Marrow adipocytes inhibit the differentiation of mesenchymal stem cells into osteoblasts via suppressing BMP-signaling
© The Author(s). 2017
Received: 13 October 2016
Accepted: 31 January 2017
Published: 7 February 2017
Reduced bone formation is associated with increased bone marrow fat in many bone-loss related diseases including aging, post-menopause, and anorexia nervosa. Several lines of evidence suggested the regulation of osteogenesis and adipogenesis of the bone marrow-derived mesenchymal (skeletal) stem cells (BMSCs) by paracrine mediators. This study aimed to investigate the impact of adipocytes-secreted factors on the cell proliferation and osteoblast differentiation of BMSCs.
Serum free conditioned medium (CM-Adipo) was collected from stromal ST2 cells-derived adipocytes. Cell viability, quantitative alkaline phosphatase (ALP) activity assay, Alizarin red staining for matrix mineralization and osteogenic gene array expression were performed to determine the effect of CM-Adipo on cell proliferation and osteoblast differentiation of primary murine BMSCs (mBMSCs). Regulation of BMPs and NF-κB signaling pathways by CM-Adipo were detected by Western blot analysis and gene reporter assay.
CM-Adipo showed no effect on cell viability/proliferation of primary mBMSCs as compared to CM-control. On the other hand, CM-Adipo significantly inhibited the commitment of mBMSCs into osteoblastic cell lineage in dose-dependent manner. CM-Adipo was found to dramatically inhibit the BMP2-induced osteoblast differentiation and to activate the inflammatory NF-κB signaling in mBMSCs. Interestingly, treatment of mBMSCs with the selective inhibitor of NF-κB pathway, BAY11-770682, showed to retrieve the inhibitory effect of CM-Adipo on BMP2-induced osteoblast differentiation in mBMSCs.
Our data demonstrated that the marrow adipocytes exert paracrine inhibitory effect on the osteoblast differentiation of mBMSCs by blocking BMPs signaling in a mechanism mediated by adipokines-induced NF-κB pathway activation.
KeywordsMesenchymal stem cells BMSCs Osteoblast Adipocyte Paracrine factors osteoblast differentiation
Bone marrow-derived mesenchymal stem cells (BMSCs, also known as bone marrow skeletal stem cells) reside in the perivascular compartment of bone marrow and can differentiate into osteoblast and adipocyte cell lineages among other mesoderm cell types [1, 2]. BMSCs hold a great promise in cell-based therapy for many degenerative diseases including osteoporosis, due to their differentiation potential, immune-modulatory functions and the secretion of paracrine factors involved in endogenous tissue regenerative capacity [3, 4].
Lineage-specific differentiation of BMSCs into either osteoblasts or adipocytes is regulated by many paracrine factors including cytokines/growth factors and hormones that act to induce intercellular signaling and subsequently activate the key transcriptional factors, core-binding factor 1(CBFA1/Runx2) , or peroxisome proliferator-activated receptor gamma 2 (PPARγ2)  for osteogenesis and adipogenesis respectively.
Adipocytes-secreted adipokines and free fatty acids affect both osteoblasts and osteoclasts formation/activity and therefore mediate skeletal homeostasis [7, 8]. Marrow fat volume was observed to be increased in animal models of ovariectomy, aging and calorie restriction and in human, it was inversely correlated with BMD in the clinical conditions of aging, post-menopause, and anorexia nervosa (for review, [9–14]. Several studies attributed this increased marrow adiposity to the shifting in the differentiation capacity of BMSCs towards adipocyte versus osteoblast cell lineage, suggesting an inverse relationship between these two lineages [10, 15–17]. In vitro and in vivo studies reported a paracrine regulatory mechanism for controlling this inverse relationship between osteoblast and adipocyte differentiation of BMSCs [7, 9, 10]. For examples: adipocytes secrete factors that inhibit osteoblastogenesis and favor adipogenesis, such as sFRP-1 , sFRP-4, and chemerin  and pro-inflammatory cytokines .
