Modulation of osteogenic and myogenic differentiation by a phytoestrogen formononetin via p38MAPK-dependent JAK-STAT and Smad-1/5/8 signaling pathways in mouse myogenic progenitor cells

Formononetin (FN), a typical phytoestrogen has attracted substantial attention as a novel agent because of its diverse biological activities including, osteogenic differentiation. However, the molecular mechanisms underlying osteogenic and myogenic differentiation by FN in C2C12 progenitor cells remain unknown. Therefore the objective of the current study was to investigate the action of FN on myogenic and osteogenic differentiation and its impact on signaling pathways in C2C12 cells. FN significantly increased myogenic markers such as Myogenin, myosin heavy chains, and myogenic differentiation 1 (MyoD). In addition, the expression of osteogenic specific genes alkaline phosphatase (ALP), Run-related transcription factor 2(RUNX2), and osteocalcin (OCN) were up-regulated by FN treatment. Moreover, FN enhanced the ALP level, calcium deposition and the expression of bone morphogenetic protein isoform (BMPs). Signal transduction pathways mediated by p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-related kinases (ERKs), protein kinase B (Akt), Janus kinases (JAKs), and signal transducer activator of transcription proteins (STATs) in myogenic and osteogenic differentiation after FN treatment were also examined. FN treatment activates myogenic differentiation by increasing p38MAPK and decreasing JAK1-STAT1 phosphorylation levels, while osteogenic induction was enhanced by p38MAPK dependent Smad, 1/5/8 signaling pathways in C2C12 progenitor cells.

FN promotes both osteogenic and myogenic potential in C2C12 progenitor cells. First, we analyzed whether FN treatment promoted osteogenic and myogenic activities in the presence of 2% horse serum with 50 µg/mL Vitamin C and 10 mM β-glycerophosphate (HSCG). FN treatment potentially increased the levels of early and delayed osteogenic markers such as ALP at 24, 48, 72 h, and calcium deposition at days 2, 4, and 6 ( Fig. 2a-c). At the same time, microscopic views showed higher numbers of multinucleated myotubes than the control cells (Fig. 3a). It prompted us to investigate the osteogenic and myogenic properties of FN simultaneously in C2C12 cells cultured in the same medium.
FN treatment enhanced the expression of osteogenic and myogenic markers. FN treatment potently enhanced both osteogenic and myogenic differentiation. Therefore, we investigated the expression of osteogenic specific transcripts, ALP, osteocalcin, RUNX 2 and the expression of specific myogenic markers myoD, myogenin, myosin heavy chain and its isoforms (MHC) at different time intervals. Quantitative-PCR analysis revealed that HSCG significantly upregulated the osteoblast markers during differentiation, while FN combined with HSCG potently enhanced the transcription of ALP, OCN, and RUNX 2 (Fig. 2d). The ALP and RUNX 2 expression levels were peaked at 24 h, whereas the OCN expression was upregulated throughout experimental periods after FN treatment at a concentration of 2.5 µM (p < 0.05).Western blot results indicated that FN treatment strongly increased the expression of myogenic markers including myoD, myogenin, and myosin heavy chain and its isoforms (MHC) at different time intervals during differentiation (Fig. 3b,c). FN treatment Increased bone morphogenetic protein isoforms (BMPs). Next, we investigated the osteogenic potential and enhancement of different forms of BMPs by FN in vitro. We found that FN treatment potently upregulated the genes encoding BMP-2, BMP-4, BMP-6 BMP-7, and BMP-9 compared with the control cells. The induction of BMP-2, BMP-4 and BMP-7 was accelerated by FN until day 6, whereas the levels of BMP-6 and BMP-9 were upregulated by day 2 and 4, respectively (Fig. 4a). Western blot results revealed that BMP-2 and BMP-4 protein expression was also induced after FN treatment compared with control on day 6 ( Fig. 4b).
The results indicate that FN treatment induced osteogenic differentiation in C2C12 cells via induction of different BMPs at different time points.
