PP2A catalytic subunit silence by microRNA-429 activates AMPK and protects osteoblastic cells from dexamethasone

https://doi.org/10.1016/j.bbrc.2017.04.111Get rights and content

Highlights

  • miR-429 putatively targets the 3′UTR of PP2A-catalic subunit (PP2A-c) mRNA.

  • miR-429 expression downregulates PP2A-c and activates AMPK in osteoblastic cells.

  • miR-429 expression alleviates dexamethasone (Dex)-induced osteoblastic cell death.

  • AMPK activation mediates miR-429-mediated osteoblast cytoprotection against Dex.

  • AMPK activation by miR-429 inhibits Dex-induced ROS production in osteoblastic cells.

Abstract

Activation of AMP-activated protein kinase (AMPK) could efficiently protect osteoblasts from dexamethasone (Dex). Here, we aim to induce AMPK activation through miRNA-mediated downregulating its phosphatase, protein phosphatase 2A (PP2A). We discovered that microRNA-429 (“miR-429”) targets the catalytic subunit of PP2A (PP2A-c). Significantly, expression of miR-429 downregulated PP2A-c and activated AMPK (p-AMPKα1 Thr172) in human osteoblastic cells (OB-6 and hFOB1.19 lines). Remarkably, miR-429 expression alleviated Dex-induced osteoblastic cell death and apoptosis. On the other hand, miR-429-induced AMPK activation and osteoblast cytoprotection were almost abolished when AMPKα1 was either silenced (by targeted shRNA) or mutated (T172A inactivation). Further studies showed that miR-429 expression in osteoblastic cells increased NADPH (nicotinamide adenine dinucleotide phosphate) content to significantly inhibit Dex-induced oxidative stress. Such effect by miR-429 was again abolished with AMPKα1 silence or mutation. Together, we propose that PP2A-c silence by miR-429 activates AMPK and protects osteoblastic cells from Dex.

Introduction

Long-term and/or excessive clinical use of glucocorticoids could lead to osteoporosis or even osteonecrosis [1]. Dexamethasone (Dex) and other glucocorticoids will induce direct damages to osteoblasts [1], [2], [3]. Indeed, osteoblast cell apoptosis is often detected in bones of Dex-taking patients [4], [5]. Our group [6], [7], [8], [9] and others [10], [11], [12] have been adding Dex to the cultured osteoblasts/osteoblastic cells, which mimics glucocorticoid-induced cell injuries [4], [7], [8], [13], [14]. This cellular model is utilized to explore the pathological mechanisms of Dex-induced osteoblast cell damages, which shall help to develop possible intervention strategies [6], [7], [8], [9].

AMP-activated protein kinase (AMPK) is a highly-conserved serine/threonine protein kinase [15]. It is a key energy sensor, which controls cell metabolism during environmental stresses [15]. Emerging evidences have implied that AMPK activation could also promote cell survival [16]. Studies, including ours [9], [17], have investigated the potential function of AMPK in osteoblasts [9], [18], [19], [20]. It was found that genetic or pharmacological activation of AMPK could efficiently protect osteoblasts from Dex [9], [18], [19], [20].

Studies have suggested that protein phosphatase 2A (PP2A) is a key AMPK phosphatase [21], [22]. Inhibition of PP2A, with a selective PP2A inhibitor (okadaic acid) or PP2A knockdown, could provoke or restore AMPK activation [21], [22]. PP2A is composed of a scaffold (PP2A-a) and a catalytic subunit (PP2A-c) [21], [22]. The 36 kDa PP2A-c is tightly associated with the 65 kDa PP2A-c to form the core structure of PP2A [21], [22]. In the current study, we aim to identify microRNA (“miRNA”) that could possibly activate AMPK via silencing PP2A-c. Our results here discover that microRNA-429 (“miR-429”) selectively targets 3’-untranslated region (UTR) of PP2A-c mRNA. Significantly, expression of miR-429 silences PP2A-c to activate AMPK signaling, therefore protecting osteoblastic cells from Dex.

Section snippets

Chemicals and reagents

Dex, neomycin and puromycin were obtained from Sigma Aldrich (Shanghai, China). Cell culture reagents were provided by Gibco (Shanghai, China). Antibodies of this study were purchased from Cell Signaling Technology (Danvers, MA).

Cell culture

The OB-6 [4] and hFOB1.19 [23] human osteoblastic cell lines were obtained from the Cell Bank of Shanghai Institute of Biological Science (Shanghai, China). Osteoblastic cells were cultured as described [4], [23].

miR-429 transfection

Osteoblastic cells were initially transfected with 30 nM

Expression of miR-429 downregulates PP2A catalytic subunit and activates AMPK in human osteoblastic cells

Our previous studies [9], [17] have demonstrated that AMPK activation could inhibit Dex-induced osteoblast cell death. In the current study, we aim to provoke AMPK activation via miRNA-mediated downregulating its phosphatase PP2A [21], [22]. TargetScan miRNA database was searched. We discovered that miR-429 putatively targets the 3′UTR of PP2A-c mRNA (Fig. 1A). To study the potential effect of miR-429 on PP2A-c, miR-429 was introduced to hFOB1.19 osteoblastic cells, and stable hFOB1.19 cell

Discussions

AMPK is mainly composed of the catalytic α subunit and β/γ regulatory subunits [27], [28]. Multiple isoforms and transcriptional variants of these subunits have been characterized [27], [28]. Thr172 phosphorylation of AMPKα1 subunit is key to AMPK activation [27], [29], [30]. Different groups have been focusing on the mechanisms of AMPKα1 phosphorylation [31]. Several AMPKα1 kinases have been identified, including LKB1 (Liver kinase B1) [30], CaMKK (calcium/calmodulin-dependent protein kinase

Conflicts of interest

The listed authors have no conflict of interests.

Acknowledgments

This work is supported by the National Natural Science Foundation (No. 81672170. To: Feng Ji).

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