was inserted into the p3XFLAG-CMV™-14 vector. To mutate the active cysteine residue in the C-terminus of UBE2Q1 into an alanine, the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) was used. Primers were designed using the online primer design tool on the Agilent Technologies website. After constructs were sequenced. Wild type UBE2Q1 gene or mutated UBE2Q1 (C351A) with 3XFLAG tag was cloned into CD513B-1 lentivirus expression vector. A total of 293ft cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS. When cells reached 60% to 80% confluence, 4.5 μg CD513B-1 plasmid that contains either WT or mutated UBE2Q1 (empty plasmid as control) was mixed with 45 μL pPACKH1 packaging plasmids (System Biosciences, Palo Alto, CA) in DMEM. Lipofectamine LTX and Plus Reagent (Life Technologies, Carlsbad, CA) was used to transfect 293ft cells.

Construction of UBE2Q1 short hairpin RNA

UBE2Q1 short hairpin RNA (shRNA) DNA templates were designed using an online tool (siDirect and Ambion), and synthesized commercially (Sigma-Aldrich). The following primers were used to amplify desired UBE2Q1 shRNA sequence: 5′-GAGCTGGAGCCATGCGAATTC-3′ (sense) and 5′-GACGAGTCGACGAGGTACCGG-3′ (antisense). PCR products of predicted size were digested with EcoRI and AgeI, and ligated with pLKO.1 shRNA cloning vector (from Dr. Jianping Jin, University of Texas Health Science Center at Houston). Colonies were verified for UBE2Q1 shRNA sequence prior to further use.

Lentiviral particles were produced following similar procedures mentioned above. A total of 293ft cells (ATCC) were plated onto 150 cm² plates and cultured in DMEM supplemented with 10% FBS. When cells reached 60% to 80% confluence, 4.5 μg pLKO.1 empty plasmid or pLKO.1 plasmid that contains UBE2Q1 shRNA was mixed with packaging plasmids (from Dr Jianping Jin) in DMEM. Lipofectamine LTX and Plus Reagent (Life Technologies) was used to transfect 293ft cells.

Detection of calcification in SMC

Calcification in cultured SMC was induced by adding NaH₂PO₄ (final concentration: 3.6 mM, pH 7.4) in 5% FBS DMEM. Calcium deposition was determined using Alizarin Red S staining and a quantitative calcium assay. For Alizarin Red S staining, cultured medium was aspirated and cells were washed with PBS three times prior to fixation using 2.5% glutaraldehyde for 15 minutes. After extensive rinsing rinsed with PBS (pH adjusted to 4.1–4.3), cells were incubated with 2% Alizarin Red S solution at 37°C for 20 minutes. Excess dye was washed off and staining was observed under a microscope. For the quantitative calcium assay, culture medium was aspirated, and cells were washed with PBS three times. Calcium deposits were dissolved using overnight incubation with 0.6 mol/L HCl. The amount of calcium in the supernatant was determined using a colorimetric calcium assay at 575-nm wavelength against a standard curve and normalized to total protein.

Treatment of vascular cells with miR inhibitor

After 4-hour incubation in serum-free medium, cells were incubated with a synthetic miR-322 inhibitor or scrambled miR control (both from Amibon) using siPORT NeoFX Transfection Agent (Amibon).

Quantitative real-time PCR analysis

Total RNA was extracted from aorta tissues and cell cultures using TRIzol reagent (Ambion, Waltham, MA). Quantitative real-time PCR (qRT-PCR) analysis of UBE2Q1 and GAPDH mRNA was performed with specific primers for UBE2Q1 (sense: 5′-ATCCACTGCAACATCACGGAA-3′; antisense: 5′-ATCCACTGCAACATCACGGAA-3′) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sense: 5′-GGTGAAGGTCGGTGTGAACG-3′; antisense: 5′-CTCGCTCCTGGAAGATGGTG-3′) (Sigma-Aldrich, St. Louis, MO) and iQ SYBR Green Supermix kits (Bio-Rad, Hercules, CA). The relative mRNA abundance was determined following normalization to GADPH mRNA.

Western blot analysis

Equal amounts of proteins were loaded on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, separated by electrophoresis and electroblot to polyvinylidene fluoride (PVDF) membrane. Primary antibodies used include primary antibodies anti-SM22 1:5000 (Abcam, Cat#10135), anti-α-SMA 1:1600 (Abcam, Cat#5694), anti-Runx2 1:1000 (Cell Signaling, Cat#8486), anti-UBE2Q1 1:1000 (Sigma-Aldrich, St. Louis, MO), anti-FLAG 1:1000 (Sigma-Aldrich, St. Louis, MO, U3509), anti-GAPDH 1:30,000 (Abcam, Cat#8245). After incubated with secondary antibodies, immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Amersham, Slough, UK).

