miR-125b/Ets1 axis regulates transdifferentiation and calcification of vascular smooth muscle cells in a high-phosphate environment

doi:10.1016/j.yexcr.2014.01.025

Experimental Cell Research

Volume 322, Issue 2, 1 April 2014, Pages 302-312

https://doi.org/10.1016/j.yexcr.2014.01.025 Get rights and content

Highlights

•
β-glycerophosphoric acid can induce rat aortic SMCs transdifferentiated to osteoblasts.
•
microRNA 125b regulated VSMCs phenotypic transition and calcification.
•
Ets1 was upregulated during the VSMCs phenotypic transition and regulated by miR-125b.
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Knockdown Ets1 in VSMCs inhibited VSMCs phenotypic transition and calcification induced by β-glycerophosphoric acid.
•
The expressions of miR-125b were down-regulated in human calcified radial arteries.

Abstract

Objectives

Vascular calcification is highly prevalent in patients with chronic kidney disease (CKD) and contributes to increased risk of cardiovascular disease and mortality. Accumulated evidences suggested that vascular smooth muscle cells (VSMCs) to osteoblast-like cells transdifferentiation (VOT) plays a crucial role in promoting vascular calcification. MicroRNAs (miRNAs) are a novel class of small RNAs that negatively regulate gene expression via repression of the target mRNAs. In the present work, we sought to determine the role of miRNAs in VSMCs phenotypic transition and calcification induced by β-glycerophosphoric acid.

Approach and results

Primary cultured rat aortic VSMCs were treated with β-glycerophosphoric acid for different periods of time. In VSMCs, after β-glycerophosphoric acid treatment, the expressions of cbf β1, osteocalcin and osteopontin were significantly increased and SM-22β expression was decreased. ALP activity was induced by β-glycerophosphoric acid in a time or dose dependent manner. Calcium deposition was detected in VSMCs incubated with calcification media; then, miR-125b expression was detected by real-time RT PCR. miR-125b expression was significantly decreased in VSMCs after incubated with β-glycerophosphoric acid. Overexpression of miR-125b could inhibit β-glycerophosphoric acid-induced osteogenic markers expression and calcification of VSMCs whereas knockdown of miR-125b promoted the phenotypic transition of VSMCs and calcification. Moreover, miR-125b targeted Ets1 and regulated its protein expression in VSMCs. Downregulating Ets1 expression by its siRNA inhibited β-glycerophosphoric acid-induced the VSMCs phenotypic transition and calcification.

Conclusion

Our study suggests that down-regulation of miR-125b after β-glycerophosphoric acid treatment facilitates VSMCs transdifferentiation and calcification through targeting Ets1.

Introduction

Vascular calcification is highly prevalent in patients with chronic kidney disease (CKD) and contributes to increased risk of cardiovascular disease and mortality [1], [2]. Medial artery calcification is more prevalent in CKD and ESRD (end stage renal disease) patients, and this phenotype is also known as Monckeberg׳s medial sclerosis [3], [4]. Vascular smooth muscle cells (VSMCs) are the predominant cells in the tunica media of arteries. They play a critical role in regulating the blood vessel tone, which in turn influences blood pressure. VSMCs are of mesenchymal origin and under stress could differentiate to different mesenchymal-derived cell types such as osteoblasts, chondrocytes, and adipocytes, leading to calcification, altered matrix production, and lipid accumulation in the vessel wall [4], [5]. In response to high phosphate stimulus, VSMCs increase expression of osteogenic genes such as cbfα1, osteopontin, and osteocalcin, meanwhile decrease expression of VSMCs differentiation marker genes including smooth muscle α-actin (α-SMA), SM-22α and calponin. Therefore, instead of a passive process involving spontaneous calcium phosphate precipitation in necrotic tissue, calcification is an active regulated process of osteoblastic differentiation of VSMCs. However, the specific pathways governing this active process is still unknown.

MicroRNAs are a novel class of small RNAs that negatively regulate gene expression via repression of the target mRNAs [6], [7]. Numerous miRNAs have been identified or predicted; however, their cellular targets, biologic roles and disease relevance are still under investigation. In VSMCs, key miRNAs such as miR-21, miR-143, miR-145, and miR-221 have recently been shown to play important roles in phenotypic changes, migration, proliferation, and neointimal thickening [8], [9], [10], [11]. Besides, miR-125b has been described as a marker in vascular calcification [12], and has been demonstrated as inhibitors of osteoblastic differentiation [13]. Another study demonstrated a novel upstream role for miR-125b in the epigenetic regulation of inflammatory genes in VSMC of db/db mice [14]. In the present study, we sought to determine whether microRNAs (miRNAs) play a role during the process of VSMCs phenotypic transition and calcification induced by β-glycerophosphoric acid (β-GP). β-GP as a donor of organic phosphate has been confirmed to induce VSMCs transdifferentiate into osteoblasts via several mechanisms [5], [15], [16].