To answer the question whether increased marrow adipocytes is one of the main contributing factor to reduce osteoblast differentiation and bone formation in osteoporosis, we aimed in this study to investigate the impact of adipocyte-secreted factors on BMSCs proliferation and osteoblast differentiation. Thus, we studied the paracrine effect of the serum free condition medium collected from stromal ST2 cell line-derived adipocytes (CM-Adipo) on the cell proliferation and differentiation of murine BMSCs. Results showed the inhibitory effect of adipocyte-secreted factors on the differentiation of mBMSCs into osteoblastic cell lineage without affecting their proliferation. Interestingly, CM-Adipo was found to block BMP2-induced osteogenesis via activating the inflammatory NF-κB pathway.
Mouse stromal cells ST2 was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (ACC 333, Braunschweig, Germany). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) (all purchased from Gibco Invitrogen, USA).
Mouse BMSCs were isolated from wild-type 8-weeks-old male C57BL/6 J mice as previously described . In brief, the ends of mouse tibia and femur were cut and placed in special adapted Eppendorf tubes, centrifuged for 1 min at 400 g to collect the marrow cells. Cell were filtrated through a 70-μm nylon mesh filter and cultured in 175 cm2 flasks in RPMI-1640 medium supplemented with 12% FBS (Gibco Invitrogen, USA), 12 μM L-glutamine (Invitrogen) and 1% penicillin/streptomycin (P/S) (Gibco Invitrogen, USA). Non-adherent cells were removed after 24 h by washing with PBS, and adding 30 ml of fresh medium. Every 3 to 4 day, cells were washed, and fresh medium was added for a period of 4 weeks. After 4 weeks, cells were washed and trypsinized.
The NF-κB inhibitor, BAY11-7082, that inhibits IκBα [inhibitor of NF-κB (nuclear factor κB)α] phosphorylation in cells  and insulin were from Sigma-Aldrich ApS (Brondby, Denmark). Bone morphogenetic protein-2 (BMP2), recombinant PDGF-BB and recombinant murine WNT-3a were purchased from PeproTech (London, UK). The concentrations of different growth factors were selected as previously describe [23–25], or based on manual instructions.
Collection of conditioned medium (CM)
ST2 cells and primary isolated murine BMSCs were induced to differentiate into adipocytes for 12 days as described below. To avoid any influence from the adipogenic inducer factors (i.e 1-methyl-3-isobutylxanthine (IBMX), dexamethasone and insulin) in the collected CM, medium was changed with serum free DMEM containing 1% P/S, and the adipocyte-derived CM (CM-Adipo) was collected after 24 h. Serum free control CM (CM-Control) was collected from un-differentiated ST2 cells that cultured for 12 days in basal culture medium. Collected CM was centrifuged for 10 min at 1000 rpm and aliquoted into small aliquots for different experiments. Based on the experimental setting, the serum free CM was used at either 100% or diluted with DMEM at 25% or 50%. FBS and other osteogenic inducers were added freshly to the CM upon studying the effect of CM-Adipo on osteogenesis of mBMSCs. CM-Adipo obtained from ST2 cells-derived adipocytes was used throughout this study, except otherwise stated.
Cell proliferation study
Short-term in vitro cell growth was determined by culturing the cells at 2000 cells/well in 4 well plates in either CM-Adipo (100%) or CM-control (100%) supplemented with 2% FBS. Cells were trypsinized and counted by the hemocytometer.