FN regulates different signaling pathways mediating osteogenic and myogenic differentiation. As FN showed both osteogenic and myogenic potential in the HSCG medium, we investigated the mechanism of FN-mediated regulation of signal transduction pathways involved in osteogenic and myogenic differentiation of C2C12 progenitor cells. First, we analyzed the phosphorylation levels of JAK1, JAK2, STAT1, STAT2 and Smad 1/5/8 in the differentiated cells on day 4 and 6 after FN treatment in the same medium ( Fig. 5a,b). FN treatment decreased JAK1/STAT1 and increased phosphorylated Smad 1/5/8 levels compared with the control cells. However, JAK2/STAT2 phosphorylation was similar in control and FN-treated cells. We then decided to investigate the precise mechanisms underlying osteogenic and myogenic differentiation. For myogenic signaling confirmation, cells were treated with FN at a concentration of 2.5 µM in the myogenic differentiation medium containing 2% horse serum (HS) for 6 days. Results suggested that FN treatment downregulated the phosphorylated levels of JAK1/STAT1 compared with the control, while the Smad 1/5/8 phosphorylation was not altered significantly between control and FN treatment in HS medium ( Supplementary Fig. S1). It confirmed that FN treatment increased osteogenic differentiation by increasing the levels of Smad 1/5/8 phosphorylation while myogenic differentiation was promoted by decreasing the JAK1/STAT1 phosphorylation compared with control cells.
Finally, we explored the role of p38MAPK, AKT, and ERK 1/2 pathways in FN-induced osteoblast and myoblast differentiation. The levels of p38MAPK, AKT, and ERKs phosphorylation were determined by western blot with specific antibodies on day 4 and 6 following FN treatment in HSCG and HS medium (Figs 6a,b and S2). FN treatment at 2.5 µM activated p38MAPK by increasing its phosphorylation level, while the levels of p44/42 and AKT were not altered by FN when compared with the control cells. Therefore, a specific p38MAPK inhibitor www.nature.com/scientificreports www.nature.com/scientificreports/ was used in this study to establish FN-induced osteogenic and myogenic differentiation. Cells treated with p38 inhibitor alone at 10 μM in HSCG medium for 48 h decreased ALP activity, calcium deposition, and osteogenic specific genes such as ALP, RUNX 2 and osteocalcin mRNA expression compared with the control and FN-treated cells. FN treatment combined with p38 inhibitor increased ALP activity, calcium deposition and osteogenic specific genes ALP, RUNX 2 and osteocalcin mRNA expression compared with the control and cells treated with p38 inhibitor alone ( Fig. 7a-e.). In addition, cells treated with p38 inhibitor showed the reduced development of multinucleated myotubes and myogenic specific markers myoD, and myogenin proteins whereas treatment of p38 inhibitor together with FN at 2.5 μM reversed the p38 inhibitor-mediated inhibition of myotube formation and its associated myogenic markers compared with control and p38 inhibitor-treated cells in HSCG (Fig. 8a-c). Overall results suggest that FN treatment stimulated osteoblast differentiation by activating p38MAPK/Smad/ BMP signaling pathways, while myogenic differentiation was regulated by p38MAPK/JAK1/STAT1 signaling.
The key findings suggest that p38MAPK plays a significant role in both osteogenic and myogenic enhancement of C2C12 progenitor cells by FN.

Discussion
FN is a phytoestrogen known to display a diverse spectrum of biological activities. Its functional role in osteogenic and myogenic differentiation is poorly demonstrated. Hence, we explored the role of FN in C2C12 cell differentiation into osteogenic and myogenic lineage as well as the underlying signal transduction pathways. FN showed a strong osteogenic and myogenic potential in C2C12 progenitor cells. Enhancement of alkaline phosphatase, OCN, RUNX and other factors such as BMPs, is important for the differentiation of C2C12 progenitor cells into pre-osteoblasts. In mouse C2C12 cells, BMP-2, 4, 6, 7, and 9 strongly induced the expression of early osteogenic marker ALP and late osteogenic marker OCN 36 . OCN and osteopontin (OPN) are osteogenic markers mediating the intermediate to late stages of osteogenesis. They enhance calcium deposition in the late phases of osteogenesis 37 . The current study findings strongly reinforce the results discussed above. Indeed, FN treatment increased ALP production and its mRNA expression in C2C12 cells in a time-dependent manner. ALP and its mRNA expression were increased until 48hrs by FN treatment, whereas later, the levels of ALP and its mRNA expression were downregulated. ALP has been widely accepted to be a strong and early marker of osteoblast differentiation. It is responsible for the mineralization of the extracellular matrix (ECM) 38 . In addition, calcium deposition and the gene encoding OCN at later stages of differentiation were accelerated by FN treatment as compared with the control. Osteopontin and OCN are commonly used as early and late markers of osteogenic differentiation respectively 39 .