Ets1, as the first member of ets family, is a evolutionarily related, DNA-binding transcriptional factor, and is involved in regulating a wide variety of biological processes, including cellular growth, migration and differentiation [17], [18]. During the process of bone formation, Ets1 is expressed in proliferating preosteoblastic cells and mainly promotes osteoblasts proliferation, differentiation and mineralization. Previous studies have shown that Ets1 plays critical role in mediating vascular inflammation and remodeling [19], [20]. However, the role of Ets1 on VSMCs phenotypic transition has not been elucidated in details. Zhang et al. reported that Ets1 was a novel direct target of miR-125b and explored the role of miR-125b/Ets1 axis in breast cancer [21]. However, the contribution of miR-125b/Ets1 axis to vascular calcification has not been explored.

Evidences from our studies suggest that β-GP induces osteoblastic transition of VSMCs, but down-regulates miR-125b expression in VSMCs. Ectopic expression of miR-125b inhibits VSMCs transdifferentiation and calcification induced by high phosphorus. Manipulating the expression of miR-125b changes the expression of Ets1, a reported target, in vitro. Our studies identify miR-125b as a pivotal mediator for regulating VSMCs calcification.

Section snippets

Cell culture and treatment

Rat aortic SMCs were cultured as described previously [22], [23]. Cell cultures were maintained in DMEM/F12 containing 20% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and neomycin. Cells were grown to confluence and used from passages 3–8. For high phosphorus treatment, cells were incubated with 10 mmol/l β-glycerophosphoric acid (Sigma-Aldrich), after different time of treatment, cells were harvested for real-time RT-PCR or Western blotting. MiR-125b mimics, inhibitors

High phosphorus induces VSMCs transdifferentiation to osteoblast-like cells

The initial aim of the present studies was to determine whether our cultured rat aortic SMCs had an ability to convert into osteogenic cells under high phosphate concentration, as described previously by other laboratories [4], [5], [15], [28], [29], [30], [31]. We first examined the effect of phosphorus on osteogenic gene expression by incubating rat aortic SMCs with β-glycerophosphoric acid (10.0 mmol/l) for 72 h. As shown in Fig. 1a, β-glycerophosphoric acid induced cbfα1, collagen I and

Discussion

Accumulated evidences suggested that VSMCs to osteoblast-like cells transdifferentiation (VOT) plays a crucial role in promoting vascular calcification [4], [5], [15]. Many recent studies have shown that elevated serum phosphorus level is one of the causative agents of vascular calcification [33], [35]. However, the precise mechanism involved in phosphorus-induced VOT remained undefinite. In our study, we presented persuasive evidence suggesting that through repressing Ets1 expression, miR-125b

Disclosures

None.

Acknowledgments and sources of funding

This work was supported by Scientific Research Fund of Jiangsu Provincial Science and Technology Department to Yang J (BL2013037). Natural Science Foundation of Jiangsu Province of China (BK2012870) to W. He.

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Research ArticlemiR-125b/Ets1 axis regulates transdifferentiation and calcification of vascular smooth muscle cells in a high-phosphate environment

Highlights

Abstract

Objectives

Approach and results

Conclusion

Introduction

Section snippets

Cell culture and treatment

High phosphorus induces VSMCs transdifferentiation to osteoblast-like cells

Discussion

Disclosures

Acknowledgments and sources of funding

Kidney Int.

Cell

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Kidney Int.

Am. J. Pathol.

Eur. J. Pharmacol.

Chronic kidney disease and mortality risk: a systematic review

J. Am. Soc. Nephrol.

Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality

Nephrol. Dial. Transplant

Beta-glycerophosphate accelerates calcification in cultured bovine vascular smooth muscle cells

Arterioscler. Thromb. Vasc. Biol.

Mir-145 and mir-143 regulate smooth muscle cell fate and plasticity

Nature

MicroRNA expression signature and antisense-mediated depletion reveal an essential role of microRNA in vascular neointimal lesion formation

Circ. Res.

A necessary role of mir-221 and mir-222 in vascular smooth muscle cell proliferation and neointimal hyperplasia

Circ. Res.

Smooth muscle cell phenotypic transition associated with calcification: upregulation of cbfa1 and downregulation of smooth muscle lineage markers

Circ. Res.

The ets-domain transcription factor family

Nat. Rev. Mol. Cell. Biol.

Opposing effects of ets and id proteins on p16ink4a expression during cellular senescence

Nature

Ets-1 is a critical regulator of ang ii-mediated vascular inflammation and remodeling

J. Clin. Invest.

Regulated expression of the ets-1 transcription factor in vascular smooth muscle cells in vivo and in vitro

Circ. Res.

Research Article
miR-125b/Ets1 axis regulates transdifferentiation and calcification of vascular smooth muscle cells in a high-phosphate environment