Ex vivo calcification assays

Male mice (8–10 weeks old) were sacrificed by cervical dislocation under isoflurane anesthesia. The aorta (from aortic arch to the abdominal aorta) were cut into rings (2–3 mm in length), rinsed with sterile PBS containing 200 U/mL penicillin and 200 μg/mL streptomycin, and cultured in individual wells of 24-well plates in DMEM containing 100 U/mL penicillin, 100 μg/mL streptomycin, 5% FBS, and 1 mmol/L phosphate. Calcification was induced with exposure to high phosphate (3.6 mmol/L) for 9 days. Medium was changed every 3 days. Upon completion of the experiment, aortic ring was treated with 0.6 mol/L HCl at 37°C for 24 hours. The amount of calcium was determined colorimetrically with an o-cresolphthaleincomplexone kit from Abcam (Cambridge, UK, Cat#102505), and normalized to the dry weight.

Bioinformatics and miRNA target analysis

Analysis of predicted targets of miR-322 was based on the TargetScan prediction program (http://www.targetscan.org).

Luciferase reporter gene constructs

To generate miR-322 expression vector (pLVX-miR322), the fragment containing the miR-322 genomic sequence was amplified by PCR from C57BL/6J mouse genomic DNA and was cloned into a pLVX-shRNA2 vector (Clontech, Shiga, Japan) between BamHI and EcoRI sites. A fragment coding for UBE2Q1 3′UTR containing putative miRs targeting sites was amplified by PCR and cloned into the psiCHECK2 vector (Promega, Madison, WI) between XhoI and NotI sites downstream of the Renilla luciferase gene (Luciferase-UBE2Q1-3′UTR-WT). The negative control plasmid contains the luciferase gene alone (Luciferase-Control), and the UBE2Q1 3′UTR negative control plasmid has the insert of the luciferase reporter gene plus UBE2Q1 3′UTR mutated sequence (Luciferase-UBE2Q1-3′UTR-Mut).

Dual-luciferase reporter assays

After overnight culturing in 48-well plates (4 × 10⁴ cells in each well), HEK293 cells were co-transfected with 50 ng psiCHECK2-3′UTR-UBE2Q1 vectors and 150 ng pLVX-miRs expression vector. Cells were harvested 48 hours later, and dual-luciferase assays were conducted using the Dual-Glo Luciferase Assay System (Promega). Renilla luciferase activity was normalized to Firefly luciferase expression.

Statistical analysis

Quantitative data were presented as means ± SD. The normality of data was checked by Shapiro-Wilk test before performing comparisons with Student t test for experiment that involved only two groups or by one-way analysis of variance (ANOVA) followed by Dunnett multiple comparisons test if three or more groups were involved. P < 0.05 (2-side) was considered statistically significant.

RESULTS

UBE2Q1 deficiency promotes cardiac ischemia, arterial hyperplasia, and calcification.

In comparison to the WT control (Figure 1A), the aortic wall of UBE2Q1^−/− mice (Figure 1B) contained osteocyte-like cells with increased volume of eosin-stained cytoplasma and large hematoxylin-stained nuclei. In ex vivo experiments with aortic rings, the increase of calcium content upon incubation with Pi was more pronounced in UBE2Q1^−/− mice than either the WT or UBE2Q1 heterozygous (UBE2Q1^+/−) control (Figure 1C).

Figure 1.: Histochemistry showing that UBE2Q1 deficiency promotes arterial hyperplasia and calcification in mice. Aorta were harvested, fixed, and paraffin sectioned. Staining with H&E was performed for routine examination. H&E staining. A, WT aortic section; B, UBE2Q1^−/− aortic section. The segments of aortas dissected freshly from WT, heterozygous (UBE2Q1^+/−) and homozygous (UBE2Q1^−/−) mice were incubated with 3.6 mmol/L Pi for 9 d, and then subjected to calcium contents assays. C, Calcification of murine aortic rings with Pi exposure. Data represent means ± SD. n = 4 per group. *P < 0.05 versus WT, ^# P < 0.05 versus UBE2Q1^+/−. Arrows indicate osteocyte-like cells.H&E: hematoxylin and eosin; Pi: inorganic phosphate; UBE2Q1: ubiquitin-conjugating enzyme E2 Q1; WT: wild type.

