Elsevier

Bone

Volume 74, May 2015, Pages 125-133
Bone

Original Full Length Article
AMP-activated protein kinase (AMPK) activity negatively regulates chondrogenic differentiation

https://doi.org/10.1016/j.bone.2014.12.001Get rights and content

Highlights

  • Treatment with metformin significantly reduced cartilage matrix formation.

  • Activated AMPK inhibits sox9, sox6, col2a1, and acp expressions.

  • Chondrocyte differentiation is functionally associated with decreased AMPK activity.

Abstract

Chondrocytes are derived from mesenchymal stem cells, and play an important role in cartilage formation. Sex determining region Y box (Sox) family transcription factors are essential for chondrogenic differentiation, whereas the intracellular signal pathways of Sox activation have not been clearly elucidated. AMP-activated protein kinase (AMPK) is a serine–threonine kinase generally regarded as a key regulator of cellular energy homeostasis. It is known that the catalytic alpha subunit of AMPK is activated by upstream AMPK kinases (AMPKKs) including liver kinase B1 (LKB1). We have previously reported that AMPK is a negative regulator of osteoblastic differentiation. Here, we have explored the role of AMPK in chondrogenic differentiation using in vitro culture models. The phosphorylation level of the catalytic AMPK alpha subunit significantly decreased during chondrogenic differentiation of primary chondrocyte precursors as well as ATDC-5, a well-characterized chondrogenic cell line. Treatment with metformin, an activator of AMPK, significantly reduced cartilage matrix formation and inhibited gene expression of sox6, sox9, col2a1 and aggrecan core protein (acp). Thus, chondrocyte differentiation is functionally associated with decreased AMPK activity.

Introduction

AMP-activated protein kinase (AMPK) is a serine–threonine kinase, which maintains the balance between production and consumption of ATP in eukaryotic cells [1]. AMPK is a heterotrimeric enzyme composed of the catalytic α subunit and regulatory β and γ subunits in a 1:1:1 stoichiometric ratio [2]. AMPK is activated by the elevation of intracellular AMP/ATP ratio caused by energy restriction. Subsequently, ATP-generating pathways are activated, while ATP-consuming mechanisms are inhibited, thereby restoring the normal cellular AMP/ATP ratio. Thus, AMPK is generally considered as an essential regulator of energy homeostasis of cells, and termed as a metabolic “energy sensor” of the biological system. The activation of AMPK is induced by phosphorylation of the catalytic α subunit [3]. The most characterized upstream AMPK kinase (AMPKK) is liver kinase B1 (LKB1), which is a reported target of metformin, a type 2 diabetes drug [4].

Recent reports have revealed that AMPK has non-metabolic functions as well. In vivo studies of Drosophila have demonstrated that AMPK is essentially involved in cell polarity and mitosis [5]. On the other hand, activation of AMPK has been proposed as one of the regulatory mechanisms of mammal longevity [6]. Notably, several lines of evidence have indicated the involvement of AMPK in the regulation of cellular differentiation. Activation of AMPK has been suggested to be inhibitory to the differentiation of adipocytes [7], [8], myoblasts [9], and osteoblasts [10]. In contrast, it has also been reported that AMPK is promotive of the differentiation of endothelial progenitor cells [11].

In the earliest event of chondrogenesis, recruited mesenchymal cells proliferate and aggregate. Subsequently, the cells mature into chondrocytes, producing cartilage matrix proteins. After differentiation into hypertrophic cells, they are finally replaced by bone tissue [12]. Deregulation of chondrocyte differentiation has been described in various chronic skeletal diseases including osteoarthritis (OA) [13], [14], [15]. Chondrocytes produce a characteristic cartilage matrix that consists of collagen and proteoglycan [16]. During mouse embryogenesis, a transcription factor, Sox9, is expressed in all chondrocytes and their progenitor cells [12]. Heterozygous sox9 mutants exhibited delayed chondrogenic mesenchymal condensation and enlargement of the hypertrophic zone [17]. Sox9 regulates col2a1 gene, which encodes one component of collagen type II, in the early stage of chondrogenesis [18]. Two other Sox family transcription factors, Sox5 and Sox6, are co-expressed with Sox9 [19] and are required for the expression of aggrecan, which is a cartilage-specific proteoglycan [20]. Inactivation of Sox9 before mesenchymal condensation results in the absence of Sox5 and Sox6, as well as other chondrogenic marker gene expression [21], indicating that Sox9 is the essential master regulator of chondrocyte differentiation.