Real time-polymerase chain reaction (RT-PCR)
RNA was extracted using TRIzol according to the manufacturer’s instructions (Invitrogen) and the first strand cDNA was synthesized from 1 μg of total RNA using a Revert Aid™ H minus first strand cDNA synthesis kit (Fermentas, St Leon-Rot, Germany). RT-qPCR was performed using an ABI StepOne™ Real-TIME PCR machine (Life Technologies/Applied Biosystems) with using Fast SYBR® Green Master Mix (Applied Biosystems, California, USA). The targeted primers and reference genes are shown in Additional file 1: Table S1. The data were normalized to the geometric means of the reference genes β-actin and HPRT. The relative expression levels of each target gene were calculated using a comparative CT method [(1/(2ΔC T) formula, where ΔCT is the difference between CT-target and CT-reference] with Microsoft Excel 2007®.
PCR array analysis
Total RNA was extracted from mBMSCs induced to osteoblast differentiation in either CM-Control or CM-Adipo. Osteogenic RT2 Profiler™ PCR array, containing 84 osteoblast-related genes (Qiagen Nordic), was performed for each sample in triplicates using SYBR® Green quantitative PCR method on Applied Biosystems 7500 real-time PCR system and data were analyzed according to the manufacturer’s instructions.
Cells were plated at 15,000 cells/cm2 and cultured for 12 days in adipogenic-induction medium (AIM; DMEM supplemented with 9% horse serum, 450 μM 1-methyl-3-isobutylxanthine (IBMX), 100nM dexamethasone, 5 μg/mL insulin (Sigma-Aldrich) and 1 μM rosiglitazone (BRL 49653, Cayman Chemical, Ann Arbor, Michigan). The media was changed every three days.
Osteoblast differentiation was performed in cells plated at 10,000/cm2 in osteoblast-induction media (OIM) containing DMEM supplemented with 10% FBS 10 mM beta glycerophosphate (Calbiochem-Merck, Germany), 50 μg/mL L-ascorbic acid-2-phosphate (Wako Chemicals GmbH, Germany) and 10nM dexamethasone (Sigma-Aldrich, Denmark). The medium was changed every three days during induction period.
Alkaline phosphatase (ALP) activity and quantification
ALP activity was performed after 6 days of osteoblastic induction. Cell viability was determined using the Cell Titer-Blue cell viability assay according to the manufacturer’s instructions (Promega, USA) and the viability measured at 560Ex/590Em nm using a FLUO star Omega plate reader (BMG Laboratories). ALP activity was determined following incubation with 1 mg/ml of P-nitro phenyl phosphate in 50 mMNAHCO3 and 1 mM MgCl2 buffer (pH 9.6) at 37 °C for 20 min. The activity was stopped by addition of 3 M NaOH. The reaction absorbance was measured at 405 nm using a FLUO star Omega plate reader and ALP activity corrected for cell viability.
Alkaline phosphatase (ALP) staining
After 6 days of osteoblast differentiation, cells were fixed with acetone/10 mM citrate buffer pH4.2 (1.5:1 ratio) at room temperature for 5 min and incubated for 1 h at room temperature with ALP substrate staining solution containing 0.2 mg/ml Naphtol-AS-TR-phosphate dissolved in distilled water (1:5) and 0.417 mg/ml Fast Red dissolved in 0.1 M Tris buffer.
Oil Red O staining and quantification
At day 12 of adipocyte induction, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and then stained with Oil Red O (0.5 g in 100% isopropanol) (Sigma, USA). Lipid accumulations were quantified by elution of Oil Red O in absolute isopropanol for 10 min at room temperature. The absorbance of the extracted dye was detected at 490 nm.
Alizarin Red S staining and quantification
Calcium deposition, at day 12 of osteoblastic differentiation, was measured using Alizarin Red staining. Osteoblasts were fixed with 70% ice-cold ethanol for 1 h at -20 °C before addition of AR-S (40 mM; Sigma-Aldrich) dissolved in distilled water, pH 4.17. The cells were stained for 10 min at room temperature. The level of calcium deposition was quantified by elution of AR-S following incubation in 10% cetylpyridinium chloride (Sigma-Aldrich) for 1 h at room temperature. The absorbance of the eluted dye was assessed at 570 nm in a FLU Ostar Omega plate reader.
