Enhancing Activity of Pleurotus sajor-caju (Fr.) Sing β-1,3-Glucanoligosaccharide (Ps-GOS) on Proliferation, Differentiation, and Mineralization of MC3T3-E1 Cells through the Involvement of BMP-2/Runx2/MAPK/Wnt/β-Catenin Signaling Pathway

Osteoporosis is a leading world health problem that results from an imbalance between bone formation and bone resorption. β-glucans has been extensively reported to exhibit a wide range of biological activities, including antiosteoporosis both in vitro and in vivo. However, the molecular mechanisms responsible for β-glucan-mediated bone formation in osteoblasts have not yet been investigated. The oyster mushroom Pleurotus sajor-caju produces abundant amounts of an insoluble β-glucan, which is rendered soluble by enzymatic degradation using Hevea glucanase to generate low-molecular-weight glucanoligosaccharide (Ps-GOS). This study aimed to investigate the osteogenic enhancing activity and underlining molecular mechanism of Ps-GOS on osteoblastogenesis of pre-osteoblastic MC3T3-E1 cells. In this study, it was demonstrated for the first time that low concentrations of Ps-GOS could promote cell proliferation and division after 48 h of treatment. In addition, Ps-GOS upregulated the mRNA and protein expression level of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor-2 (Runx2), which are both involved in BMP signaling pathway, accompanied by increased alkaline phosphatase (ALP) activity and mineralization. Ps-GOS also upregulated the expression of osteogenesis related genes including ALP, collagen type 1 (COL1), and osteocalcin (OCN). Moreover, our novel findings suggest that Ps-GOS may exert its effects through the mitogen-activated protein kinase (MAPK) and wingless-type MMTV integration site (Wnt)/β-catenin signaling pathways.


Introduction
Osteoporosis is a global disease resulting from an imbalance of bone homeostasis. An increase in the ratio of bone resorption to bone formation causes low bone mass and strength, which leads to posseses various biological activities, including antimicroorganisms, antitumor, antioxidant, antihypertension, antidiabetic, and anti-inflammation. According to its various biological activities, many biologically active compounds have also been studied regarding its role on therapeutic applications such as β-glucans, proteoglycan, phenolic acids, terpenes, proteins, and sterols [29]. Extracted Pleurotus sajor-caju β-glucan has a high molecular weight and low water solubility. The biological activities of β-glucans usually depend on physicochemical properties, source, purity, primary structure, water solubility, and molecular weight [30,31]. Insoluble particulate β-glucans have limited potential for medicinal applications, but may have more applicability following partial hydrolysis. For example, curdlan, which is a water-insoluble microbial linear exo-polysaccharide (1→3) β-d-glucan, has recently been effectively digested with a novel recombinant endo-β-1→3-glucanase to obtain a water soluble glucanoligosaccharide [32]. Here, we have used Hevea β-1,3-glucanase isozymes GI and GII, previously reported to specifically hydrolyse glucans such as laminarin [33], containing a low frequency of β-1,3-d-glucosidic linkages. We have digested particulate β-glucans to obtain a hydrolysate, Pleurotus sajor-caju glucanoligosaccharide (Ps-GOS), with shorter chain length, lower molecular weight, and higher water solubility (Ratthajak et al., manuscript in preparation).
The aim of this study is to investigate the effect of Ps-GOS on the proliferation and differentiation of the murine pre-osteoblastic cell line MC3T3-E1. We show that Ps-GOS promotes MC3T3-E1 cell proliferation, and induction of BMP-2 and Runx-2, which act coordinately, together with components of MAPK and Wnt/β-catenin signaling pathways, to enhance both gene expression of osteogenic bone markers and mineralization.

Preparation of Water Soluble P. sajor-caju Glucanoligosaccharide (Ps-GOS)
Ps-GOS was obtained from an investigation under the Center of Excellence in Natural Rubber Latex Biotechnology Research and Development (CERB) (additional details are under patent and will be published elsewhere). Briefly, the insoluble β-glucan fraction was isolated from the fruiting bodies of grey oyster mushroom (Pleurotus sajor-caju) according to modified Freimund's method [34,35]. The β-glucan assay was conducted according to the method of McClear and Glennie-Holmes [36], using the Mushroom and Yeast β-glucan kit (K-YBGL) (Megazyme International Ireland Ltd., Wicklow, Ire-land). The soluble β-glucan from P. sajor-caju (Ps-GOS) was prepared by mixing the insoluble β-glucan (mean particle size as 646.6 ± 299.3 µm determined using a Beckman Coulter LS 230 Laser Diffraction Particle Size Analyser) with the concentrate enzyme fraction contained 0.66 nkat ml −1 of glucanase activity (ratio of 1.5%, w/v). The optimized Hevea glucanase conditions of the concentrate enzyme fraction were chosen according to report of Churngchow et al., [33]. After complete digestion, the Ps-GOS derived after centrifugation was freeze-dried (mean particle size as 122.7 ± 147.0 µm). Both insoluble β-glucan and Ps-GOS were subjected to FTIR spectroscopy for structural characterization (data not shown). Data from electrospray ionization mass spectrometry (ESI-MS) demonstrated the mass of insoluble β-glucan, and Ps-GOS was distributed from 20 and 5 kDa, respectively.