In this present study, we have explored the role of AMPK in chondrogenic differentiation by in vitro chondrocyte differentiation models. It was found that the phosphorylation level of AMPKα was progressively decreased during chondrogenic differentiation. Stimulation with metformin during chondrogenic differentiation significantly decreased gene expression of sox9 and sox6 along with other chondrogenic differentiation markers including col2a1, and aggrecan core protein (acp). Conversely, knock-down of AMPKα expression by siRNA increased sox9, col2a1, and acp mRNA Thus, our present data indicate that differentiation of chondrocytes is functionally associated with decreased AMPK activity.

Section snippets

Reagents and antibodies

Sodium selenite, transferrin human, metformin, sodium salicylate, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) and insulin from bovine pancreas were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies specifically recognizing AMPKα1/2, AMPKβ1/2, phospho-AMPKα (Thr172), and EGR1 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against AMPKγ1/2/3 and β-Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Monolayer and micro-mass culture

ATDC-5, a well-characterized

The phosphorylation level of AMPKα decreased during chondrogenic differentiation

In order to assess AMPK activity during the course of chondrocyte differentiation, we analyzed phosphorylation of AMPKα and the expression levels of AMPK α/β/γ subunits during chondrogenic differentiation by Western blot and qPCR analyses. ATDC-5 cells were induced to differentiate in the presence of IST, and total cell lysates and RNA were isolated. It was found that the protein levels of AMPKα, β, and γ subunits and mRNA expression levels of prkaa, prkab, and prkag (respectively coding AMPKα,

Discussion

Previous reports have demonstrated that cellular AMPK activity is functionally associated with differentiation of several cell types. More specifically, activation of AMPK has been reported to be inhibitory to the differentiation of preadipocytes [7], [8], myoblasts [9], and osteoblasts [10]. In this present study, metformin, an established activator of AMPK, clearly inhibited the synthesis of cartilage matrix (Figs. 3A and 4A) and the expression of chondrogenic differentiation marker genes (

Conclusion

In summary, our present study has suggested a negative regulatory role of AMPK in chondrogenic differentiation through the suppression of sox9, sox6, col2a1, and acp expressions. This study may provide insights into a new list of target molecules for regenerative therapies of cartilage lesions, such as OA and articular cartilage injuries.

Acknowledgments

This work was supported by Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant 23592739, Grant 23592740, Grant 23592741, and Grant 25462895). We thank Ms. Momoko Uemura for the secretarial assistance.

References (42)

  • I. Asahina et al.

    Human osteogenic protein-1 induces chondroblastic, osteoblastic, and/or adipocytic differentiation of clonal murine target cells

    Exp Cell Res

    (1996)
  • M. Ushita et al.

    Transcriptional induction of SOX9 by NF-kappaB family member RelA in chondrogenic cells

    Osteoarthritis Cartilage

    (2009)
  • T. Ikeda et al.

    Identification and characterization of the human SOX6 promoter

    Biochem Biophys Res Commun

    (2007)
  • L. Tan et al.

    Egr-1 mediates transcriptional repression of COL2A1 promoter activity by interleukin-1beta

    J Biol Chem

    (2003)
  • D.G. Hardie

    AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy

    Nat Rev Mol Cell Biol

    (2007)
  • P.C. Cheung et al.

    Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding

    Biochem J

    (2000)
  • R.J. Shaw et al.

    The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin

    Science

    (2005)
  • J.H. Lee et al.

    Energy-dependent regulation of cell structure by AMP-activated protein kinase

    Nature

    (2007)
  • T. Kasai et al.

    Osteoblast differentiation is functionally associated with decreased AMP kinase activity

    J Cell Physiol

    (2009)
  • X. Li et al.

    AMP-activated protein kinase promotes the differentiation of endothelial progenitor cells

    Arterioscler Thromb Vasc Biol

    (2008)
  • H. Akiyama

    Control of chondrogenesis by the transcription factor Sox9

    Mod Rheumatol

    (2008)
  • Cited by (0)

    View full text