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International Journal of Clinical and Experimental Pathology logoLink to International Journal of Clinical and Experimental Pathology
. 2017 Aug 1;10(8):8544–8552.

Leptin inhibits AMPKα2 down-regulation induced decrease in the osteocytic MLO-Y4 cell proliferation and the expression of osteogenic markers

Qing Fan 1,*, Hao Li 1,*, Zhu Liu 1, Zhiqiang Zhang 1, Hai Li 1, Jing Ding 1, Ziming Zhang 1
PMCID: PMC6965451  PMID: 31966708

Abstract

AMP-activated protein kinase (AMPK) is of biological and clinical importance for regulating cellular and systemic energy homeostasis. Although AMPKα1, one of the two AMPK’s catalytic subunit α, expresses in the bone and stimulates bone nodule formation, the role of AMPKα2 in osteogenesis remains incompletely understood. The aim of this study was to determine the role of AMPKα2 in osteocytic MLO-Y4 cellproliferation and the expression of osteogenic markers. The current study silenced AMPKα2 in MLO-Y4 cells by transfection with pLKO.1-AMPKα2-shRNA vector and analyzed cell proliferation and the expression of osteogenic markers in MLO-Y4 cells with or without 100 μg/ml leptin treatment through CCK-8, Real-time PCR, Western blot and RNA-seq assay. We found that knockdown of AMPKα2 significantly decreased the mRNA level of AMPKα2 and the cell proliferation of MLO-Y4 cellsas well as the mRNA and protein levels of OPG, OCN, OPN, ALP and BMP6 and the protein expression of p-Smad5/Smad5. However, leptin treatment increased the MLO-Y4 cell proliferation and the expression of these osteogenic markers in MLO-Y4 cells with or without AMPKα2 silencing. Furthermore, RNA-seq assay showed 1019 transcriptors decreased in AMPKα2-silencing group and 995 transcriptors increased in leptin group compared with control group, respectively. 737 transcriptors decreased in AMPKα2-silencing group and 1282 transcriptors increased inleptin group compared with AMPKα2-silencing+leptin group, respectively. These findings suggest that AMPKα2 knockdown inhibited MLO-Y4 cell proliferation and osteogenic marker expressions, which implicates an important role of AMPKα2 in osteogenesis in vitro.

Keywords: AMPKα2, osteogenesis, leptin, RNA-seq, GSEA

Introduction

Bone tissue is constantly renewed by the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteocytes are the most abundant cell type in bone and regulate the activity of both osteoblast and osteoclast of great importance for bone remodeling as well as generate inhibitory signal through their cell processes to osteoblasts for recruitment to enable bone formation [1,2]. In addition, osteocytes regulate bone resorption by regulating receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio [2] and inhibit osteoblast activity by blocking Wnt/β-catenin pathway [3].

Hormones is secreted by bone cells and plays an important role in bone remodeling, food intake and maintaining the energy metabolic balance and homeostasis of glucose and fat metabolism [4], suggesting the close association between osteoporosis, obesity, and diabetes-associated bone diseases [5], which bring us back into the hot issue in the control of bone mass and its relationship to energy homeostasis. Therefore, it is of great biological and clinical importance for clarifying the underlying molecular mechanism of hormones in energy metabolism of skeleton as well as for regulating hormone production. Communication between fat and bone has been debated for a long time, recent evidence showed that subcutaneous administration of leptin, an adipocyte-secreted hormone, increased bone growth, osteoblast-lined bone perimeter and bone formation ratein ob/ob mice [6]. Another mutual relation between bone and energy metabolism was borne out by a feedback control of energy homeostasis exerted by the skeleton through the release of the osteoblast-secreted molecule osteocalcin (OCN) [7].