FN treatment increased myogenic differentiation via upregulation of specific myogenic markers in a time-dependent manner. Differentiation of myogenic cells is a highly organized process that is regulated by the members of MyoD family including, MyoD and myogenin as well as the proteins in the myocyte enhancer factor Protein lysates were prepared and used for immunoblotting using specific antibodies against key myogenic markers myoD, myogenin, myosin heavy chains (MHC) and α-tubulin. (c) The intensity of protein reacted bands was determined by densitometry using ImageJ software. Bars represent mean ± SEM for three replicates and statistical analysis was performed using a general linear model with multivariate, post hoc test and comparisons with respective controls. p < 0.05 level was considered significance between treatment and non-treatment. (2019) 9:9307 | https://doi.org/10.1038/s41598-019-45793-w www.nature.com/scientificreports www.nature.com/scientificreports/ (MEF2) family. The process of differentiation is highly complex and involves cell cycle termination, expression of myogenic specific genes, and multinucleate myotube formation 2,40,41 . Myogenin plays an essential role in myoblast differentiation. It acts at later stages of myogenesis to control their fusion 42 . The terminal differentiation of myoblasts into skeletal myocytes and fusion into myotubes is mediated by the controlled increase in the expression of MyoD, Myf5, myogenin and MRF4, and the decreased the activity of cell cycle regulatory factors 43 . The activation of myogenic regulatory factors (MRFs) including MyoD, myogenic factor 5 (Myf5), MRF4, and myogenin, also regulates the expression of several muscle-specific genes, such as myosin heavy chain (MyHC) and creatinine kinase in muscle fiber type maturation [44][45][46] .
Accordingly, FN treatment increased the formation of multinucleated myotubes in C2C12 cells compared with the control, without affecting the cell viability or morphology. These results based on cell morphology and analysis of specific myogenic markers suggests that FN increased the fusion of myoblasts into multinucleated myotubes. Myogenin is a key factor required for muscle cell differentiation 45 . Its level is increased during the early stages of differentiation, and decreased in fully differentiated cells. Also, MyHC and its isoforms are increased during different stages of cellular differentiation 47 . The expression of myogenic specific proteins such as myoD, myogenin, and muscle-specific genes myosin heavy chain and myosin heavy chain IIA during the differentiation phase was analyzed. Our data suggested that FN treatment increased the expression levels of myoD, myogenin, myosin heavy chain and myosin heavy chain IIA in differentiated cells compared with the control cells.
Bone morphogenetic protein induction and Smad1/5/8 signaling in osteogenic differentiation by FN were examined, because BMPs are known to mediate bone formation via Smad signaling 21,48,49 . BMPs including, BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 promote osteoblast differentiation of mesenchymal stem cells. BMP-2 50 , BMP-7, BMP-4, BMP-6, and BMP-9 exhibit strong osteogenic potential in C2C12 cells 51 . BMP-2 and BMP-7 induce rapid bone formation and increase the endogenous expression of BMP-4 52 . BMP9 is recognized as one of the most osteogenic BMPs. It promotes osteoblastic differentiation of mesenchymal stem cells (MSCs) both in vitro and in vivo 36,[53][54][55][56][57] . Bone morphogenetic protein -9 might provides a useful clinical strategy for the augmentation of bone regeneration and healing compared with other BMPs 11 . FN treatment significantly accelerated the expression of BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9. Furthermore, the levels of Smad 1/5/8 phosphorylation were increased compared with the control cells. Osteogenic activities of BMPs are known to activate Smad-Runx2 58 . Our results corroborate the findings of previous reports suggesting that FN treatment activates the Smad1/Smad 5/Smad8 expression by increasing its levels of phosphorylation. In addition, FN treatment increased the mRNA expression of Runx2 compared with the control suggesting that FN activated Smads/1/5/8 signaling during osteogenic differentiation by inducing BMP transcriptional activity. www.nature.com/scientificreports www.nature.com/scientificreports/ As FN showed both osteogenic and myogenic potential, we investigated its role in the regulation of signaling pathways involved in osteogenic and myogenic differentiation of C2C12 cells. First, we analyzed the phosphorylation levels of JAK1, JAK2, STAT1, and STAT2 in differentiated cells on day 4 and 6 post-treatment with FN. It is known that the JAKs/STATs pathway plays an essential role in myogenic differentiation. The JAK1/STAT1/ The intensity of protein bands was quantified by densitometry using ImageJ software. Bars display mean ± SEM of three experimental replicates. *p < 0.05 represents a statistically significant difference between control and treatment. www.nature.com/scientificreports www.nature.com/scientificreports/ STAT3 axis is involved in myoblast proliferation, which prevents premature differentiation into myotubes 59 . JAK2/STAT2/STAT3 expression appears to positively regulate differentiation, indicating that STAT3 elicits specific responses at various times during myogenesis. Inhibition of JAK2 expression abrogates myogenic differentiation. At the same time, JAK1 knockdown accelerates myogenic differentiation, while proliferation is inhibited in C2C12 cells and primary myoblasts 60 . In addition, STAT1 knockdown promotes myogenic differentiation in both primary and immortalized myoblasts 59 . Therefore, we analyzed whether FN altered JAK/STAT signaling pathways involved in the differentiation. Our data showed that FN treatment downregulated JAK1/STAT1 expression by decreasing their phosphorylation level. However, the phosphorylation level of JAK2/STAT2 was not altered significantly between control and FN-treated cells. These results confirm that FN regulated myogenic differentiation via inhibition of JAK1/STAT1 by decreasing their phosphorylation.