Pi induces calcification as well as inhibits expression of UBE2Q1 mRNA and protein expression in SMC

Elevation of extracellular Pi levels has been reported to increase mineralization and calcium deposition in cultured SMC^[11]. In our experiments, incubation of SMC with Pi increased calcium deposits, as evidenced by increased staining with Alizarin Red S dye; such an increase was seen as early as after 3 days and was more pronounced after 9 days (Figure 2A). Exposure to Pi decreased the cellular levels of UBE2Q1 mRNA (Figure 2B and 2D) and protein (Figure 2C and 2E). Significant reductions in the levels of UBE2Q1 mRNA and protein were detected on days 3 and 6, respectively, and persisted to day 9.

Figure 2.: RT-PCR and Western blot assays for UBE2Q1 expression in vascular smooth muscle cells undergoing Pi-induced calcification. SMC cultured from murine aortas were incubated with 3.6 mmol/L Pi. Total RNA and proteins were extracted from SMC and subjected to RT-PCR and Western blot analysis, respectively, for assessing UBE2Q1 expression. Agarose gel electrophoresis of PCR products from GTAP and GAPDH reactions using gene-specific primers.A, Pi-treated SMC with calcium deposits stained with Alizarin Red S (red color) up to 9 d; B, RT-PCR bands in agarose gels from UBE2Q1 mRNA of Pi-exposed SMC; C, Western blot bands of UBE2Q1 protein in SMC under the same treatment as (B); D, densitometry of RT-PCR bands as shown in (B); E, Densitometry of Western blot bands as shown in (C). Data represent means ± SD. n = 3, *P < 0.05 versus day 0 controls, **P < 0.01 versus day 0 controls.GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GTAP: galactosyltransferase 1-associating protein; Pi: inorganic phosphate; RT-PCR: real-time polymerase chain reaction; SMC: smooth muscle cell; UBE2Q1: ubiquitin-conjugating enzyme E2 Q1.

UBE2Q1 but not its loss-of-functional mutant regulates calcification in SMC induced by Pi

SMC with or without UBE2Q1 overexpression were examined for calcification by incubation with Pi-rich media. Alizarin Red S staining showed apparent calcium deposits in UBE2Q1^WT and UBE2Q1^C351A overexpressing SMC upon exposure to Pi (Figure 3). SMC with UBE2Q1^WT overexpression (Figure 3A) displayed less Alizarin Red S staining and calcium accumulation than the loss-of-functional UBE2Q1^C351A mutant (Figure 3B). Quantitative comparison in the calcium contents of SMC with control vector (Mock), UBE2Q1 wild type (UBE2Q1WT) and UBE2Q1C351A mutant expression demonstrated that SMC with the loss-functional mutant UBE2Q1C351A had similar contents of calcification as the mock controls (Figure 3C). In comparison to cells transduced with control shRNA (Figure 3), SMC with UBE2Q1-specific shRNA exhibited far stronger Alizarin Red S staining as well as increased calcium content (Figure 3D).

Figure 3.: Assessment of Pi-induced calcification in murine aortic SMC with or without UBE2Q1-mutation or UBE2Q1 down-regulation. SMC carrying UBE2Q1^C351A mutant or UBE2Q1 down-regulation by shRNA were induced to calcification with Pi for 9 d, identified by Alizarin Red S staining or calcium content measurement.A, Mock cDNA vector transfected SMC,UBE2Q1^WT cDNA transfected SMC andUBE2Q1^C351A mutant cDNA transfected SMC stained with Alizarin Red S (red color); B, SMC treated with non-specific control shRNA and SMC treated with UBE2Q1-specific shRNA stained with Alizarin Red S; C, Quantitative comparison in the calcium contents of SMC with control vector (Mock), UBE2Q1 wild type (UBE2Q1^WT) and UBE2Q1^C351A mutant expression; D, Comparison in the calcium contents between SMC with or without UBE2Q1 down-regulation by UBE2Q1-specific shRNA. Data represent mean ± SD of three independent experiments, *P < 0.05 versus Mock or Ctrl shRNA, **P < 0.01 versus Ctrl shRNA, ^# P < 0.05 versus UBE2Q1^WT.GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GTAP: galactosyltransferase 1-associating protein; Pi: inorganic phosphate; SMC: smooth muscle cell; UBE2Q1: ubiquitin-conjugating enzyme E2 Q1; WT: wild type.

