Inhibition of adenosine monophosphate-activated protein kinase suppresses bone morphogenetic protein-2-induced mineralization of osteoblasts <i>via</i> Smad-independent mechanisms

Abstract

Abstract. Previous studies showed that adenosine monophosphate-activated protein kinase (AMPK), which plays as an intracellular energy sensor, promotes the differentiation and mineralization of osteoblasts via enhancing expression of bone morphogenetic protein (BMP)-2, which is a potent inducer of osteoblastogenesis. Thus, the aim of this study was to examine the roles of AMPK in BMP-2-induced osteoblastogenesis. We used a murine osteoblastic cell line MC3T3-E1 and a murine marrow stromal cell line ST2. BMP-2 (50 and 100 ng/mL) stimulated alkaline phosphatase (ALP) activity and enhanced mineralization of MC3T3-E1 cells, while the effects of BMP-2 were partly abolished by an inhibitor of AMPK, ara-A (0.1 mM). Real-time PCR showed that BMP-2 significantly increased the mRNA expressions of Alp, osteocalcin (Ocn), Runx2, Osterix and Dlx-5 in MC3T3-E1 cells, while co-incubation of ara-A significantly decreased the BMP-2-stimulated expression of Alp, Ocn, and Runx2. Moreover, co-incubation of ara-A suppressed the BMP-2-induced upregulation of Alp and Ocn in ST2 cells. Western blot analysis showed that BMP-2 phosphorylated Smad1/5 although it did not affect AMPK phosphorylation in MC3T3-E1 cells. Furthermore, a BMP receptor inhibitor LDN-193189 inhibited the phosphorylation of Smad1/5, but did not affect AMPK. In addition, co-incubation of ara-A did not affect BMP-2-induced phosphorylation of Smad1/5. These findings suggest that the inhibition of AMPK activation reduces the osteo-inductive effects of BMP-2 by decreasing the expression of Alp, Ocn, and Runx2 through Smad-independent mechanisms in osteoblastic cells. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine kinase and associated with regulation of energy homeostasis [3,4]. Accumulating evidence has shown that AMPK is involved in bone remodeling. AMPK is activated during early osteoblast differentiation, and it is reported that the AMPK activation is required for normal bone formation and remodeling [5][6][7]. We and other researchers have previously demonstrated that AMPK activation promotes osteoblast differentiation and mineralization [5,[8][9][10][11][12][13]. Moreover, the effect of AMPK on osteoblast differentiation is mediated by increasing expression of 10,14]. Huang et al. showed that adiponectin, an adipokine, increased the expression of BMP-2, but not other BMPs, through AMPK activation in human osteoblast-like cell

Salvianolic acid C promotes osteogenic differentiation of bone marrow mesenchymal stem cells in osteoporotic rats through activation of AMPK/SIRT1 pathway

“…Combined with the results of this study, SAC affects the expression of Runx2, Osx, and OCN by activating the AMPK/SIRT1 signaling pathway, thereby promoting bone development. 26,27 Therefore, this study suggests that SAC can promote osteogenic differentiation of BMSCs in osteoporotic rats by activating the AMPK/SIRT1 pathway.…”

Focal adhesion kinase promotes BMP2‐induced osteogenic differentiation of human urinary stem cells via AMPK and Wnt signaling pathways

“…We found that 10 −6 mol/L of BMP2 concentration exhibited optimum stimulation, and FAK was demonstrated to be overexpressed following BMP2-induced osteogenic differentiation. Next, cell proliferation and metabolism were evaluated, and our findings revealed (Takeno, Kanazawa, Notsu, Tanaka, & Sugimoto, 2018). Another study showed that the BMP pathway inhibitor could block the polydatin-induced osteogenesis-potentiating effects through the inhibition of the Wnt/β-catenin pathway in BMSCs (X. J. .…”

“…Studies have confirmed that AMPK and Wnt signaling pathways play important roles in BMP‐induced osteogenic differentiation. It has been reported that inhibition of AMPK activation could significantly decrease the expression of OCN, Runx2, and ALP following BMP2‐enhanced mineralization of osteoblasts (Takeno, Kanazawa, Notsu, Tanaka, & Sugimoto, ). Another study showed that the BMP pathway inhibitor could block the polydatin‐induced osteogenesis‐potentiating effects through the inhibition of the Wnt/β‐catenin pathway in BMSCs (X. J. Chen et al, ).…”

Systematic Analysis of Plasma Exosome MiRNA in Patients with Postmenopausal Osteoporosis

“…Consistent with previous studies, miRNAs are involved in some key processes of PMOP. Studies have shown that by inhibiting adenosine monophosphate-activated protein kinase, the mineralization of osteoblasts induced by bone morphogenetic protein-2 (BMP-2) can be inhibited through the non-Smad mechanism [18] . Adenosine monophosphate-activated protein kinase (AMPK), a serine/threonine kinase, is involved in bone remodelling.…”