At last, we determined the role of FN on p38MAPK, AKT and ERK1/2 signaling pathways involved in osteogenic and myogenic differentiation of C2C12 cells. Extracellular signals regulating both osteogenic and myogenic signals are transduced to the nucleus by mitogen-activated-kinases. Inhibition of p38 prevents the differentiation mechanism in myogenic cell lines and human primary myocytes. Inhibition of p38 also prevents induction of early markers such as myogenin, p21, and late (MHC) myogenic markers 8 . In addition, p38 MAPK phosphorylation plays a key role in the regulation of ALP production during MC3T3-E1 cells differentiation. Inhibition of p38 MAPK by specific inhibitors decreased the ALP expression and mineral deposition in MC3T3-E1 cells 61 . Another study reported that p38 MAPK was required for the expression of ALP and osteocalcin while ERKs were necessary for OC expression only 62 . Several investigators have reported that the ERK groups of MAPKs also play a role in myogenic differentiation. While some investigators have indicated that ERK members inhibit differentiation 63,64 others reported that ERKs are positive regulators of myogenesis 65 . Akt signaling also plays a major function in hypertrophy and contributes to the myotubes size increases in C2C12 cells 21 . In addition, IGF-phosphoinositide 3-kinase (PI3K)-Akt signaling has been shown to induce myogenic differentiation by stimulating genes specific for myogenic markers such as myogenin, MyoD and MEF2 66,67 . The current study demonstrates that treatment of FN significantly increased the p38 MAPK expression at 2.5 μM of FN without altering Akt or ERKs phosphorylation levels. These results suggest that FN enhanced both osteogenic and myogenic induction via p38 signaling without altering Akt or ERKs pathways. Furthermore, the augmentation of the p38 www.nature.com/scientificreports www.nature.com/scientificreports/ pathway by FN treatment in differentiated cells was investigated using specific inhibitors. Cells were treated with SB-203580; a p38 inhibitor potently reduced both osteogenic and myogenic differentiation by downregulating specific myogenic markers such as myogenin, myoD, and osteogenic markers including the level of ALP, calcium accumulation and the specific gene expression ALP, RUNX2 and OCN in cells. By contrast, FN treatment with p38 inhibitor accelerated both osteogenic and myogenic specific genes and protein expression.

Conclusion
In conclusion, the current data suggest that FN treatment significantly increases ALP activity, calcium deposition, and the expression of osteogenic key markers including ALP, RUNX2, OCN and myogenic specific genes such as myogenin, MyoD, myosin heavy chains. BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 levels were enhanced by the FN treatment in a concentration and time-dependent manner. FN treatment activates myogenic differentiation by increasing p38MAPK and decreasing JAK1-STAT1 phosphorylation levels, while osteogenic differentiation was enhanced by p38MAPK dependent Smad, 1/5/8 signaling pathways in C2C12 progenitor cells. FN might represent a potential lead compound to promote osteogenic and myoblast differentiation in C2C12 progenitor cells, especially regulating the progression of osteogenic enhancement and myotube morphology.  www.nature.com/scientificreports www.nature.com/scientificreports/ Determination of cell viability. Ez-cytox assay kit (iTSBiO, Seoul, Korea) was used to determine the effects of FN on cell viability. In details, the cells (C2C12-ATCC, USA) at the density of 1 × 10 4 were treated with different concentrations of FN after 24 h seeding in 96 well plates and incubated at 37 °C with 5% CO 2 for 24 h and 48 h. After incubation, ten microliters of WST reagent was added to each well and incubated at 37 °C with 5% CO 2 for 1 to 2 h. The cell viability was measured at 450 nm using a Packard SpectraCount Absorbance Microplate Reader [Packard Instrument Co., Downers Grove, IL].

