Bone integrity is retained by a dynamic process of bone remodeling, which maintains the balance between bone formation by osteoblasts and bone resorption by osteoclasts. Osteoblasts are resultant from bone marrow-derived mesenchymal stem cells (BMSCs), while osteoclasts are derived from hematopoietic stem cells in bone marrow [1
]. An excess bone resorption rate and/or dysregulation of bone formation results in osteoporosis [3
]. To date, the treatment for osteoporosis is mainly based on anti-resorptive drugs that target osteoclast-mediated bone resorption such as bisphosphonates, calcitonin, estrogen and Denosumab, a humanized monoclonal antibody against the receptor-activator of nuclear factor kappa-b ligand (RANKL) [6
]. Conversely, there are few existing anabolic bone therapeutics for osteoporosis including, parathyroid hormone (PTH) and, recently, Romosozumab, a humanized-neutralizing monoclonal antibody against sclerostin, an antagonist of Wnt signaling secreted by an osteocyte [8
]. Accordingly, there is a need for developing new anabolic bone drugs with safe processes to directly target the stimulation of bone formation in bone-loss related diseases.
Osteoblast differentiation of BMSCs involves sequential stages of lineage commitment, proliferation, extracellular matrix maturation, and mineralization [12
]. Several signaling pathways have been reported to be crucial for mediating the commitment and differentiation of BMSCs into osteoblasts, including Bone morphogenetic protein (BMP), Wnt signal, transforming growth factor beta (TGF-β), adenosine monophosphate (AMP)-activated protein kinase (AMPK), and mitogen-activated protein kinases (MAPKs) [13
]. The MAPK family consists of three major subfamilies: the extracellular signal-regulated kinases (ERKs), the p38 kinases and the Jun, N-terminal kinases (JNKs), that regulate a variety of cellular programs such as cell proliferation, differentiation, and survival [16
]. Moreover, ERK is identified as a crucial signal for controlling the lineage commitment of BMSCs into osteoblast or adipocyte, osteoblast proliferation, apoptosis and differentiation by regulating the expression of Runx2
, ALP activity and cell cycle regulators [17
]. Additionally, the stimulatory effect of anabolic bone factors, such as platelet-derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I) on osteoblastogenesis is mediated by the ERK signaling pathway [20
]. Therefore, identifying compounds that target the osteogenic differentiation signal of BMSCs, such as the ERK pathway, could provide a pharmacologic approach for developing a new anabolic bone drug.
Butein (3,4,2′,4′-tetrahydroxychalcone) is a phytochemical product belonging to the chaloconoid, which is a subclass of the flavonoids family. Butein is a major compound in Toxicodendron vernicifluum
, Caragana jubata
and Rhus verniciflua Stokes
]. In vitro and in vivo studies reveal the biological activity of butein as an anti-oxidant [25
], anti-fibrogenic [26
], anti-inflammatory [27
], anti-cancer [28
] and anti-adipogenic [30
] compound. Therefore, butein shows a high potential for the treatment of inflammatory diseases, cancer, and metabolic disorders (including obesity and diabetes) in many preclinical studies [27
]. Based on its process, butein exerts anticancer activity by regulating ERK signaling. Butein is demonstrated to inhibit the proliferation of breast cancer cells and the migration and invasion by bladder cancer cells and hepatocarcinoma cells via the modulating ERK signaling pathway [34
], for example.
Interestingly, butein has been reported to inhibit receptor activators of nuclear factor-kappaB (NF-κB) ligand (RANKL)-induced osteoclastogenesis [37
] and to reduce the progression of osteoarthritis in rodents [38
]. However, despite this potential therapeutic effect of butein for bone loss prevention, no studies have been published on the effect of butein on the differentiation of BMSCs into the osteoblastic cell lineage. We hypothesize that butein may regulate osteogenesis via modulating ERK signaling. Thus, we aim to investigate the effects of butein on mBMSC differentiation into osteoblasts and adipocytes, and to study the role of MAPK/ERK signaling pathways in mediating the effects of butein on osteogenesis. Our data identify butein as a new nutraceutical compound that promotes osteogenesis via activating ERK1/2 signaling.
