Butein Promotes Lineage Commitment of Bone Marrow-Derived Stem Cells into Osteoblasts via Modulating ERK1/2 Signaling Pathways.

Butein is a phytochemical that belongs to the chalcone family of flavonoids and has antitumor, anti-inflammatory, and anti-osteoclastic bone resorption activities. This study aims to investigate the effects of butein on the differentiation potential of mouse primary bone marrow-derived mesenchymal stem cells (mBMSCs) into osteoblast and adipocyte lineages. Primary cultures of mBMSCs are treated with different doses of butein during its differentiation. Osteoblast differentiation is assessed by alkaline phosphatase (ALP) activity quantification and Alizarin red staining for matrix mineralization, while adipogenesis is assessed by quantification of lipid accumulation using Oil Red O staining. Osteoblastic and adipocytic gene expression markers are determined by quantitative real-time PCR (qPCR). Western blot analysis is used to study the activation of extracellular signal-regulated kinase (ERK1/2). Interestingly, butein promotes the lineage commitment of mBMSCs into osteoblasts, while suppressing their differentiation into adipocytes in a dose-dependent manner. A similar effect of butein is confirmed in human (h) primary BMSCs. Occurring at the molecular level, butein significantly upregulates the mRNA expression of osteoblast-related genes, while downregulating the expression of adipocyte-related genes. The mechanism of butein-induced osteogenesis is found to be mediated by activating the ERK1/2 signaling pathway. To conclude, we identify butein as a novel nutraceutical compound with an osteo-anabolic activity to promote the lineage commitment of BMSCs into osteoblast versus adipocyte. Thus, butein can be a plausible therapeutic drug for enhancing bone formation in osteoporotic patients.


Introduction
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 [1,2]. An excess bone resorption rate and/or dysregulation of bone formation results in osteoporosis [3][4][5]. 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) [6,7]. 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][9][10][11]. 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.
Molecules 2020, 25 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][14][15]. 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][18][19]. 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 [20,21]. 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.
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.

Butein Induces Osteoblast Differentiation of mBMSCs
We examined the cytotoxicity of butein on primary cultures of mBMSCs. The effect of different concentrations of butein on cell viability of mBMSCs was measured using an MTT assay. Shown in Figure 1A, butein displayed toxicity on cell viability at concentrations above 30 µM. Similarly, a cell proliferation assay revealed the inhibitory effect of butein on the cell proliferation of mBMSCs at concentrations above 30 µM, as assessed by counting cell numbers after 3 and 6 days in culture. Thus, we used butein at concentrations between 1-30 µM throughout this study ( Figure 1B).
We examined the effect of butein on the differentiation of mBMSCs into osteoblasts. Butein exerts a stimulatory effect on the osteogenesis of mBMSCs in a dose-dependent manner, as demonstrated by increased levels of ALP activity ( Figure 1C) and Alizarin Red staining for matrix mineralization ( Figure 1D).

Butein Upregulates the mRNA Expression of Osteoblast-Related Genes in mBMSCs
Treatment of mBMSCs with butein during an osteoblast differentiation course showed significant upregulation early (Runx2, Alp, Col1a1, Osx) and late (Ocn and Opn) of osteoblastic markers by (≥ 2 fold, p < 0.005), as compared to control induced cells with DMSO) (Figure 2A,B). Further, qPCR-based osteogenic gene array analysis revealed significant up-regulation of the gene expression of ossification and matrix-related genes (≥ 2 fold, p < 0.05) by butein in mBMSCs ( Figure  2C, Table 1).

Butein Promotes Osteogenesis of mBMSCs at Early Commitment Stage
To get insight into the mechanism underlying the stimulatory effect of butein on osteogenesis, we examined the effect of butein on mBMSCs at different stages during osteoblast-lineage commitment. Shown in Figure 2D, treatment of mBMSCs with butein during osteogenesis at early (days 0-9) and middle (days 3-9) stages showed a significant increase in ALP activity levels, while late treatment of mBMSCs with butein (days 6-9) was not effective in increasing ALP activity.

Butein Upregulates the mRNA Expression of Osteoblast-Related Genes in mBMSCs
Treatment of mBMSCs with butein during an osteoblast differentiation course showed significant upregulation early (Runx2, Alp, Col1a1, Osx) and late (Ocn and Opn) of osteoblastic markers by (≥2 fold, p < 0.005), as compared to control induced cells with DMSO) (Figure 2A,B). Further, qPCR-based osteogenic gene array analysis revealed significant up-regulation of the gene expression of ossification and matrix-related genes (≥2 fold, p < 0.05) by butein in mBMSCs ( Figure 2C, Table 1).

Butein Promotes Osteogenesis of mBMSCs at Early Commitment Stage
To get insight into the mechanism underlying the stimulatory effect of butein on osteogenesis, we examined the effect of butein on mBMSCs at different stages during osteoblast-lineage commitment. Shown in Figure 2D, treatment of mBMSCs with butein during osteogenesis at early (days 0-9) and middle (days 3-9) stages showed a significant increase in ALP activity levels, while late treatment of mBMSCs with butein (days 6-9) was not effective in increasing ALP activity.
Cells were induced to differentiate into osteoblast without (DMSO) or with 30 µM butein for 6 days. A mouse osteogenesis RT 2 Profiler™ PCR array was performed for each cDNA sample using the SYBR ® Green quantitative PCR method. Up-regulated genes by butein (≥2 fold, p < 0.05) in mBMSCs were represented as a fold change over differentiated cells without butein after normalization with reference genes.