Western blot assays
Cells were collected at specific time points post treatment, and washed in cold PBS buffer before being lysed in cell lysis buffer supplemented with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Twenty μg of protein was separated on 8 to 12% NuPAGE® Novex® Bis-Tris gel systems (Invitrogen). The membrane was blocked and probed with antibodies and incubated with peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Aarhus, Denmark). Antibodies for (total or phosphor) specific Smad1/5/8 and NF-κB p65 were obtained from Cell Signaling Technology (Leiden, Netherland).
Luciferase reporter assay
The activation of NF-κB pathway was determined by using Cignal™ NF-κB luciferase Reporter Assay Kit (QIAGEN Ltd., Manchester, UK). HEK 293 or mBMSCs cells were cultured in 96-well plates and transfected with a mixture of NF-κB luciferase reporter negative control or positive control, along with Renilla construct (as an internal control) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 24 h, medium was changed with either CM-Adipo or CM-Control and cells were cultured for 24 h. Luciferase activities were determined using the Dual-Luciferase Assay System (Promega, Southampton, UK). Reporter activities were represented as arbitrary units after normalization to the internal Renilla reporter.
All experiments were performed in 3–6 replicates and in at least 3 independent experiments. The data were presented as the mean ± SD. Students t-test was used for comparison between two groups. Differences were considered statistically significant at *P <0.05, and **P < 0.005.
CM-Adipo does not affect the cell viability or cell proliferation of mBMSCs
CM-Adipo inhibits the differentiation of mBMSCs into osteoblastic cell lineage
Down-regulation of osteogenic genes expression in mBMSCs treated with CM-Adipo as compared to CM-Control
Ossification and matrix molecules
Alkaline phosphatase, liver/bone/kidney
Bone gamma carboxyglutamate protein
Collagen type I alpha 1
Collagen type I alpha 2
Collagen type V alpha 1
Secreted phosphoprotein 1 (Osteopontin)
BMP signaling pathway
Bone morphogenetic protein 2
Bone morphogenetic protein 7
Bone morphogenetic protein receptor. type 1A
MAD homolog 5 (Drosophila)
Cell Adhesion Molecules
Integrin beta 1 (fibronectin receptor beta)
Integrin alpha 2
Integrin alpha 2b
Osteogenic growth factors
Fibroblast growth factor receptor 2
Insulin-like growth factor 1
Insulin-like growth factor I receptor
Platelet derived growth factor. alpha
Osteogenic transcription factors
Distal-less homeobox 5
Runt related transcription factor 2
Sp7 transcription factor 7
CM-Adipo inhibits BMP2-induced osteoblast differentiation of mBMSCs
The inhibitory effect of CM-Adipo on BMP2-induced osteogenesis is mediated by NF-κB activation
In this study, we demonstrated the paracrine inhibitory effect of marrow adipocytes on the differentiation potential of mBMSCs into osteoblasts by blocking BMP2-induced osteogenesis. This effect is mediated via a mechanism involved the activation of the NF-κB signaling pathway by adipokines.
In this study, the ST2 cell line  was selected as a source of adipocytes for collecting the CM-Adipo due to the following reasons; a) ST2 cell line is a clone of homogenous stromal cell population that derived from the mouse bone marrow with a characteristics phenotype of pre-adipocytic cells; b) ST2 cells can efficiently differentiate into homogenous population of adipocytes; c) ST2 cells are suitable for collecting the serum free conditioned medium due to their ability to maintain their viability in serum free medium for up to 48 h.
To avoid any undesirable effects from the residual amount of adipogenic inducers (i.e Dexamethasone, IBMX and insulin) in the collected CM, we changed the adipogenic medium with serum and adipogenic inducers free medium prior to the collection of CM. This strategy eliminated the plausible competing effects between such adipogenic inducers (exist at relatively high concentrations) and the adipocytes-secreted factors (exist at low concentrations) on the osteoblast differentiation of mBMSCs. In supporting to this notion, the high concentration of dexamethasone-induced adipogenic differentiation of BMSCs was reported to suppress the proliferation of osteoblasts .
