Biochemical and Biophysical Research Communications
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
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.
References (31)
- et al.
New insights into osteoclastogenic signaling mechanisms
Trends Endocrinol. Metab.
(2012) - et al.
Regulation of Wnt/β-catenin signaling within and from osteocytes
Bone
(2013) - et al.
Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling
J. Biol. Chem.
(2005) - et al.
AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism
Cell Metab.
(2005) - et al.
AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass
Bone
(2010) - et al.
AMP kinase acts as a negative regulator of RANKL in the differentiation of osteoclasts
Bone
(2010) - et al.
Metformin enhances the differentiation and mineralization of osteoblastic MC3T3-E1 cells via AMP kinase activation as well as eNOS and BMP-2 expression
Biochem. Biophys. Res. Commun.
(2008) - et al.
Activation of AMP-activated protein kinase protects against homocysteine-induced apoptosis of osteocytic MLO-Y4 cells by regulating the expressions of NADPH oxidase 1 (Nox1) and Nox2
Bone
(2015) - et al.
Genetic deletion of catalytic subunits of AMP-activated protein kinase increases osteoclasts and reduces bone mass in young adult mice
J. Biol. Chem.
(2013) - et al.
Osteogenic actions of the anti-diabetic drug metformin on osteoblasts in culture
Eur. J. Pharmacol.
(2006)