Methods
Osteogenic differentiation. C2C12 progenitor cells were seeded into 6-well (or) 12-well cell culture plates at a density of 5 × 10 4 or 2.5 × 10 4 cells/well, respectively. Cells were cultured in 10% FBS in DMEM (ATCC30-2002) medium and incubated at 37 °C with 5% CO 2 . Osteogenic induction was performed according to the previous method 68 with modifications. The growth medium was replaced by osteogenic differentiation medium containing vitamin C (50 µg/mL) and β-glycerophosphate (10 mM) in the presence of 2% horse serum (HSCG) medium after cells reached 80-90% confluence. FN at different concentrations was exposed to the cells in the HSCG medium for every 48 h during the experimental periods.
Myogenic differentiation. C2C12 cells were seeded into 6-well (or) 12-well cell culture plates at a density of 5 × 10 4 or 2.5 × 10 4 cells/well, respectively. The cells were cultured in 10% FBS in DMEM DMEM (ATCC30-2002) medium and incubated at 37 °C with 5% CO 2 . Myogenic differentiation was induced with myogenic differentiation medium consisting of DMEM with 2% horse serum (HS) after cells reached 80-90% confluence. The growth medium was replaced by 2% HS medium for every 48 h with different concentration of FN until the end of the experimental periods 69 .
ALP quantification. Experimental cells were harvested at different time points and washed with cold PBS three times. Cells were suspended in 500 µL of assay buffer and homogenized using a homogenizer. Cell lysates were centrifuged at 12000 g, 4 °C for 15 min. The supernatants were collected and stored on ice for further assay. The alkaline phosphatase activity of samples was measured using the ALP assay kit (Abcam, Cambridge, MA, USA).
Calcium staining and quantification. The culture medium was aspirated and washed with PBS twice.
Cells were fixed with 1 mL of 4% paraformaldehyde in PBS for 15 min at room temperature. Subsequently, after carefully removing the fixative, the cells were washed three times with dH2O. After draining the water completely, 1 mL of 2% Alizarine Red S stain solution was slowly added to each well. Plates were then incubated at the room for 30 min temperature. After removing excess dye, plates were washed 3-5 times with dH 2 O followed by the addition of water 1 mL to each well. Images were obtained using an inverted microscope (CKX41, Olympus Corporation, Tokyo Japan]. Calcium deposition in differentiated cells was quantified using the Alizarin Red S staining quantification kit according to the manufacturer's protocol (Science Cell, Carlsbad, CA).
Protein extraction and immunoblotting. Proteins lysates were prepared from the cells using Radioimmunoprecipitation assay buffer (RIPA) (Rockland, Limerick, PA) with 1X protease and phosphatase inhibitors (Roche, Basel, Switzerland and Sigma Aldrich, St. Louis, USA). Cells were washed three times with PBS. After the addition of the required volume of RIPA lysis buffer based on the plate types, cells were incubated at 4 °C for 5 min. Cells were scraped rapidly with a cell scraper (TPP, Trasadingen, Switzerland) to remove and lyse residual cells. The cell lysate was transferred to a 2 mL tube and centrifuged at 8000 g for 10 min at 4 °C. Protein concentration was quantified by the Pierce BCA protein assay kit (Thermofisher Scientific, Massachusetts, USA). Protein samples were separated by pre-casting-SDS-PAGE (4-12%, Biorad-California, USA) and blotted onto polyvinylidene difluoride (PVDF) membranes (Trans-blot Turbo transfer system, Biorad, California, USA). Immunoblotting was performed according to the western breeze chemiluminescence kit (Invitrogen, Massachusetts, USA) using rabbit monoclonal and polyclonal antibodies. All primary antibody reactions were carried out at 4 °C for overnight (Cell Signaling Technology antibodies 1:1000; Santa Cruz Biotechnology antibodies 1.500; Abcam antibodies 1:1000) against specific proteins 69 . The HRP-conjugated secondary antibody was used for the detection of primary antibodies (Cell Signaling Technology, Danvers, MA, USA). Band signals were analysed with an enhanced chemiluminescence kit (Bio-Rad-California, USA) on a chemiluminescence imaging system (Davinch gel imaging system, Seoul, South Korea). The intensity of immunoreactive bands was quantified with ImageJ software -1.49 version(32 bit), (Wayne Rasband, National Institute of Health, USA).