Dietary nutraceuticals are highly attractive therapeutic compounds for the treatment of acute and chronic disorders of human diseases due to their effectiveness, low toxicity, and reduced side effects. Here, we demonstrate that the nutraceutical compound, butein, can effectively promote the early commitment of murine and human BMSCs into osteoblasts, while suppressing their differentiation into adipocytes. The regulatory effect of butein on BMSCs differentiation is mediated by an ERK1/2-dependent signaling pathway.
Butein is a multi-targeted flavonoid with a potential therapeutic effect against several chronic diseases including cancers, inflammatory diseases, obesity, and diabetes [33
]. Butein displays an anti-cancer effect by stimulating an anti-proliferative effect via, for example, inducing cell cycle arrest at the G2/M phase or by stimulating a pro-apoptotic effect via activation of mitochondria-dependent caspase-3 [41
]. Several studies report the anti-inflammatory activity of butein and its capacity to inhibit inflammatory reactions via suppression of NF-κB, stimulation of heme oxygenase-1, inhibition of iNOS-derived NO production, and downregulation of IL-6, IL-1β, interferon (IFN)-γ and MMP-9 expression [24
]. Butein is found to exert anti-adipogenic effects and improve glucose tolerance in diet-induced obese and leptin-deficient mice models via the inhibition of central IκB kinase β (IKKβ)/nuclear factor-κB (NF-κB) pathways [44
]. Furthermore, butein reduces hyperglycemia-induced diabetic complications in rodents [45
] and inhibits fibrosis in carbon tetrachloride (CCl4)-induced liver fibrosis in rats [47
Our data identify butein as a novel stimulator of osteoblast differentiation of BMSCs. Previous studies demonstrate the potential therapeutic activity of butein for some bone-related diseases. Butein has been reported to inhibit tumor cell (including myeloma cells) -induced osteoclastogenesis [37
] and to reduce the progression of cartilage degradation in a mouse osteoarthritis model [38
], for example.
Osteoblast differentiation is initiated by the activation of the master transcriptional regulator, Runt-related transcription factor 2 (Runx2/Cbfa1
), which regulates the expression of specific genes required for osteoblast maturation and function, including alkaline phosphatase (ALP), type I collagen, osteocalcin, bone sialoprotein, and osteopontin [48
]. Consistently, our data show the stimulatory effect of butein on upregulating the expression of Runx2
and its downstream targets.
The result of BMSCs into osteoblasts or adipocytes is reported to be regulated by the MAPK/ERK signaling pathway [13
]. Based on this, we demonstrate that the stimulatory effect of butein on the differentiation of mBMSCs into osteoblasts versus adipocytes is mediated via the activating ERK1/2 signaling pathway. Similarly, increasing ERK1/2 phosphorylation is found to be involved in mediating the differentiation result of BMSCs toward osteoblasts or adipocytes [52
]. Additionally, cell shape-dependent control of the lineage commitment of BMSCs through RhoA/ROCK signaling is associated with the ERK/MAPK activation [56
Our results demonstrate the inhibitory effect of butein on adipogenesis of BMSCs. Consistently, butein is reported to inhibit adipocyte differentiation, reduce fat mass and enhance browning of white adipose tissue [57
]. Further, we show that the inhibitory effect of butein on adipogenesis is mediated via ERK1/2-dependent signaling. Other signaling molecules are reported to mediate the inhibitory effect of butein on adipogenesis. These include the activation of p38 mitogen-activated protein kinase/nuclear factor erythroid 2-related factor 2 pathways in pre-adipocyte 3T3-L1 cell lines (p38 MAPK/Nrf2 pathway) [57
] and the stimulation of the transforming growth factor-β pathway, followed by STAT3 signaling in the C3H10T1/2 cell line [30
]. Thus, the effect of butein on the modulating signaling pathway is cell type-dependent.
Butein is found to possess estrogenic activity with a high binding affinity to estrogen receptors (ERs) [60
]. Interestingly, phytoestrogens that exert estrogen-like biological effects are shown to stimulate osteogenesis and prevent bone loss via ERK1/2 signal pathways. The two phytoestrogens, Genistein and Icariin, induce osteogenesis of osteoprogenitor cells and BMSCs, respectively, via the activation of the ERK1/2 signal pathways [61
], for example. Additionally, Puerarin, which possesses an estrogen-like structure, suppresses osteoblast apoptosis in an ERK-dependent manner [63
]. Thus, it is plausible that the stimulatory effect of butein on osteogenesis via ERK signaling involves the contribution of ERs. However, this hypothetical model needs further experimental work.
