Original Full Length ArticleAMP-activated protein kinase (AMPK) activity negatively regulates chondrogenic differentiation
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.
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