Our data demonstrated the paracrine inhibitory effect of CM-Adipo on osteoblast differentiation of mBMSCs. Few studies have investigated the paracrine effect of adipocytes on osteoblast differentiation. For example, Benayahu et al.  demonstrated the inhibitory effect of adipocyte CM on osteogenesis of marrow-derived osteoblastic cells, MBA-15 , while Maxson S, et al.,  showed the stimulatory effect of adipocyte CM on the osteoblast differentiation of BMSCs. On the other hand, mammary adipose tissue-derived CM did not affect osteoblast differentiation of primary human osteoblasts . These contradictory data can be attributed to the use of different sources of adipocytes for collecting CM. In addition, these studies apply the traditional method for collecting CM, which does not exclude the presence of adipogenic inducers. Indeed, when such inducers were omitted in a co-culture system, adipocytes were shown not only to inhibit osteoblast differentiation of BMSCs [33, 34] but also to induce their trans-differentiation into adipocytes .
Our data identified the BMPs signaling as the most distinct osteogenic pathway to be inhibited by the adipocytes CM. In consistence with our finding, the impairment of BMPs signaling was reported in BMSCs derived from osteoporotic postmenopausal women (the condition of increased marrow adipocytes) . Recently, the activation of NF-κB was shown to suppress the BMP2-induced osteogenesis. In this mechanism, the inflammatory environment-stimulated NF-κB activity suppresses BMPs pathway via either activating the Toll-like receptor-4 and its intracellular adaptor protein My88 (TLR4/MyD88) dependent pathway  or interfering with DNA binding of the Smad complex [37, 38]. The interaction between BMPs and NF-κB signaling pathways was further confirmed by demonstrating that the inhibition of local inflammation is effective to promote the BMP-2 induced bone regeneration in vivo [39, 40]. Considering, that the adipocytes secret several inflammatory cytokines including IL-6, TNF-a, IL-1β, MCP1, CCL2 and PAI-1 known to be involved in the activation of the NF-κB [27, 41], our data demonstrated the stimulatory effect of CM-Adipo on the NF-κB signaling activation. In addition, we showed that the blocking of NF-κB signaling in mBMSCs rescued the inhibitory effect of CM-Adipo on BMP2-induced osteogenesis, suggesting that the inhibitory effect of CM-Adipo on osteoblast differentiation is mediated at least in part by NF-κB activation. Taken together, the inhibitory effect of CM-Adipocyte on osteogenesis is mediated by a group of secreted factors (Adipokines) rather than one individual factor and targeting the inhibition of adipocyte differentiation could be beneficiary for enhancing bone formation
Marrow adiposity is inversely correlated with bone mass in many osteoporotic clinical conditions. The differentiation potential of BMSCs into either osteoblastic or adipocytic cell lineage was shown to be regulated by local and secreted factors in bone marrow microenvironment. Here, we studied the paracrine effect of adipocytes on the differentiation capacity of mBMSCs into osteoblasts. Our data demonstrated the paracrine inhibitory effect of adipocytes-secreted factors on the osteoblast differentiation of mBMSCs by blocking BMPs signaling in a mechanism mediated by the activation of NF-κB pathway. This study identified a novel mechanism that controlling the paracrine inhibitory effect of adipocytes on osteoblast differentiation of BMSCs.
Adipocyte lipid-binding protein 2
Bone morphogenetic protein
CCAAT/enhancer-binding protein alpha
Collagen type 1
Nuclear factor kappa-light-chain-enhancer of activated B cells
Peroxisome proliferator-activated receptor gamma 2
Runt-related transcription factor 2
Secreted Frizzled-Related Protein 4
Professor Moustapha Kassem is thanked for providing the research facilities at KMEB laboratory, OUH, Denmark to perform some of the experiments of this study. The author is grateful to Bianca Jørgensen and Catherine Reilly for excellent technical assistance.