Interestingly, our data confirm the stimulatory effect of butein on osteogenesis of mBMSCs in human primary BMSCs, suggesting the plausible potential use of butein as a therapeutic drug for enhancing bone formation in osteoporotic patients. However, pre-clinical studies are needed to prove the osteo-anabolic effect of butein in the bone-loss animal model.
4. Materials and Methods
4.1. Cell Cultures and Reagents
Primary male C57BL/6J mouse BMSCs were isolated from 8-weeks-old as previously described [64
]. Cells were cultured in an RPMI-1640 medium supplemented with 12% FBS (Thermo Fisher Scientific GmbH, Dreieich, Germany), 12 μM L-glutamine (Thermo Fisher Scientific GmbH, Dreieich, Germany) and 1% penicillin/streptomycin (P/S) (Thermo Fisher Scientific GmbH, Dreieich, Germany). After 24 h, non-adherent cells were removed and cultured in 60 cm2
. The medium was changed every 3–4 days and cells were washed and regularly sub-cultured.
Human BMSC cells (hBMSCs) were purchased from Cell Applications Inc. (San Diego, CA, USA). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM)/low glucose (Sigma-Aldrich GmbH, Hamburg, Germany) containing 10% FBS (Thermo Fisher Scientific GmbH, Dreieich, Germany) and 1% penicillin/streptomycin according to the manufacturer’s instruction. The medium was changed every 2–3 days.
Butein (Cat. No. B178) and U0126 (Cat. No. U120) were purchased from (Sigma-Aldrich, Hamburg, Germany).
4.2. Cell Toxicity Assay
The cell toxicity of butein was determined by measuring cell viability using an MTT cell proliferation assay kit (Sigma-Aldrich, Hamburg, Germany) according to the manufacturer’s instruction kit. Cells were incubated with an MTT solution to metabolize to formazan, and absorbance was measured at a wavelength of 550 nm for the MTT assay. Values were represented as a fold change for control non-treated cells.
4.3. Cell Proliferation Study
The effect of butein on mBMSC proliferation was determined by culturing the cells at 2000 cells/well in 4 well plates. Cells were counted after 3 and 6 days using a hemocytometer. We measured 4–6 biological replicates for each concentration at each time point.
4.4. Osteoblast Differentiation
Osteoblast differentiation was stimulated in mBMSCs using an osteogenic induction medium (OIM), which consisted of α-minimum essential medium (α-MEM; Thermo Fisher Scientific GmbH, Dreieich, Germany) supplemented with 10% FBS, 10 mM β-glycerol-phosphate, 100 U/mL of penicillin, 100 mg/mL of streptomycin (Sigma-Aldrich, Hamburg, Germany), and 50 mg/mL of vitamin C (Sigma-Aldrich, Hamburg, Germany). The medium was changed every third day during osteogenesis.
4.5. Adipocyte Differentiation
Cells were induced to differentiate into adipocytes with an adipogenic-induction medium (AIM) consisting of DMEM supplemented with 9% horse serum, 450 µM 1-methyl-3-isobutylxanthine (IBMX), 250 nM dexamethasone, and 5 µg/mL insulin (Sigma-Aldrich, Hamburg, Germany) and 1 µM rosiglitazone (BRL 49653, Cayman Chemical, Ann Arbor, MI, USA). The medium was changed every 2–3 days.
4.6. Alkaline Phosphatase (ALP) Activity Assay
Cells were induced with OIM in a 96 well plate. ALP activity was determined by incubating the cells with 1 mg/mL of P-nitro phenyl phosphate in 50 mM NAHCO3 and 1 mM MgCl2 buffer (pH 9.6) at 37 °C for 20 min. Absorbance was measured at 405 nm. Cell viability was determined using the CellTiter-Blue® cell viability assay according to the manufacturer’s instruction. The value of ALP activity was normalization to the value of cell viability and represented as a fold change over the control. Each sample was measured in 6 biological replicates.
