Runx2/miR-3960/miR-2861 Positive Feedback Loop Is Responsible for Osteogenic Transdifferentiation of Vascular Smooth Muscle Cells

We previously reported that Runx2/miR-3960/miR-2861 regulatory feedback loop stimulates osteoblast differentiation. However, the effect of this feedback loop on the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) remains unclear. Our recent study showed that miR-2861 and miR-3960 expression increases significantly during β-glycerophosphate-induced osteogenic transdifferentiation of VSMCs. Overexpression of miR-2861 or miR-3960 in VSMCs enhances β-glycerophosphate-induced osteoblastogenesis, whereas inhibition of miR-2861 or miR-3960 expression attenuates it. MiR-2861 or miR-3960 promotes osteogenic transdifferentiation of VSMCs by targeting histone deacetylase 5 or Homeobox A2, respectively, resulting in increased runt-related transcription factor 2 (Runx2) protein production. Furthermore, overexpression of Runx2 induces miR-2861 and miR-3960 transcription, and knockdown of Runx2 attenuates β-glycerophosphate-induced miR-2861 and miR-3960 transcription in VSMCs. Thus, our data show that Runx2/miR-3960/miR-2861 positive feedback loop plays an important role in osteogenic transdifferentiation of VSMCs and contributes to vascular calcification.


Introduction
Medial artery calcification is a common and serious problem with ageing of the population. It is a nonocclusive process which leads to decreased vessel elasticity, increased blood pressure, and higher risk of cardiovascular mortality [1][2][3]. Medial artery calcification is now recognized as a highly regulated process which is similar in many ways to bone mineralization. Osteoblast-like phenotypes transformation of vascular smooth muscular cells (VSMCs), the predominant cells in the tunica media of arteries, is currently considered responsible for the formation of medial artery calcification [4][5][6]. However, the specific mechanism governing this process is still elusive.
MicroRNAs (miRNAs) are endogenous, small (16∼25 nucleotides), single-stranded noncoding RNAs [7]. MicroR-NAs mediate translational repression or degradation of target transcript by binding to sites of complementarity in the 3 -UTRs of target mRNAs. During the past decade, they have emerged as powerful posttranscriptional regulators of gene expression [8]. To date, more than 3% of the genes in the human genome have been found to encode for miRNAs, and over 30% of genes in the human genome are estimated to be regulated by miRNAs [9]. It has been documented that miRNAs participate in cellular proliferation, differentiation, migration, and apoptosis [10][11][12]. However, their roles in osteogenic transdifferentiation or calcification of VSMC have just begun to be understood.

BioMed Research International
Recently, we reported that Runx2/miR-3960/miR-2861 positive feedback loop is responsible for osteoblast differentiation [13]. Osteoblast differentiation signals lead to the activation of Runx2 transcription factor in stromal cells. In addition to induction of genes essential for osteoblast differentiation, Runx2 transactivates miR-3960/miR-2861. In turn, miR-3960 and miR-2861 maintain the levels of Runx2 mRNA and protein via repressing Homeobox A2 (Hoxa2) and histone deacetylase 5 (HDAC5) and stabilizing the osteoblast differentiation. The objective of this study was to elucidate the effect of Runx2/miR-3960/miR-2861 feedback loop on regulation of the osteogenic transdifferentiation of VSMCs.

Cell Culture and Transfection.
To isolate primary cells, eight-week-old C57BL/6 male wild type mice received a 150 mg/kg intraperitoneal dose of pentobarbital sodium prior to euthanasia which was confirmed by the absence of a heartbeat. Vascular smooth muscle cells (VSMCs) were isolated from aorta as previously described [14]. VSMCs were cultured in DMEM supplemented with 10% FBS. To induce osteogenic transdifferentiation, VSMCs between passages 3 and 7 were cultured in DMEM supplemented with 10 mM -glycerophosphate. For transient transfection of miR-2861, miR-3960, anti-miR-2861, anti-miR-3960, and Runx2 siRNA, a mixture of each of them or their control with Lipofectamine 2000 (Invitrogen) was added to cells in 24-well plates at a density of 3 × 10 4 cells per well for 48 hours.

