Activation of AMP-activated protein kinase decreases receptor activator of NF-κB ligand expression and increases sclerostin expression by inhibiting the mevalonate pathway in osteocytic MLO-Y4 cells

https://doi.org/10.1016/j.bbrc.2015.12.072Get rights and content

Highlights

  • There are no studies on effects of AMPK activation on RANKL and sclerostin expressions in osteocytes.

  • There are no studies on the roles of mevalonate pathway in osteocytes.

  • AMPK activation decreased RANKL and increased sclerostin expression in MLO-Y4 cells by inhibiting mevalonate pathway.

  • Simvastatin decreased RANKL expression and increased sclerostin expression in MLO-Y4 cells.

Abstract

Background

AMP-activated protein kinase (AMPK) plays important roles in bone metabolism; however, little is known about its role in osteocytes. This study investigated the effects of AMPK activation on the expression of receptor activator of NF-κB ligand (RANKL) and sclerostin in osteocytes.

Results

Real-time PCR showed that AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) significantly decreased the expression of Rankl in a dose- and time-dependent manner and significantly increased the expression of Sost, the gene encoding sclerostin, in osteocytic MLO-Y4 cells. Western blotting confirmed that AICAR decreased RANKL protein levels and increased sclerostin levels. In addition, suppression of AMPKα1 by siRNA significantly increased the expression of Rankl on 4 days after the transfection of siRNA, while Sost expression was not changed. Simvastatin, an inhibitor of HMG-CoA reductase, significantly decreased Rankl expression and increased Sost expression in MLO-Y4 cells. Supplementation with mevalonate or geranylgeranyl pyrophosphate, which are downstream metabolites of HMG-CoA reductase, significantly reversed the effects of AICAR.

Conclusion

These findings indicated that AMPK regulated RANKL and sclerostin expression through the mevalonate pathway in osteocytes.

Introduction

Bone tissue is constantly renewed by a balanced between bone formation and bone resorption. Several studies have shown that osteocytes play multifunctional roles in orchestrating bone remodeling by regulating both osteoblast and osteoclast functions [1], [2]. A recent study showed that osteocytes expressed much higher levels of receptor activator of nuclear factor-κB ligand (RANKL) and had a great capacity to support osteoclastogenesis [3]. Previous studies have indicated that osteocyte-derived RANKL plays a key role in bone remodeling in response to mechanical loading [3], [4], [5]. Thus, osteocytes are the main cells involved in the initiation of bone remodeling. In addition, osteocytes produce osteoprotegerin (OPG), a decoy receptor for RANKL. Thus, osteocytes regulate bone resorption by regulating RANKL/OPG ratio [2]. Osteocytes also produce sclerostin, a protein encoded by Sost, that inhibits osteoblast activity by blocking Wnt/beta-catenin pathway [6], [7].

AMP-activated protein kinase (AMPK) is a crucial regulator of energy and metabolic homeostasis at the cellular and whole-organism levels [8], [9]. AMPK is a heterotrimeric complex containing a catalytic α subunit and regulatory β and γ subunits and functions as a serine/threonine kinase [10]. An increase in cellular AMP/ATP ratio activates AMPK through the phosphorylation of the α subunit (Thr 172). Once activated, AMPK inactivates several metabolic enzymes involved in ATP-consuming cellular events, including cholesterol and protein synthesis, by inhibiting HMG-CoA reductase [11].

Increasing evidence indicates that osteoporosis is a disorder of energy metabolism. Recent studies have shown that the AMPK signaling pathway plays pivotal roles in bone physiology [12]. AMPK subunits are expressed in bone tissue and cells, with AMPKα1 subunit being the dominant catalytic isoform expressed in the bone [13]. A study showed that mice lacking the AMPKα1 subunit (AMPKα1−/− mice) experienced a significant reduction in bone mass [14], suggesting that this subunit played a major role in skeletal metabolism. Activated AMPK inhibits osteoclast formation and bone resorption in vitro [15]. We previously showed that AMPK activation stimulated the differentiation and mineralization of osteoblastic MC3T3-E1 cells by inhibiting mevalonate pathway [16], [17], [18]. Moreover, we recently reported that AMPK activation exerted protective effects against homocysteine-induced apoptosis of osteocytic MLO-Y4 cells [19].

However, the effects of AMPK activation on RANKL and sclerostin expression in osteocytes are unclear. This is the first study to show that AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) decreased RANKL expression and increased sclerostin expression by inhibiting the mevalonate pathway in osteocytic MLO-Y4 cells.

Section snippets

Reagents

Cell culture medium and supplements were purchased from Gibco-BRL (Rockville, MD). AICAR and antibodies against total AMPKα and phosphorylated AMPKα were purchased from Cell Signaling (Beverly, MA). Antibodies against AMPKα1 and α2 subunits were purchased from Abcam (Tokyo, Japan). Simvastatin, mevalonate, and geranylgeranyl pyrophosphate (GGPP) were purchased from Sigma–Aldrich (St. Louis, MO). Antibodies against RANKL and sclerostin were purchased from Santa Cruz Biotechnology (Santa Cruz,

AMPK activation increases RANKL expression and decreases sost expression in MLO-Y4 cells

We have previously shown that all AMPK subunits are expressed in MLO-Y4 cells [19]. In this study, we confirmed the mRNA expression of the AMPKα1 and AMPKα2 subunits, the catalytic subunit (Fig. 1A). Moreover, the protein levels of AMPKα1 and AMPKα2 subunits were examined in mouse stromal ST2, mouse osteoblast-like MC3T3-E1, and MLO-Y4 cells (Fig. 1B). The protein expression of AMPKα2 was relatively low in MLO-Y4 cells compared to other cells. We also confirmed that 1.0 mM AICAR treatment

Discussion

We recently showed that AMPK subunits are expressed in osteocytic MLO-Y4 cells and that AMPK exerts antiapoptotic effects against homocysteine-induced oxidative stress in these cells [19]. In the present study, we confirmed protein expression of the AMPKα1 and AMPKα2 subunits and phosphorylation by AICAR. Moreover, we observed that AMPK activation regulated RANKL and Sost expression in MLO-Y4 cells, suggesting that AMPK plays important roles in bone metabolism.

A previous study showed that

Disclosure summary

The authors have nothing to disclose.

Conflicts of interest

None.

Acknowledgments

This study was partly supported by a Grant-in-Aid for Scientific Research (C) (15K09433). Authors' roles: Study design and conduct: MY and IK. Performed the experiments and analyzed the data: MY and AT. Contributed equipment/materials: IK, MN, KT, and TS. Wrote the paper: MY and IK. Approving final version: all authors. IK takes responsibility for the integrity of the data analysis. The authors thank Keiko Nagira for technical assistance.

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