AMP-activated protein kinase (AMPK) functions as a conserved serine/threonine heterotrimeric kinase consisting of a catalytic α subunit and two regulatory noncatalytic β and γ subunits [8] and plays a pivotal role as an intracellular energy sensor in the regulation of energy homeostasis [9] as well as bone physiology [10]. Interestingly, there has been conflicting evidence for the AMPK regulation of osteoblast differentiation. AMPK activation stimulates the differentiation and mineralization of mouse osteoblastic cell line MC3T3-E1 [11] and prevents against apoptosis of MLO-Y4 cells by decreasing the expressions of Nox1 and Nox2 [12], and knockout of AMPK resulted in a significant reduction in bone mass [4], suggesting a positive effect of AMPK in bone formation. However, this is clearly contrary to the recent research from Kasai et al. [13] which demonstrate decreased the phosphorylation of AMPK during osteoblastic differentiation in both primary osteoblasts and MC3T3-E1 cells. AMPKα comprises α1 and α2 isoforms. The α1 subunit is widely expressed in osteocytes, osteoblasts and osteoclasts [14,15], whereas the α2 subunit is expressed in osteoblasts and osteocytes, but was not ex-pressed in osteoclasts and chondrocyte [4,16]. siRNA-AMPKα1 increases RANKL expressionbut did not affect Sost expression and siRNA-AMPKα2 did not affect the expression of both RANKL and Sost in MLO-Y4 cells [17]. Moreover, cells overexpressing AMPKα2 showed higher osteogenesis potential than AMPKα1 in MC3T3-E1 cells, primary osteoblasts and bone marrow stromal cells (BMSCs) [18].

However, the effect of AMPKα2 on osteogenesisin mouse osteocyte-like MLO-Y4 cells is unclear. This is the first study to show that an aberrant downregulation of AMPKα2 was significant inhibited MLO-Y4 cell proliferation and the expression of osteogenic markers. Additionally, multiple genes and pathways predicted to be correlated to AMPKα2 expression were also investigated in the present study.

Materials and methods

Cell cultures

Mouse osteocyte-like MLO-Y4 cells purchased from Shanghai Honsun Biological Technology Co.,Ltd. (Shanghai, China) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO2.

Lentiviral transduction

Oligonucleotides encoding shRNA directed against human AMPKα2 (point 103-125 shRNA-1, 5’-CCAATTGACAGGCCATAAA-3’, point 290-312 shRNA-2, 5’-GGAGGTGAATTGTTCGACT-3’, and point 444-466 shRNA-3, 5’-CCCAGATGAACGCTAAGAT-3’) and scramble shRNA were purchased from Sangon Biotech, Shanghai. The shRNA sequences were cloned into the pLKO.1 using AgelIand EcolI. The scramble shRNA cloned into the pLKO.1 vector was used as a negative control (NC). The constructs containing 1 μg pLKO.1-AMPKα2-shRNA-1, -2, or -3, 0.9 μg psPAX2 and 0.1 μg pMD2G were then co-transfected into HEK 293T cells when reached 80-90% confluence according to the manufacturer’s instruction. After incubation in a CO2 incubator at 37°C, lentivirus was collected 48 h after transfection.

CCK-8 assay

The cell proliferation of MLO-Y4 cells was measured by CCK-8 assay. Briefly, the MLO-Y4 cells (1-5×103 cells/well) were cultured with 5% CO2 at 37°C overnight. After treatment for 0, 12, 24, 48 and 72 h, the cells were added with 10 μl CCK-8 solution with 5% CO2 at 37°C for 1 h. The cell proliferation was calculated using a microplate reader (ELX 800; Bio-tek Instruments, Winooski, VT, USA) at a wavelength of 450 nm.

Real-time PCR

Total RNA was extracted using TriZol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and quantified. First strand complementary DNAs (cDNAs) were synthesized from isolated RNA by using the PrimeScript RT reagents Kit and used as templates for real-time PCR. All gene transcripts were measured by Real-time PCR using SYBR Green (Takara, Otsu, Shiga, Japan) qPCR kit (Takara, Otsu, Shiga, Japan). Data collection was performed using an Applied Biosystems 7300 Fast Real-Time PCR System (Thermo Fisher Scientific) and relative quantification of gene expression was calculated by the 2-ΔΔCt method. To compare relative mRNA expression levels, the expression levels of CTHRC1, Bcl-2, Bax, Caspase-3 and MMP-13 are given as ratios to GAPDH. The primers were designed by the Primer Express software and were listed as followed: AMPKα2, Forward: 5’-TGGCTCTGATAGAAGTTCAC-3’, Reverse: 5’-TGTTCCTCACGGTATTACTG-3’; OPG, Forward: 5’-AGGGCATACTTCCTGTTG-3’, Reverse: 5’-TTCCTGGGTTGTCCATTC-3’; OCN, Forward: 5’-GAGGGCAATAAGGTAGTG-3’, Reverse: 5’-GGTCTTCAAGCCATACTG-3’; OPN, Forward: 5’-ATCTCACCATTCGGATGAGTCT-3’, Reverse: 5’-TGTAGGGACGATTGGAGTGAAA-3’; ALP, Forward: 5’-GGCTGGAGATGGACAAATTCC-3’, Reverse: 5’-CCGAGTGGTAGTCACAATGCC-3’; Smad5, Forward: 5’-CAGTATCGCCATTGGGAGGAC-3’, Reverse: 5’-AAGTGCTGCCAGGGAAGTG-3’; BMP6, Forward: 5’-AGGTGCCTTCTGTCTACTGTTG-3’, Reverse: 5’-ACGGTCTGTCTACTTTGGGTTC-3’; GAPDH, Forward: 5’-ATCACTGCCACCCAGAAG-3’, Reverse: 5’-TCCACGACGGACACATTG-3’.