This work was funded by Novo Nordisk Foundation, Denmark (R152-A12355).
Availability of data and materials
All materials are available by the corresponding author.
BMA conceived the project, designed the study, performed experiments, analyzed data and wrote the manuscript.
The author declares that he has no competing interests.
Consent for publication
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Abdallah BM, Kassem M. Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther. 2008;15:109–16.View ArticlePubMedGoogle Scholar
- Bianco P, Robey PG. Skeletal stem cells. Development. 2015;142:1023–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Burchfield JS, Dimmeler S. Role of paracrine factors in stem and progenitor cell mediated cardiac repair and tissue fibrosis. Fibrogenesis Tissue Repair. 2008;1:4.View ArticlePubMedPubMed CentralGoogle Scholar
- Pigott JH, Ishihara A, Wellman ML, Russell DS, Bertone AL. Investigation of the immune response to autologous, allogeneic, and xenogeneic mesenchymal stem cells after intra-articular injection in horses. Vet Immunol Immunopathol. 2013;156:99–106.View ArticlePubMedGoogle Scholar
- Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 1997;89:755–64.View ArticlePubMedGoogle Scholar
- Siersbæk R, Nielsen R, Mandrup S. Transcriptional networks and chromatin remodeling controlling adipogenesis. Trends Endocrinol Metab. 2012;23:56–64.View ArticlePubMedGoogle Scholar
- Sadie-Van Gijsen H, Crowther NJ, Hough FS, Ferris WF. The interrelationship between bone and fat: from cellular see-saw to endocrine reciprocity. Cell Mol Life Sci. 2013;70:2331–49.View ArticlePubMedGoogle Scholar
- Sheu Y, Cauley JA. The Role of Bone Marrow and Visceral Fat on Bone Metabolism. Curr Osteoporos Rep. 2011;9:67–75.View ArticlePubMedPubMed CentralGoogle Scholar
- Hardouin P, Pansini V, Cortet B. Bone marrow fat. Joint Bone Spine. 2014;81:313–9.View ArticlePubMedGoogle Scholar
- Abdallah BM, Kassem M. New factors controlling the balance between osteoblastogenesis and adipogenesis. Bone. 2012;50:540–5.View ArticlePubMedGoogle Scholar
- Bredella MA, Fazeli PK, Daley SM, Miller KK, Rosen CJ, Klibanski A, Torriani M. Marrow fat composition in anorexia nervosa. Bone. 2014;66:199–204.View ArticlePubMedPubMed CentralGoogle Scholar
- Grey A, Beckley V, Doyle A, Fenwick S, Horne A, Gamble G, Bolland M. Pioglitazone increases bone marrow fat in type 2 diabetes: results from a randomized controlled trial. Eur J Endocrinol. 2012;166:1087–91.View ArticlePubMedGoogle Scholar
- Schwartz AV. Marrow Fat and Bone: Review of Clinical Findings. Front Endocrinol. 2015;6:40.View ArticleGoogle Scholar
- Schwartz AV, Sigurdsson S, Hue TF, Lang TF, Harris TB, Rosen CJ, Vittinghoff E, Siggeirsdottir K, Sigurdsson G, Oskarsdottir D, Shet K, Palermo L, Gudnason V, Li X. Vertebral Bone Marrow Fat Associated With Lower Trabecular BMD and Prevalent Vertebral Fracture in Older Adults. J Clin Endocrinol Metab. 2013;98:2294–300.View ArticlePubMedPubMed CentralGoogle Scholar