UBE2Q1 regulates expression of the osteogenic protein and contractile proteins expression in SMC via different mechanisms

UBE2Q1 contains a putative ubiquitin-binding domain with the active cysteine residue (Cys 351) vital for UBE2Q1 bioactivity^[19]. The UBE2Q1 mutant UBE2Q1^C351A did not inhibit Pi-induced SMC calcification (Figure 4). We next transduced SMC with lentivirus vector harboring the cDNA coding for the fusion proteins, 3XFLAG-UBE2Q1^WT and 3XFLAG- UBE2Q1^C351A. Western blot analysis confirmed high expression of the fusion protein (Figure 4A). UBE2Q1^WT SMC had increased levels of α-SMA and SM22α (Figure 4A–4C), but decreased level of Runx2 (Figure 4A and 4D). Runx2 expression did not differ between UBE2Q1^C351A and mock control SMC (Figure 4A and 4D). Overexpression of UBE2Q1^C351A enhanced, slightly but not significantly, the expression of α-SMA in SMC (Figure 4B). There was a significant increase in expression of SM22α in UBE2Q1^WT SMC, and to a lesser extent in UBE2Q1^C351A SMC (Figure 4A and 4D). Western blot analysis revealed lower α-SMA (Figure 5A and 5B), lower SM22α (Figure 5A and 5C), but higher level of Runx2 (Figure 5A and 5D) in SMC with UBE2Q1 knockdown than the negative shRNA control.

Figure 4.: Comparison in expression of osteogenic and myogenic proteins between UBE2Q1 overexpressing and UBE2Q1 ^C351A mutant SMC. Total proteins were extracted from SMC with UBE2Q1 overexpression (UBE2Q1^WT) and UBE2Q1^C351A mutant and analyzed by Western blot for expression of UBE2Q1 and osteogenic or myogenic proteins. A, Representative Western blot of FLAG, α-SMA, SM22α, and Runx2 protein expressions were shown of SMC overexpressing UBE2Q1. B–D, Quantitative densitometry was performed. Data represent mean ± SD. n = 3, *P < 0.05 versus Mock. α-SMA: smooth muscle α-actin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GTAP: galactosyltransferase 1-associating protein; Runx2: runt-related transcription factor 2; SM22α: smooth muscle 22α; SMC: smooth muscle cells; UBE2Q1: ubiquitin-conjugating enzyme E2 Q1; WT: wild type.

Figure 5.: Comparison in expression of osteogenic and myogenic proteins in SMC with UBE2Q1-specific and control shRNA. Total cellular proteins were extracted from SMC with or without UBE2Q1-shRNA knockdown via lentiviral infection and analyzed by Western blot for smooth muscle myogenic proteins (α-SMA and SM22α) and osteogenic protein (Runx2). A, Western blot bands of α-SMA, SM22α and Runx2 proteins. B–D, Densitometry of protein bands in SMC treated with UBE2Q1-specific and control shRNA.

Luciferase reporter assay indicates that miR-322 blocks UBE2Q1 mRNA 3′UTR function

In co-transfection experiments with luciferase-UBE2Q1-3′UTR-WT and pLVX-miR322 expression vector, the sense miR-322 reduced the luciferase enzymatic activity by nearly 35%. In the cells co-transfected with pLVX-miR-322 expression vector and luciferase-UBE2Q1-3′UTR-Mut, overexpression of miR-322 in SMC did not alter luciferase activity (Figure 6A and 6B).

Figure 6.: Luciferase reporter assays for the activity of miR-322 targeting UBE2Q1 mRNA 3′UTR and anti-miR-322 impact on SMC calcification by upregulating UBE2Q1 expression. HEK293 cells transfected with psiCHECK2 luciferase reporter vectors containing miR-322 targeting site downstream of the Renilla luciferase gene were transfected with pLVX-miR-322 expression vector for assessing miR-322/UBE2Q1 targeting, and the internal Firefly luciferase gene was used to normalize for transfection efficiency. Dual-luciferase assays were performed after transfection.A, Schematic representation of psiCHECK2 luciferase reporter constructs. The UBE2Q1 mRNA 3′UTR sequence containing miR-322 targeting site (UBE2Q1-3′UTR WT) or its mutant sequence (UBE2Q1-3′ UTR mut) were inserted into the psiCHECK2 reporter vector at the site downstream of the luciferase reporter gene. B, Activity of miR-322 targeted UBE2Q1 3′ UTR or 3′ UTR mutant determined by luciferase reporter assays. SMC cultured from aortas were incubated with 100 nM anti-miRNA-322. C and D, UBE2Q1 miRNA and proteins expression were analyzed by qRT-PCR and Western blot, respectively. E and F, Alizarin S staining of calcification were observed in SMC incubated with or without anti-miR-322. G, The calcium content was quantified. Data represent means ± SD. n = 3, *P < 0.05 versus Ctrl.α-SMA: smooth muscle α-actin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GTAP: galactosyltransferase 1-associating protein; Ctrl: control; miRNA: microRNA; Runx2; pLVX-miR-322: miR-322 expression vector: qRT-PCR: Quantitative real-time polymerase chain reaction; Runt-related transcription factor 2; SM22α: smooth muscle 22α; SMC: smooth muscle cells; UBE2Q1: ubiquitin-conjugating enzyme E2 Q1.