Protein extraction and western blot analysis

Total protein from the MLO-Y4 cells was extracted with RIPA lysis buffer supplemented with protease inhibitors. 15 μl of total protein wasseparated through 10% or 15% SDS-PAGE, and the gel was electrophoretic transferred onto polyvinylidene difluoride membrane. Blots were incubated with anti-OPG, anti-OCN, anti-OPN, anti-ALP, anti-BMP6, anti-Smad5, anti-p-Smad5, and anti-GAPDH antibody overnight at 4°C, followed by 3 washes of 5 to 7 minute seach in TBST. Blots were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature, washed 3 times for 5 to 7 min in TBST. The blots were detected using enhanced chemiluminescence according to the manufacturer’s instructions (Pierce, Rockford, IL, USA) and exposed to X-ray filmand quantified in Chemi Doc XRS Imaging System, Bio-Rad (USA). All the primary antibodies were purchased from Abcam (Cambridge, MA, USA) except ALP (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (Cell Signaling Technology, Inc., Danvers, MA, USA). All the second antibodies were purchased from Beyotime Institute of Biotechnology (Haimen, China).

RNA-seq and gene set enrichment analysis

RNA-seq was performed as previously described [19]. Data were normalized by log2 and gene expression changes were considered. Changes in gene expression with P-value less than 0.05 and fold change ≥ 1.5 were considered as statistical significance. To gain further insight into the biological pathways, a Gene set enrichment analysis (GESA) was performed on KEGG (Kyoto Encyclopedia of Genes and Genomes) and REACTOME pathway database. The pathways used in this analysis were selected to the one which the selected genes belong. The gene sets showing P<0.05 were considered enriched between different groups, including control, AMPKα2 silencing, leptin, AMPKα2 silencing+leptin group.

Statistical analysis

All experiments were performed in triplicate. Data are presented as mean ± standard deviation. Statistical evaluations for differences between groups were performed using one-way ANOVA and unpaired two-tailed Student t testwith GraphPad Prism software version 5 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was defined as statistically significant.

Results

Depletion of AMPKα2 inhibits MLO-Y4 cell proliferation

To demonstrate the role of AMPKα2 in osteogenesis in vitro, the mouse osteocyte-like MLO-Y4 cells were transfected with three pLKO.1-AMPKα2-shRNAs lentiviral vector. As shown in Figure 1A, pLKO.1-AMPKα2-shRNA-1, -2 and -3 lentiviral vector significantly decreased the mRNA expression of AMPKα2 by 87.0%, 78.3% and 46.8% in MLO-Y4 cells, respectively, with the lowest expression detected in pLKO.1-AMPKα2-shRNA-1 group. This shRNA was therefore used for subsequent experiments.

Figure 1.

Figure 1

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Knockdown of AMPKα2 inhibits the cell proliferation of MLO-Y4 cells. (A) The mRNA expression of AMPKα2 in MLO-Y4 cells with pLKO.1-AMPKα2-shRNA-1, -2 or -3 transfection was measured by Real-time PCR. The cell proliferation and morphology of MLO-Y4 cells with pLKO.1-AMPKα2-shRNA-1 transfection in the absence or presence of leptin were measured by CCK-8 assay (B) and microscopy (C), respectively. **P<0.01 compared with control.