- Berendsen AD, Olsen BR. Osteoblast-Adipocyte Lineage Plasticity in Tissue Development, Maintenance and Pathology. Cell Mol Life Sci. 2014;71:493–7.View ArticlePubMedGoogle Scholar
- Hardouin P, Rharass T, Lucas S. Bone Marrow Adipose Tissue: To Be or Not To Be a Typical Adipose Tissue? Front Endocrinol. 2016;7:85.View ArticleGoogle Scholar
- Li J, Liu X, Zuo B, Zhang L. The Role of Bone Marrow Microenvironment in Governing the Balance between Osteoblastogenesis and Adipogenesis. Aging Dis. 2016;7:514–25.View ArticlePubMedGoogle Scholar
- Taipaleenmaki H, Abdallah BM, Aldahmash A, Saamanen AM, Kassem M. Wnt signalling mediates the cross-talk between bone marrow derived pre-adipocytic and pre-osteoblastic cell populations. Exp Cell Res. 2011;317:745–56.View ArticlePubMedGoogle Scholar
- Muruganandan S, Roman AA, Sinal CJ. Role of chemerin/CMKLR1 signaling in adipogenesis and osteoblastogenesis of bone marrow stem cells. J Bone Miner Res. 2010;25:222–34.View ArticlePubMedGoogle Scholar
- Shapses SA, Sukumar D. Bone Metabolism in Obesity and Weight Loss. Annu Rev Nutr. 2012;32:287–309.View ArticlePubMedPubMed CentralGoogle Scholar
- Peister A, Mellad JA, Larson BL, Hall BM, Gibson LF, Prockop DJ. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood. 2004;103:1662–8.View ArticlePubMedGoogle Scholar
- Lee J, Rhee MH, Kim E, Cho JY. BAY 11-7082 Is a Broad-Spectrum Inhibitor with Anti-Inflammatory Activity against Multiple Targets. Mediators Inflamm. 2012;2012:416036.PubMedPubMed CentralGoogle Scholar
- Abdallah BM, Ditzel N, Laborda J, Karsenty G, Kassem M. DLK1 Regulates Whole-Body Glucose Metabolism: A Negative Feedback Regulation of the Osteocalcin-Insulin Loop. Diabetes. 2015;64:3069–80.View ArticlePubMedGoogle Scholar
- Andersen RK, Zaher W, Larsen KH, Ditzel N, Drews K, Wruck W, Adjaye J, Abdallah BM, Kassem M. Association between in vivo bone formation and ex vivo migratory capacity of human bone marrow stromal cells. Stem Cell Res Ther. 2015;6:196.View ArticlePubMedPubMed CentralGoogle Scholar
- Zeng Z, Yin X, Zhang X, Jing DA, Feng XUE. Cyclic stretch enhances bone morphogenetic protein-2-induced osteoblastic differentiation through the inhibition of Hey1. Int J Mol Med. 2015;36:1273–81.PubMedPubMed CentralGoogle Scholar
- Huang R-L, Yuan Y, Zou G-M, Liu G, Tu J, Li Q. LPS-Stimulated Inflammatory Environment Inhibits BMP-2-Induced Osteoblastic Differentiation Through Crosstalk Between TLR4/MyD88/NF-κB and BMP/Smad Signaling. Stem Cells Dev. 2014;23:277–89.View ArticlePubMedGoogle Scholar
- Mancuso P. The role of adipokines in chronic inflammation. Immunotargets Ther. 2016;5:47–56.View ArticlePubMedPubMed CentralGoogle Scholar
- Ogawa M, Nishikawa S, Ikuta K, Yamamura F, Naito M, Takahashi K, Nishikawa S. B cell ontogeny in murine embryo studied by a culture system with the monolayer of a stromal cell clone, ST2: B cell progenitor develops first in the embryonal body rather than in the yolk sac. EMBO J. 1998;7:1337–43.Google Scholar
- Walsh S, Jordan GR, Jefferiss C, Stewart K, Beresford JN. High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro: relevance to glucocorticoid‐induced osteoporosis. Rheumatology. 2001;40:74–83.View ArticlePubMedGoogle Scholar