Butein Suppresses the Differentiation of mBMSCs into Adipocytes
Since osteoblasts and adipocytes in bone marrow are originated from the same precursor BMSCs [39], we examined the effect of butein on adipocyte differentiation of mBMSCs. Butein was shown to inhibit the commitment of mBMSCs into adipocytic cell lineage in a dose-dependent manner, as assessed by Oil Red O quantification and staining for lipid accumulation ( Figure 3A,B). Further, butein down-regulated the mRNA expression of both early (Pparγ2 and C/ebpα) and late (aP2, and Lpl) adipogenic markers in mBMSCs, as measured by qPCR analysis ( Figure 3C).

Butein Activates ERK1/2 Signaling Pathway during Osteogenesis.
To understand the mechanism mediating the stimulatory effect of butein on osteogenesis, we examined the possible regulation of mitogen-activated protein kinases (MAPKs) by butein. Western blot analysis of MAPK expression revealed a dose-dependent stimulatory effect of butein on ERK1/2 phosphorylation without affecting the expression of either p38 or JNK protein phosphorylation ( Figure 4A). Further, butein significantly stimulated ERK1/2 phosphorylation during osteoblast differentiation of mBMSCs, as assessed by Western blot analysis ( Figure 4B).

Butein Stimulates mBMSCs Differentiation into Osteoblast Versus Adipocyte in ERK1/2-Dependent Pathway
To ensure the involvement of ERK1/2 in mediating the effects of butein on mBMSC differentiation, we examined the effect of the blocking of ERK1/2 signaling on osteoblast and adipocyte differentiation of mBMSCs in the presence of butein. The ERK1/2-specific inhibitor, U0126, significantly inhibited ERK1/2 phosphorylation ( Figure 5A). Shown in Figure 5B,C, the butein-induced osteogenesis in mBMSCs was significantly abolished by U0126 treatment, as assessed by inhibiting levels of ALP activity and Alizarin Red staining (for matrix mineralization) by 59.3% and 65.4%, respectively, compared to non-treated cells with U0126 ( Figure 5B,C). Conversely, pre-treatment of mBMSCs with U0126 rescued the inhibitory effect of butein on the adipogenesis of mBMSCs, as shown by a significantly increased level of lipid accumulation in cells treated with butein compared to non-treated cells ( Figure 5D).

Butein Activates ERK1/2 Signaling Pathway during Osteogenesis.
To understand the mechanism mediating the stimulatory effect of butein on osteogenesis, we examined the possible regulation of mitogen-activated protein kinases (MAPKs) by butein. Western blot analysis of MAPK expression revealed a dose-dependent stimulatory effect of butein on ERK1/2 phosphorylation without affecting the expression of either p38 or JNK protein phosphorylation ( Figure 4A). Further, butein significantly stimulated ERK1/2 phosphorylation during osteoblast differentiation of mBMSCs, as assessed by Western blot analysis ( Figure 4B).

Butein Induces the Differentiation of Human (h) BMSCs into Osteoblasts
We further examined the effects of butein on osteoblast and adipocyte differentiation of hBMSCs. Interestingly, butein dose-dependently induced the differentiation of hBMSCs into osteoblasts, while suppressing their differentiation into adipocytes, as assessed by significantly increased ALP activity levels and Alizarin Red staining for osteogenesis, (Figure 6A,B) and reduced levels of Oil Red O staining for adipogenesis ( Figure 6C).

Butein Induces the Differentiation of Human (h) BMSCs into Osteoblasts
We further examined the effects of butein on osteoblast and adipocyte differentiation of hBMSCs. Interestingly, butein dose-dependently induced the differentiation of hBMSCs into osteoblasts, while suppressing their differentiation into adipocytes, as assessed by significantly increased ALP activity levels and Alizarin Red staining for osteogenesis, (Figure 6A,B) and reduced levels of Oil Red O staining for adipogenesis ( Figure 6C).

Discussion
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 [33,40]. 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 mitochondriadependent caspase-3 [41,42]. 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 [24,27,43]. Butein is found to exert anti-adipogenic

Discussion
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 [33,40]. 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 [41,42]. 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 [24,27,43]. 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 [45,46] 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 [48,49]. 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 [13,48,50,51]. 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][53][54][55]. 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][58][59]. 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 [61,62], 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.

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.

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.

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 NAHCO 3 and 1 mM MgCl 2 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.

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.

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.

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 and Hprt 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 ® [65].

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 RT 2 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.

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.

Conclusions
Here, we identified butein as a new nutraceutical compound with a potential therapeutic effect for enhancing bone formation in bone-loss related diseases. Our data demonstrated the stimulatory effect of butein on the lineage commitment of BMSCs into osteoblasts on the process of adipocytes. The dual effects of butein for stimulating osteogenesis and inhibiting adipogenesis of BMSCs was found to be mediated via activating ERK1/2 phosphorylation. Thus, butein has a potential to be used as an osteo-anabolic drug for the treatment of osteoporotic patients. Table S1: List of primers used for qPCR.