To further investigate the function of AMPKα2 in MLO-Y4 cells, pLKO.1-AMPKα2-shRNA-1 was transfected into the MLO-Y4 cells with or without 100 μg/ml leptin treatment. We found that knockdown of AMPKα2 significantly decreased the MLO-Y4 cell proliferation by 10.9%, 17.3%, 34.5%, and 48.7% compared with control at 12, 24, 48 and 72 h, respectively (Figure 1B). Moreover, leptin treatment for 12, 24, 48 and 72 h significantly inhibited the decreased the proliferation of MLO-Y4 cells induced by AMPKα2 silencing by 19.3%, 19.7%, 36.4%, and 63.5%, respectively. The similar effect of AMPKα2 and leptin was also detected by microscopy (Figure 1C). These results suggest that leptin inhibits AMPKα2 knockdown-induced decrease of MLO-Y4 cell proliferation.

Depletion of AMPKα2 inhibits the expression of osteogenic markers in MLO-Y4 cells

After transfection of MLO-Y4 cells with pLKO.1-AMPKα2-shRNA-1, the expression of ALP and the osteogenesis markers OPG, OCN, OPN, BMP6, and Smad5 was assessed by Real-time PCR and Western blotting. Compared with control group, the AMPKα2 silencing group had a significant downregulation in the mRNA level of OPG, OCN, OPN, ALP, and BMP6 (Figure 2A and 2B) but had no effect on Smad5 expression (data not shown). Consistent with these results, the protein level of OPG, OCN, OPN, ALP, BMP6, and p-Smad5/Smad5 was also decreased in AMPKα2 silencing group compared with control group (Figure 2C-E). More importantly, a significant upregulation in the expression of OPG, OCN, OPN, ALP, BMP6, and p-Smad5/Smad5 was found in leptin-treated MLO-Y4 cells with or without AMPKα2 silencing (Figure 2A-E).

Figure 2.

Figure 2

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Knockdown of AMPKα2 inhibits the expression of osteogenic markers in MLO-Y4 cells. After treatment of MLO-Y4 cells with pLKO.1-AMPKα2-shRNA-1 transfection and/or leptin treatment, the mRNA expression of OPG, OCN, OPN, ALP and BMP6 was measured by Real-time PCR (A, B), and the protein expression of these markers as well as p-Smad5 and Smad5 was measured by Western blotting (C-E). **P<0.01 compared with control. #P<0.05, ##P<0.01 compared with shRNA.

Analysis of gene expression pattern inMLO-Y4 cells

We compared the gene expression patterns in MLO-Y4 cells with or without AMPKα2 silencing or leptin treatment. We found that 2501 specific genes with significantly fold changes, among which 1482 genes were up-regulated and 1019 genes were down-regulated in AMPKα2 silencing group compared with control MLO-Y4 cells (P<0.05). Meanwhile, top 50 up-regulated genes, including Svep1 (4.66-fold), Dcn (4.64-fold), Bfsp1 (4.64-fold), Gm9958 (4.64-fold) and Serpini1 (4.61-fold), and down-regulated genes, including Baat (4.80-fold), Serpinf2 (4.75-fold), Gm1123 (4.65-fold), Nlrx1 (4.62-fold) and Selplg (4.62-fold), were shown in Figure 3A.

Figure 3.

Figure 3

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Differently expressed genes in MLO-Y4 cells with AMPKα2 silencing and/or leptin treatment. A. Top 50 up-regulated and down-regulated genes in MLO-Y4 cells with AMPKα2 silencing group compared with control MLO-Y4 cells. B. Top 50 up-regulated and down-regulated genes in MLO-Y4 cells with leptin treatment group compared with control MLO-Y4 cells. C. Top 50 up-regulated and down-regulated genes in MLO-Y4 cells with AMPKα2 silencing and leptin treatment group compared with control MLO-Y4 cells. D. Top 50 up-regulated and down-regulated genes in MLO-Y4 cells with AMPKα2 silencing group compared with MLO-Y4 cells with AMPKα2 silencing and leptin treatment. E. Top 50 up-regulated and down-regulated genes in MLO-Y4 cells with leptin treatment group compared with MLO-Y4 cells with AMPKα2 silencing and leptin treatment.

1862 specific genes with significantly fold changes, among which 995 genes were up-regulated and 867 genes were down-regulated in leptin treatment group compared with control MLO-Y4 cells (P<0.05). Meanwhile, top 50 up-regulated genes, including Bfsp1 (4.68-fold), Dcn (4.66-fold), Phtf1os (4.66-fold), Muc5b (4.66-fold) and Ctla2a (4.66-fold), and down-regulated genes, including 4632427 E13Rik (4.62-fold), Tmprss2 (4.59-fold), Dpm1 (4.55-fold), Snord47 (4.55-fold) and P2rx2 (4.51-fold), were shown in Figure 3B.