4.7. Cytochemical Staining
4.7.1. Alkaline Phosphatase Staining
Osteogenic cells were fixed with an acetone/citrate buffer pH 4.2 (1.5:1) for 5 min at room temperature. Cells were stained with Napthol-AS-TR-phosphate solution (Sigma-Aldrich, Hamburg, Germany) for 1 h at room temperature. The staining solution consists of 1:1 v/v Napthol-AS-TR-phosphate solution (Napthol-AS-TR-phosphate diluted 1:5 in H2O) and Fast Red TR solution (Sigma-Aldrich ApS, Hamburg, Germany) (diluted 1:1.2 in 0.1 M Tris buffer, pH 9.0).
4.7.2. Alizarin Red S Staining and Quantification
Cells induced to osteogenic lineage were fixed with 70% ice-cold ethanol for 1 h at −20 °C and stained with Alizarin red (40 mM, pH = 4; Sigma-Aldrich, Hamburg, Germany) for 10 min at room temperature. To quantify calcium deposition, AR-S was eluted with 10% cetylpyridinium chloride (Sigma-Aldrich ApS, Hamburg, Germany) for 1 h at room temperature, and the absorbance was measured at 570 nm. Values were normalized to cell number and presented as a fold change over the control non-induced cells.
4.7.3. Oil Red O Staining and Quantification
Differentiated cells into adipocytes were fixed in 4% paraformaldehyde for 10 min at room temperature. Accumulated fat droplets were stained with Oil Red O (0.5 g in 60% isopropanol) (Sigma-Aldrich, Hamburg, Germany) for 1 h. Oil Red O staining was eluted with isopropanol for 10 min at room temperature and lipids quantified from the extracted dye were measured at an absorbance of 490 nm. Oil Red O values were normalized to cell number (measured by number of viable cells) and then represented as a fold change over the control non-induced cells.
4.8. Western Blot Analysis
Cells were collected at specific time points in a lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 1% NP-40, 0.1% SDS, 1 mM EDTA, 1 mM phenyl-methylsulfonyl fluoride, 1 mM NaF, 1 mM Na3VO4), with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Then, 30 μg of protein was separated on 8–12% NuPAGE® Novex® Bis-Tris gel systems (Thermo Fisher Scientific, Dreieich, Germany), followed by transfer to a Hybond LFP polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The membrane was blocked and incubated with a peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Heidelberg Germany). Specific antibodies for total or phosphor p38 MAPK (Thr180/Tyr 182) and JNK (Thr183/Tyr185) were purchased from Cell Signaling Technology (Leiden, Netherlands). Phospho ERK1/2 (E4, sc-7383) and total ERK2 antibodies were purchased from Santa Cruz Biotechnology, Inc. Quantification of western blots was performed using an ImageJ program.
4.9. RNA Extraction and Real-Time PCR Analysis
Total RNA was extracted from cells using a TRIzol single-step method (Thermo Fisher Scientific, Dreieich, Germany). Then, 1 µg of total RNA was used to synthesise cDNA with a revertAid H minus first strand cDNA synthesis kit (Fermentas, St Leon-Rot, Germany) according to the manufacturer’s instructions. Quantitative real time PCR (qPCR) was performed using an Applied Biosystems 7500 Real-Time system with Fast SYBR®
Green Master Mix (Applied Biosystems, city, CA, USA). Primer sequences for target genes were presented in additional file 1: Table S1
. Target gene expression was normalization to β-Actin
mRNA expression as reference genes, using a comparative CT method [(1/(2delta-CT) formula, where delta-CT was the difference between CT-target and CT-reference] with Microsoft Excel 2007®
4.10. PCR Array Analysis
mBMSCs were induced to osteoblast differentiation in the presence or the absence of butein. Total RNA was extracted after 6 days of induction. A mouse osteogenic RT2 Profiler™ PCR array, containing 84 osteoblast-related genes (Qiagen Nordic, Sollentuna, Sweden) was performed using the SYBR® Green qPCR method on an Applied Biosystems 7500 real-time PCR system. Upregulated genes by butein were represented as a fold change over the control (≥2 fold, p < 0.005) after normalization to the reference genes.
4.11. Statistical Analysis
All values were expressed as mean ± SD (standard deviation), of at least 3 independent experiments. Power calculation was performed for 2 samples using an unpaired Student’s T-test (2-tailed) assuming equal variation in the two groups. Differences were considered statistically significant at * p < 0.05, and ** p < 0.005.