- Benayahu D, Zipori D, Wientroub S. Marrow Adipocytes Regulate Growth and Differentiation of Osteoblasts. Biochem Biophys Res Commun. 1993;197:1245–52.View ArticlePubMedGoogle Scholar
- Maxson S, Burg KJL. Conditioned media cause increases in select osteogenic and adipogenic differentiation markers in mesenchymal stem cell cultures. J Tissue Eng Regen Med. 2008;2:147–54.View ArticlePubMedGoogle Scholar
- Maurin AC, Chavassieux PM, Frappart L, Delmas PD, Serre CM, Meunier PJ. Influence of mature adipocytes on osteoblast proliferation in human primary cocultures. Bone. 2000;26:485–9.View ArticlePubMedGoogle Scholar
- Zhang H, Lu W, Zhao Y, Rong P, Cao R, Gu W, Xiao J, Miao D, Lappe J, Recker R, Xiao GG. Adipocytes Derived from Human Bone Marrow Mesenchymal Stem Cells Exert Inhibitory Effects on Osteoblastogenesis. Curr Mol Med. 2011;11:489–502.View ArticlePubMedGoogle Scholar
- Liu L-F, Shen W-J, Zhang ZH, Wang LJ, Kraemer FB. Adipocytes decrease Runx2 expression in osteoblastic cells: Roles of PPARγ and adiponectin. J Cell Physiol. 2010;225:837–45.View ArticlePubMedGoogle Scholar
- Clabaut A, Delplace S, Chauveau C, Hardouin P, Broux O. Human osteoblasts derived from mesenchymal stem cells express adipogenic markers upon coculture with bone marrow adipocytes. Differentiation. 2010;80:40–5.View ArticlePubMedGoogle Scholar
- Donoso O, Pino AM, Seitz G, Osses N, Rodríguez JP. Osteoporosis-associated alteration in the signalling status of BMP-2 in human MSCs under adipogenic conditions. J Cell Biochem. 2015;116:1267–77.View ArticlePubMedGoogle Scholar
- Hirata-Tsuchiya S, Fukushima H, Katagiri T, Ohte S, Shin M, Nagano K, Aoki K, Morotomi T, Sugiyama G, Nakatomi C, Kokabu S, Doi T, Takeuchi H, Ohya K, Terashita M, Hirata M, Kitamura C, Jimi E. Inhibition of BMP2-Induced Bone Formation by the p65 Subunit of NF-κB via an Interaction With Smad4. Mol Endocrinol. 2014;28:1460–70.View ArticlePubMedGoogle Scholar
- Yamazaki M, Fukushima H, Shin M, Katagiri T, Doi T, Takahashi T, Jimi E. Tumor Necrosis Factor α Represses Bone Morphogenetic Protein (BMP) Signaling by Interfering with the DNA Binding of Smads through the Activation of NF-κB. J Biol Chem. 2009;284:35987–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Ratanavaraporn J, Furuya H, Tabata Y. Local suppression of pro-inflammatory cytokines and the effects in BMP-2-induced bone regeneration. Biomaterials. 2012;33:304–16.View ArticlePubMedGoogle Scholar
- Lee K-B, Murray SS, Taghavi CE, Song K-J, Brochmann EJ, Johnson JS, Keorochana G, Liao J-C, Wang JC. Bone morphogenetic protein binding peptide reduces the inflammatory response to recombinant human bone morphogenetic protein-2 and recombinant human bone morphogenetic protein-7 in a rodent model of soft-tissue inflammation. Spine J. 2013;11:568–76.View ArticleGoogle Scholar
- Schäffler A, Müller-Ladner U, Schölmerich J, Büchler C. Role of Adipose Tissue as an Inflammatory Organ in Human Diseases. Endocr Rev. 2006;27:449–67.View ArticlePubMedGoogle Scholar