2554 specific genes with significantly fold changes, among which 1372 genes were up-regulated and 1182 genes were down-regulated in AMPKα2 silencing+leptin treatment group compared with control MLO-Y4 cells (P<0.05). Meanwhile, top 50 up-regulated genes, including Nat8 (4.65-fold), Svep1 (4.62-fold), Omp (4.62-fold), Dcn (4.59-fold) and 2900041 M22Rik (4.56-fold), and down-regulated genes, including A3galt2 (4.79-fold), Hnf1a (4.73-fold), Adora2a (4.69-fold), 4930447 C04Rik (4.65-fold) and Pin4 (4.65-fold), were shown in Figure 3C.

1733 specific genes with significantly fold changes, among which 737 genes were up-regulated and 996 genes were down-regulated in AMPKα2 silencing+leptin treatment group compared with AMPKα2 silencing group (P<0.05). Meanwhile, top 50 up-regulated genes, including Jsrp1 (4.47-fold), Mep1a (4.47-fold), 1110020 A21Rik (4.41-fold), Clca3a2 (4.41-fold) and Six6 (4.41-fold), and down-regulated genes, including Pin4 (4.72-fold), Rasl2-9 (4.61-fold), Kcnj16 (4.57-fold), 4930447 C04Rik (4.57-fold) and 4632428 N05Rik (4.54-fold), were shown in Figure 3D.

2553 specific genes with significantly fold changes, among which 1271 genes were up-regulated and 1282 genes were down-regulated in AMPKα2 silencing+leptin treatment group compared with leptin treatment group (P<0.05). Meanwhile, top 50 up-regulated genes, including 4632427 E13Rik (4.67-fold), Mgp (4.62-fold), Mzf1 (4.52-fold), Meiob (4.47-fold) and Mir1943 (4.47-fold), and down-regulated ge-nes, including A3galt2 (4.80-fold), Nlrx1 (4.79-fold), Hgfac (4.77-fold), Gja4 (4.75-fold) and Ampd1 (4.75-fold), were shown in Figure 3E.

Multiple pathways were bioinformatically predicted to be correlated to AMPKα2

To further characterize the enrichment of pathways that correlates to AMPKα2 expression, we carried out the gene set enrichment analysis (GSEA) on KEGG (Kyoto Encyclopedia of Genes and Genomes) and REACTOME pathway databasein MLO-Y4 cells with AMPKα2 silencing in the absence or presence of leptin treatment.We found up-regulated 86 gene sets in AMPKα2 silencing group (e.g. DNA replication, Homologous recombination, Base excision repair, Kinesins, Resolution of sister chromatid cohesion, Cell cycle-Mitotic, and Cell cycle), 11 gene setsin leptin group (e.g. Respiratory electron transport-ATP synthesis by chemiosmotic coupling and heat production by uncoupling protein, O-linked glycosylation of mucins, The citric acid cycle and respiratory electron transport, Respiratory electron transport, and Signaling by Notch2), and 55 gene sets in AMPKα2 silencing+leptin group (e.g. DNA replication, DNA strand elongation, Kinesins, Resolution of sister chromatid cohesion, Mitotic prometaphase, Cell cycle, and Cell cycle-Mitotic) compared with control group, respectively (Supplementary Tables 1, 2, 3). Moreover, up-regulated 41 gene sets in AMPKα2 silencing group (e.g. Platelet homeostasis, Synthesis-secretion-deacylation of ghrelin, Hemostasis, Translation, and SRP-dependent cotranslation protein targeting to membrane) and 57 gene setsin leptin group (e.g. Circadian rhythm, Platelet homeostasis, Respiratory electron transport-ATP synthesis by chemiosmotic coupling and heat production by uncoupling proteins, Translation, and GPCR downstream signaling) were found compared with AMPKα2 silencing+leptin group (Supplementary Tables 4, 5).

Discussion

The results of the present study revealed that knockdown of AMPKα2 significantly inhibited MLO-Y4 cell proliferation and the expression of the osteogenesis markers, including OPG, OCN, OPN, ALP, BMP6 and p-Smad5/Smad5. RNA-seq assay showed 1482 genes in MLO-Y4 cells with AMPKα2 silencing and 995 genes in MLO-Y4 cells with leptin treatment were up-regulated respectively compared with control MLO-Y4 cells. Finally, the differently expressed genes in MLO-Y4 cells with AMPKα2 silencing and/or leptin treatment were involved in multiple pathways, such as DNA replication, Kinesins, Respiratory electron transport-ATP synthesis by chemiosmotic coupling and heat production by uncoupling protein, Platelet homeostasis, and Translation.