MiR-322 inhibitor increases UBE2Q1 expression and reduces Pi-induced SMC calcification

Treatment with anti-miR-322 at 100 nM markedly increased UBE2Q1 expression in SMC. This effect appeared in a dose-dependent manner as there was a less impact on the expression when exposed to the anti-miR-322 at 50 nM (Figure 6C). QRT-PCR confirmed that cells treated with anti-miR-322 contained higher levels of UBE2Q1 mRNA than the scrambled control (Figure 6D).

Accumulation of calcium deposits in SMC was detected by the calcium assay after 9 days of exposure to 3.6 mM Pi. Alizarin S staining showed significant attenuation of Pi-induced calcium deposition by 100-nM anti-miR-322 (Figure 6E and 6F). In colorimetric calcium assay, anti-miR-322 decreased calcium content by nearly 80% (Figure 6G).

DISCUSSION

SMC is the major cell type involved in vascular calcification in patients with CKD^[38,39], primarily due to elevated phosphate level caused by compromised capability of the kidneys to remove extra phosphorus from blood^[40,41]. The results from the current study indicate a pivotal role of the ubiquitin-conjugating enzyme UBE2Q1 in regulation of SMC contractile function and calcification. The results also showed miR-322 could influence SMC calcification by regulating UBE2Q1 expression. Both the ex vivo and in vitro experiments demonstrated an inhibitory effect of UBE2Q1 on calcification of vascular SMC. UBE2Q1 contains a ubiquitin-binding core (UBC) domain, an RWD domain (RING finger, WD repeats, and yeast DEAD-like helicases containing proteins) and a NH₂-terminal domain^[19]. There is a conserved cysteine residue critical for ubiquitin conjugation in the UBC domain^[19,42,43], whereas the functions of the RING finger, WD repeats, and yeast DEAD-like helicases (RWD) and NH₂-terminal domains remain unclear. Female UBE2Q1-deficient (UBE2Q1^−/−) mice have embryo implantation failure and other reproduction-associated deficits^[20]. A recent study by Wu et al.^[44] showed that estrogen inhibits vascular calcification in rats via hypoxia-induced factor-1alpha signaling, suggesting a protective role of the female hormones. Such a notion is supported by our observation of relatively low levels of calcification in female UBE2Q1^−/− mice (data not shown). The increased calcification of aortic ring in male UBE2Q1^−/− mice induced by Pi stimulation is likely due to the UBE2Q1-knockout associated elevation of pro-osteogenic factors and/or decrease of anti-calcification factors yet to be identified. In general, UBE2Q1^−/− mice have no major alteration in smooth muscle phenotypes. However, pro-osteogenic stress, such as Pi exposure, induces the osteogenic trans-differentiation of SMC null in UBE2Q1 expression, suggesting UBE2Q1 possibly plays a role in protection against vascular calcification. Strain difference could also contribute to the lack of vascular calcification in UBE2Q1^−/− mice in vivo.

Pi stimulation of calcification was accompanied by reduction in UBE2Q1 expression, suggesting that UBE2Q1 could directly mediate Pi-induced calcification in SMC. In SMC with upregulated UBE2Q1 expression, Pi-induced calcification less pronounced than in the cells with loss-of-function mutant UBE2Q1 (UBE2Q1^C351A). Thus, UBE2Q1 appears to serve as a novel regulator of vascular calcification. The finding that UBE2Q1 overexpression inhibits the expression of Runx2, an important transcription factor necessary for osteogenesis raises the possibility of UBE2Q1 as a negative regulator of Runx2 expression and function via the UPS system. Finally, UBE2Q1 enhancement promotes expression of the smooth muscle contractile proteins, such as SM22α and α-SMA. By contrast, UBE2Q1 knockout or knockdown augments expression of Runx2 and calcification. UBE2Q1-mediated ubiquitination likely contributes to Runx2 suppression in UBE2Q1-overexpressing SMC. The ubiquitin proteasomal system has been shown to regulate Runx2 expression. Thacker et al.^[45] reported that Skp2 inhibits osteogenesis by promoting ubiquitin-proteasome degradation of Runx2. Further study is warranted to determine whether UBE2Q1-mediated proteasome degradation of Runx2 is dependent on Skp2.