Western Blot.
To detect Runx2, HDAC5, and Hoxa2 protein expression, Western blot analysis was performed as previously described [16,17]. Briefly, equal amounts of protein (40 g/lane) were subjected to SDS gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, GE Healthcare Biosciences, Pittsburgh, PA, USA). The membranes were incubated with primary antibodies against Runx2 (Santa Cruz), HDAC5 (Santa Cruz), Hoxa2 (Santa Cruz), andactin (Abcam) overnight at 4 ∘ C and then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) for 1 h at room temperature. The blots then were visualized with the chemiluminescent detection method using the SuperSignal West Pico Substrate (Pierce Biotechnology).

ALP Activity and Osteocalcin Secretion Assay.
Cells were grown to confluence in 24-well plates. The cells were then washed with PBS and scraped into a solution containing 20 mM Tris-HCl, pH 8.0, and 150 mM NaCl and 1% Triton X-100, 0.02% NaN 3 , and 1 g/mL aprotinin. The lysates were homogenized; then alkaline phosphatase (ALP) activity was assayed by spectrophotometric measurement of pnitrophenol release at 37 ∘ C. Osteocalcin released into the culture media was measured using a specific radioimmunoassay kit (DiaSorin). To normalize protein expression to total cellular protein, a fraction of the lysate solution was used in a Bradford protein assay.

Statistical
Analyses. Data were presented as mean ± s.e.m. Comparisons were made using a one-way analysis of variance. All experiments were repeated at least three times, and representative experiments were shown. Differences were considered significant at < 0.05.

Increased Expression of miR-2861 and miR-3960 during the Osteogenic Transdifferentiation of VSMCs.
We performed Northern blot analysis to examine the expression of miR-2861 and miR-3960 in VSMCs during -glycerophosphateinduced osteogenesis. Both miR-2861 and miR-3960 could be detected after treatment with -glycerophosphate for 12 h and increased with time throughout osteoblastic differentiation of VSMCs ( Figure 1).  (Figure 2(a)). The transfected VSMCs were treated with -glycerophosphate for 48 h for inducing osteogenesis. Then, ALP activity, osteocalcin secretion, and Runx2 protein expression, as markers of osteoblast differentiation, were evaluated. The levels of ALP, osteocalcin, and Runx2 were all elevated in cells with overexpression of miR-2861 or miR-3960 (Figure 2(b)). These results indicated that overexpression of miR-2861 or miR-3960 promoted osteogenic transdifferentiation of VSMCs (Figures 2(b) and 2(c)). In contrast, knockdown of miR-2861 or miR-3960 reduced ALP, osteocalcin, and Runx2 levels induced byglycerophosphate (Figures 3(a) and 3(b)). All of these results suggest that both miR-2861 and miR-3960 act to promote osteogenic transdifferentiation of VSMCs.

miR-2861 and miR-3960 Posttranscriptionally Repress
HDAC5 and Hoxa2 Expression. We previously identified that HDAC5 and Hoxa2 are the direct targets of miR-2861 and miR-3960, respectively, in a stromal cell line ST2 [13]. Here, we overexpressed miR-2861 or miR-3960 to directly identify their actions on HDAC5 or Hoxa2 in VSMCs. As expected, overexpression of miR-2861 or miR-3960 resulted in a decrease in the HDAC5 or Hoxa2 protein levels (Figures 4(a)  and 4(b)). However, the HDAC5 or Hoxa2 mRNA levels were not affected (Figures 4(a) and 4(b)). These results revealed that miR-2861 and miR-3960 posttranscriptionally repressed HDAC5 and Hoxa2 protein expression by inhibiting mRNA translation in VSMCs.