During the process of bone development, a large amount of energy is consumed, and the growth and development of bone and cartilage are closely related to energy metabolism. AMPK is a complex that regulates cell energy homeostasis and the metabolism of osteoblasts and osteoclasts [20], has been therefore implicated in diverse human diseases and ageing [21,22]. However, chondrocyte-specific ablation of AMPKα1 alone has no effect on bone developmentdue to the reactive feedback of AMPKα2 [23]. The importance of AMPK in bone physiology was also demonstrated in mice lacking AMPK catalytic subunits, which results in reduced bone mass with alterations in both bone formation and bone resorption, with more remarkable in AMPKα1 deficiency mice than in AMPKα2 deficiency mice [5]. AMPKα1 deficiency stimulated differentiation and cell fusion as well as osteoclastogenesis, whereas activation of AMPK inhibited osteoclast development [24]. While other report observed greater osteogenic potential of AMPKα2 as compared to AMPKα1 [18], which contrary to another study that AMPKα2 knockout had no effect on tibial bone mass [4]. In the present study, MLO-Y4 cells were transfected with AMPKα2-silenced vector and showed decreased cell proliferation of MLO-Y4, which was inhibited by leptin treatment. In line with our findings that subcutaneous replacement of leptin increased in bone formation and osteoblast-lined perimeter in mice, suggesting that leptin increases bone formation, in part, by increasing osteoblast number. However, opposite study showed increased bone formation in leptin-deficient ob/ob mice. The possible explanations may due to the differences between cell lines and mice.

Present study showed that knockdown of AMPKα2 could inhibit ALP and the expression of osteogenic makers (OPG, OPN, OCN, and BMP6), while leptin increased these marker expressions in MLO-Y4 cells with or without AMPKα2 silencing. OPN is an early osteogenic marker and characterized in the matrix maturation and bone remodeling phase [25]. OPG is a competitive inhibitor of RANKL, which as a critical gene in osteoclastogenesis induces osteoclast differentiation, activation, and survival upon interaction with its receptor RANK [26]. Expression of OPG and OCN were strongly induced following culture with BMP6, which significantly contributes to both osteogenic and chondrogenic differentiation of murine adipose-derived mesenchymal cells depending on culture conditions [27]. Smad5 signaling induces osteogenesis through mediating osteoblastic differentiation and maturation [28].

The RNA sequencing analysis is a potent approach for analyses of genomic gene expression patterns based on ultra-high throughput sequencing of total RNA and systematic counts of all expressed transcripts [29]. In the present study, we identified 2501 differently expressed genes in MLO-Y4 cells with AMPKα2 silencing, 1862 differently expressed genes in MLO-Y4 cells with leptin treatment, and 2254 differently expressed genes in MLO-Y4 cells with AMPKα2 silencing and leptin treatment, compared with control MLO-Y4 cells (P<0.05; fold change ≥ 1.5). Moreover, these differently expressed genes are involved in 214 pathways such as DNA replication, Cell cycle, and Translation, etc. which measured by GSEA based on KEGG and REACTOME pathway database. These findings suggest that AMPKα2 as well as leptin is associated with an unspecific and wider gene activation profile.

In summary, our findings show that knockdown of AMPKα2 inhibits MLO-Y4 cell proliferation and the expression of osteogenic makers. There are clear molecular differences between MLO-Y4 cells with AMPKα2 silencing or leptintreatment and normal controls MLO-Y4 cells, and gene signatures associated with AMPKα2 silencing or leptin treatment in MLO-Y4 cells were also identified. However, the differently expressed genes and involved pathways should be further clarified in our further investigation.

Acknowledgements

This work was funded by the National Natural Science Foundation of China (81372001).

Disclosure of conflict of interest

None.