Replacing the active cysteine with alanine (UBE2Q1^C351A) alleviated calcification in SMC. This mutation also decreased protein expression of Runx2 moderately, though not as much as the UBE2Q1^WT, in SMC, suggesting that other than the UBC domain, the RWD or NH2-terminal domain may also contribute to the regulation of calcification process. A study by Alontaga et al.^[46] indeed demonstrated interaction of the RWD domain with Ubc9, an E2 ubiquitin-conjugating enzyme involved in SUMOylation, one of the modifications similar to ubiquitination. Enzymatic assays revealed that the RWD domain may decrease the inhibitory effect of Ubc9 on the catalysis of SUMO-activating enzyme. Our previous findings suggest that the localization of UBE2Q1 in the nucleus of embryonic stem cells (ESCs) partly depends on the NH2-terminal domain of UBE2Q1 (data not shown). The NH2-terminal domain of UBE2Q1 is comprised of a unique sequence with multiple proline and gutamine residues, and may be involved in cell differentiation^[47]. These domains are potentially important for protein transport, compartmentalization, and interaction with chaperone proteins^[48,49]. Hence, we speculate the negative regulation of calcification by UBE2Q1 probably depends on these domains as well as the UBC domain.

The action of miRNAs involves incorporation of the single-stranded miRNA into the RNA-induced silencing complex and subsequent binding of the miRNA to the 3′UTR of its target mRNA through exact complementarity with its 5′ end 7 to 8 nt^[50–52]. In this way, miRNAs produce translational inhibition. MiR-322 a member of miR-322/424-503 cluster. Initially, this cluster was linked to myogenic stem cell growth and differentiation. Shen et al.^[36] performed miR microarray analysis and found miR-322-503 cluster was expressed in the earliest cardiac progenitor cells and drives cardiomyocyte specification. MiR-322 was also reported as a new marker of disease progression in pulmonary arterial hypertension^[35]. The comparison between the 3′UTR sequence of UBE2Q1 mRNA and miR-322 shows matching segments. Our luciferase reporter experiments showed that miR-322 could selectively target the UBE2Q1 mRNA 3′UTR sequence and reduce luciferase enzymatic activity. These results suggest that miR-322 directly targets the 3′UTR of UBE2Q1 mRNA and promotes the mRNA degradation. The anti-miR-322 significantly attenuated Pi-induced SMC calcification. As shown in Figure 6, UBE2Q1 expression at mRNA and protein levels were upregulated extensively by anti-miR-322. Decreased miR-322 expression causes an upregulation of UBE2Q1 proteins perhaps due to a removal of post-transcriptional inhibition by the miR action.

CONLUSIONS

UBE2Q1 protects the arterial wall against degenerative calcification. Mice lacking UBE2Q1 expression have increased calcification and cardiovascular dysfunction. The UBE2Q1 inhibition of vascular osteogenic gene Runx2 expression may lead to the resistance to Pi-stimulated SMC calcification. In contrast, suppression of UBE2Q1 enhances Pi-induced SMC calcification. Our results also suggest that miR-322 could regulate UBE2Q1 expression and UBE2Q1-associated calcification in SMC. Limitations are present in the current manuscript, including the small sample size and lack of the information about the potential disparity of gender in terms of vascular calcification.

FUNDING

This work was supported by research funds from Hermann Foundation and various private donations.

AUTHOR CONTRIBUTIONS

GSS and GS participated in the research design. All authors participated in the performance of the research. All authors contributed new reagents or analytic tools. GSS and GS participated in the data analysis. GSS, GS, and GYJ participated in the writing of the paper.

CONFLICTS OF INTEREST STATEMENT

The authors declare that they have no conflict of interest with regard to the content of this manuscript.

DATA SHARING STATEMENT

The data, analytic methods, and study materials are available for onsite audit by third parties for purposes of reproducing the results or replicating the procedure.