Runx2 Stimulates miR-2861 and miR-3960 Expression in Vascular Smooth
Muscle. We previously demonstrated that Runx2 directly induces the expression of the miR-3960/miR-2861 cluster by binding to the putative binding site of its promoter in ST2 cells [13]. Here, in order to identify the direct impact of Runx2 on miR-2861 and miR-3960 gene expression in VSMCs, we changed the functional levels of Runx2. First, we used a pcDNA-driven expression vector to overexpress Runx2 protein levels, as confirmed by Western blot (Figure 5(a)). The results showed that Runx2 overexpression could induce miR-2861 and miR-3960 expression in VSMCs (Figure 5(a)). Furthermore, we transfected siRNA designed against Runx2 into -glycerophosphate-induced VSMCs to specifically silence Runx2 expression. Western blot analysis revealed that si-Runx2 blocked Runx2 expression compared with the control (Figure 5(b)). The expression of miR-3960 and miR-2861 was inhibited by the knockdown of Runx2 (Figure 5(b)). Thus, these results suggest a direct stimulatory action of Runx2 on the expression of miR-2861 and miR-3960 in VSMCs.

Discussion
In our previous study, we cloned both miR-2861 and miR-3960 and identified that miR-2861 induces osteoblast differentiation by repressing HDAC5, an enhancer of Runx2 degradation, and miR-3960 induces osteoblast differentiation by repressing Hoxa2, a repressor of Runx2 expression [13,15]. miR-2861 binds to the coding domain sequence (CDS) of HDAC5 mRNA with complementarity to the miR-2861 seed region and posttranscriptionally represses HDAC5 protein expression by inhibiting mRNA translation and not by mRNA degradation [15]. Similarly, miR-3960 directly targets Hoxa2 by specifically binding with the target CDS of Hoxa2 and represses Hoxa2 expression at the posttranscriptional level [13]. Thus, miR-3960 and miR-2861 can coregulate the Runx2 during osteoblast differentiation. In turn, Runx2 binds to the miR-2861/miR-3960 cluster promoter to transcriptionally induce the expression of miR-2861 and miR-3960 [13]. Runx2/miR-3960/miR-2861 is a critical positive feedback loop for osteoblast differentiation.
Recently, several miRNAs have been reported to be involved in osteogenic transdifferentiation and calcification of VSMCs [18][19][20][21][22]. It has been reported that miR-221 and miR-222 increase runt-related transcription factor 2 (Runx2) expression and calcium deposition in VSMCs [18]. On the contrary, miR-204, miR-133a, and miR-125b suppress osteoblastic differentiation of VSMCs by inhibiting Runx2 protein expression, whereas miR-204, miR-133a, and miR-125b inhibition enhance osteoblastic differentiation of VSMCs [19][20][21][22]. In this study, we show that Runx2/miR-3960/miR-2861 is also responsible for stimulating osteogenic transdifferentiation of VSMCs. At first, we demonstrated that the expression of both miR-2861 and miR-3960 was significantly increased during -glycerophosphate-induced osteogenic transdifferentiation of VSMCs, indicating that miR-2861 and miR-3960 might play a role in vascular calcification. To prove whether miR-2861 and miR-3960 are directly associated with the osteogenic transdifferentiation of VSMCs, we evaluated the effect of miR-2861 and miR-3960 in the process of -glycerophosphate-induced osteogenic transdifferentiation of VSMCs. Runx2 has been identified as a "master transcription factor" simulating osteogenic transdifferentiation of precursor cells [4,23]. It plays an essential role in the osteogenic transdifferentiation of VSMCs [24,25]. Knockdown of Runx2 significantly inhibits the ALP expression and the calcification in the senescent VSMCs [26]. ALP is a prophase (proliferative phase) marker of osteoblast differentiation. Osteocalcin is a metaphase (bone matrix formation period) marker of osteoblast differentiation. Both miR-2861 and miR-3960 promoted osteogenic transdifferentiation of VSMCs, as indicated by increased levels of ALP activity, osteocalcin secretion, and Runx2 protein expression.