Supporting Information

ijcep0010-8544-f4.pdf (191.4KB, pdf)

References

  • 1.Dallas SL, Prideaux M, Bonewald LF. The osteocyte: an endocrine cell ... and more. Endocr Rev. 2013;34:658–690. doi: 10.1210/er.2012-1026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Bonewald LF. The amazing osteocyte. J Bone Miner Res. 2011;26:229–238. doi: 10.1002/jbmr.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Burgers TA, Williams BO. Regulation of Wnt/beta-catenin signaling within and from osteocytes. Bone. 2013;54:244–249. doi: 10.1016/j.bone.2013.02.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Shah M, Kola B, Bataveljic A, Arnett TR, Viollet B, Saxon L, Korbonits M, Chenu C. AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass. Bone. 2010;47:309–319. doi: 10.1016/j.bone.2010.04.596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Kang H, Viollet B, Wu D. Genetic deletion of catalytic subunits of AMP-activated protein kinase increases osteoclasts and reduces bone mass in young adult mice. J Biol Chem. 2013;288:12187–12196. doi: 10.1074/jbc.M112.430389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Turner RT, Kalra SP, Wong CP, Philbrick KA, Lindenmaier LB, Boghossian S, Iwaniec UT. Peripheral leptin regulates bone formation. J Bone Miner Res. 2013;28:22–34. doi: 10.1002/jbmr.1734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Lee NK, Sowa H, Hinoi E, Ferron M, Ahn JD, Confavreux C, Dacquin R, Mee PJ, McKee MD, Jung DY, Zhang Z, Kim JK, Mauvais-Jarvis F, Ducy P, Karsenty G. Endocrine regulation of energy metabolism by the skeleton. Cell. 2007;130:456–469. doi: 10.1016/j.cell.2007.05.047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Salminen A, Hyttinen JM, Kaarniranta K. AMPactivated protein kinase inhibits NF-kappaB signaling and inflammation: impact on healthspan and lifespan. J Mol Med. 2011;89:667–676. doi: 10.1007/s00109-011-0748-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Steinberg GR, Kemp BE. AMPK in health and disease. Physiol Rev. 2009;89:1025–1078. doi: 10.1152/physrev.00011.2008. [DOI] [PubMed] [Google Scholar]
  • 10.Jeyabalan J, Shah M, Viollet B, Chenu C. AMPactivated protein kinase pathway and bone metabolism. J Endocrinol. 2012;212:277–290. doi: 10.1530/JOE-11-0306. [DOI] [PubMed] [Google Scholar]
  • 11.Kanazawa I, Yamaguchi T, Yano S, Yamauchi M, Sugimoto T. 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;375:414–419. doi: 10.1016/j.bbrc.2008.08.034. [DOI] [PubMed] [Google Scholar]
  • 12.Takeno A, Kanazawa I, Tanaka K, Notsu M, Yokomoto M, Yamaguchi T, Sugimoto T. 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;77:135–141. doi: 10.1016/j.bone.2015.04.025. [DOI] [PubMed] [Google Scholar]
  • 13.Kasai T, Bandow K, Suzuki H, Chiba N, Kakimoto K, Ohnishi T, Kawamoto S, Nagaoka E, Matsuguchi T. Osteoblast differentiation is functionally associated with decreased AMP kinase activity. J Cell Physiol. 2009;221:740–749. doi: 10.1002/jcp.21917. [DOI] [PubMed] [Google Scholar]
  • 14.Kim JE, Ahn MW, Baek SH, Lee IK, Kim YW, Kim JY, Dan JM, Park SY. AMPK activator, AICAR, inhibits palmitate-induced apoptosis in osteoblast. Bone. 2008;43:394–404. doi: 10.1016/j.bone.2008.03.021. [DOI] [PubMed] [Google Scholar]
  • 15.Quinn JM, Tam S, Sims NA, Saleh H, McGregor NE, Poulton IJ, Scott JW, Gillespie MT, Kemp BE, van Denderen BJ. Germline deletion of AMP-activated protein kinase beta subunits reduces bone mass without altering osteoclast differentiation or function. FASEB J. 2010;24:275–285. doi: 10.1096/fj.09-137158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Bohensky J, Leshinsky S, Srinivas V, Shapiro IM. Chondrocyte autophagy is stimulated by HIF-1 dependent AMPK activation and mTOR suppression. Pediatr Nephrol. 2010;25:633–642. doi: 10.1007/s00467-009-1310-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Yokomoto-Umakoshi M, Kanazawa I, Takeno A, Tanaka K, Notsu M, Sugimoto T. Activation of AMP-activated protein kinase decreases receptor activator of NF-kappaB ligand expression and increases sclerostin expression by inhibiting the mevalonate pathway in osteocytic MLO-Y4 cells. Biochem Biophys Res Commun. 2016;469:791–796. doi: 10.1016/j.bbrc.2015.12.072. [DOI] [PubMed] [Google Scholar]
  • 18.Wang YG, Han XG, Yang Y, Qiao H, Dai KR, Fan QM, Tang TT. Functional differences between AMPK alpha1 and alpha2 subunits in osteogenesis, osteoblast-associated induction of osteoclastogenesis, and adipogenesis. Sci Rep. 2016;6:32771. doi: 10.1038/srep32771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Toung JM, Morley M, Li M, Cheung VG. RNAsequence analysis of human B-cells. Genome Res. 2011;21:991–998. doi: 10.1101/gr.116335.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Indo Y, Takeshita S, Ishii KA, Hoshii T, Aburatani H, Hirao A, Ikeda K. Metabolic regulation of osteoclast differentiation and function. J Bone Miner Res. 2013;28:2392–2399. doi: 10.1002/jbmr.1976. [DOI] [PubMed] [Google Scholar]
  • 21.Bujak AL, Crane JD, Lally JS, Ford RJ, Kang SJ, Rebalka IA, Green AE, Kemp BE, Hawke TJ, Schertzer JD, Steinberg GR. AMPK activation of muscle autophagy prevents fasting-induced hypoglycemia and myopathy during aging. Cell Metab. 2015;21:883–890. doi: 10.1016/j.cmet.2015.05.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Wang S, Dale GL, Song P, Viollet B, Zou MH. AMPKalpha1 deletion shortens erythrocyte life span in mice: role of oxidative stress. J Biol Chem. 2010;285:19976–19985. doi: 10.1074/jbc.M110.102467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Morizane Y, Thanos A, Takeuchi K, Murakami Y, Kayama M, Trichonas G, Miller J, Foretz M, Viollet B, Vavvas DG. AMP-activated protein kinase suppresses matrix metalloproteinase-9 expression in mouse embryonic fibroblasts. J Biol Chem. 2011;286:16030–16038. doi: 10.1074/jbc.M110.199398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Yamaguchi N, Kukita T, Li YJ, Kamio N, Fukumoto S, Nonaka K, Ninomiya Y, Hanazawa S, Yamashita Y. Adiponectin inhibits induction of TNF-alpha/RANKL-stimulated NFATc1 via the AMPK signaling. FEBS Lett. 2008;582:451–456. doi: 10.1016/j.febslet.2007.12.037. [DOI] [PubMed] [Google Scholar]
  • 25.Balint E, Lapointe D, Drissi H, van der Meijden C, Young DW, van Wijnen AJ, Stein JL, Stein GS, Lian JB. Phenotype discovery by gene expression profiling: mapping of biological processes linked to BMP-2-mediated osteoblast differentiation. J Biol Chem. 2003;89:401–426. doi: 10.1002/jcb.10515. [DOI] [PubMed] [Google Scholar]
  • 26.MacMillan AK, Lamberti FV, Moulton JN, Geilich BM, Webster TJ. Similar healthy osteoclast and osteoblast activity on nanocrystalline hydroxyapatite and nanoparticles of tri-calcium phosphate compared to natural bone. Int J Nanomed. 2014;9:5627–5637. doi: 10.2147/IJN.S66852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Kemmis CM, Vahdati A, Weiss HE, Wagner DR. Bone morphogenetic protein 6 drives both osteogenesis and chondrogenesis in murine adipose-derived mesenchymal cells depending on culture conditions. Biochem Biophys Res Commun. 2010;401:20–25. doi: 10.1016/j.bbrc.2010.08.135. [DOI] [PubMed] [Google Scholar]
  • 28.Dai J, Li Y, Zhou H, Chen J, Chen M, Xiao Z. Genistein promotion of osteogenic differentiation through BMP2/SMAD5/RUNX2 signaling. Int J Biol Sci. 2013;9:1089–1098. doi: 10.7150/ijbs.7367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Chu CL, Zhao CH, Zhang ZW, Wang MW, Zhang ZH, Yang AQ, Ma BB, Lu CF, Wu M, Gu MZ, Cui RJ, Xin ZX, Huang T, Zhou WL. Identification and validation of gene expression patterns in cystitis glandularis patients and controls. SLAS Discov. 2017;22:743–750. doi: 10.1177/2472555216685519. [DOI] [PubMed] [Google Scholar]

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