Cell Proliferation
MTT assay was performed as an indicator for cell proliferation based on the ability of cells to convert MTT into a formazan reaction product. MC3T3-E1 cells were seeded and then treated with Ps-GOS at various concentrations for 24 and 48 h. At the end of treatment, cultured cells were washed with PBS. Next, 5 mg/mL of MTT and RPMI-1640 phenol red-free medium were added and incubated at 37 • C. After 3 h, the mixture was removed and the formazan product was solubilized by acidic isopropanol. The absorbance was measured by microplate reader at 570 nm.

Alkaline Phosphatase (ALP) Activity Assay
Cells were seeded in 24 well plates and then treated with different concentrations of Ps-GOS for 14 days. After treatment, the cells were washed with PBS and lysed with lysis buffer containing 50 mM Tris-HCL (pH 7.4) and 1% Triton X-100 to collect supernatant for determining ALP activity and protein concentration. ALP activity was determined with 4 mg/mL of 4-nitrophenyl phosphate (4NPP) in 0.2 M of 2-amino-2-methyl-1-propanol with 4 mM of MgCl 2 as a substrate for 30 min at 37 • C. The reaction was stopped by 0.1 M NaOH, and the yellow solution was measured at 405 nm. Protein concentration was measured using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). ALP activity was normalized to the concentration of protein and showed in the term of µmole/min/mg of protein.

Alizarin Red S Staining
Alizarin red S staining was used to determine calcium accumulation. In brief, cells were grown in 24 well plates and treated with Ps-GOS for 14 and 21 days. After incubation, cells were washed with PBS and then fixed with 10% formaldehyde 15 min. The cells were stained with 40 mM Alizarin red S solution (pH 4.1-4.3) for 30 min with gentle shaking at the room temperature. To quantify the dye, nonspecific staining was removed by washing with distilled water five times (5 min/time) and then solubilizing the stain with cetylpyridinium chloride (CPC) by shaking, and the absorbance at 550 nm was measured.

Cell Cycle Analysis
Cell cycle assay was performed by flow cytometry to analyze the cell cycle progression while treating with Ps-GOS for 48 h. Cells were trypsinized and fixed in 70% ethanol at 4 • C for 30 min, followed by incubation with propidium iodide containing RNase staining solution (Merck, Branchburg, NJ, USA) for 30 min in the dark to detect DNA content in the cells. The percentage of cells in G0/G1, S, and G2/M phases was analyzed by flow cytometry Guava easyCyteTM HT (Hayward, CA, USA).

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA from growing cells incubated with Ps-GOS for 24, 48, and 72 h was extract using the TriPure Isolation Reagent (Roche, Buonas, Switzerland). To generate cDNA, total RNA (1 µg) was used as a template to transcribe into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Buonas, Switzerland) according to the manufacturer's instruction. This cDNA was used to determine the gene expression for the genes of interest using the gene specific primers detailed in Yodthong's study [37] and GAPDH as the house keeping control gene. qRT-PCR reactions were amplified using the EvaGreen HRM Mix (Solis Biodyne, Tartu, Estonia), and then relative expression ratio was calculated using the 2 −∆∆Ct method.

Western Blot Analysis
Cells were grown and treated with Ps-GOS in six well plates. After indicated time, the cells were lyzed with lysis buffer and then centrifuged at 14,000× g for 15 min at 4 • C to collect supernatant for using as total protein extract. The concentration of protein was quantified using a protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein from each sample were loaded onto 10% gels for SDS-PAGE, transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Amersham Buckinghamshire, UK), blocked with 5% nonfat dry milk solution for 1 h, and then incubated with antiBMP-2 (Abcam, Milton, UK), antiRunx2 (Cell Signaling Technology, Beverly, MA, USA), or antiβ-Actin (Sigma-Aldrich, St. Louis, MO, USA). After washing three times with TBS-Tween, the blots were probed with an Alexa infrared dye-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h and detected the bands by Odyssey Infrared Imaging System (LI-CORE) in accordance with the manufacturer's instruction. The relative of the intensity of the protein interesting bands and the intensity of β-Actin band were compared.

Immunofluorescence Microscopy
The cells treated with Ps-GOS were grown on sterile collagen-coated coverslips in 24 well plates. In brief, coverslips were fixed with 4% paraformaldehyde 20 min, quenched with 50 mM NH 4 Cl for 2 × 10 min, washed with PBS, and permeabilized with 0.1% Triton X-100 for 10 min at RT. After blocking with 0.5% FSG in PBS for 20 min, each coverslip was incubated overnight at 4 • C with polyclonal rabbit antimouse BMP-2 (Abcam, Milton, UK), followed by removing primary antibody and washing 3 × 5 min in blocking solution before incubation for 1 h with FITC-conjugated secondary antirabbit IgG (Alexa Fluor 680) (Thermo Fisher Scientific, Waltham, MA, USA) and DAPI counterstain (Thermo Fisher Scientific, Waltham, MA, USA). Coverslips were washed 3 × 5 min in blocking solution and mounted on to slides using Prolong TM Gold antifade reagent (Invitrogen, Carlsbad, CA, USA). Fluorescence images were acquired with Nikon Eclipse Ti fluorescence microscope.

Statistical Analysis
Representative data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using the SPSS 23 statistical software (SPSS Inc., Chicago, IL, USA). The significant results were analyzed by one-way analysis of variance (ANOVA), followed by Duncan's multiple range test. Different significances are indicated (* = p < 0.05, ** = p < 0.01).

Effect of Ps-GOS on Proliferation of MC3T3-E1 Cells
To determine the dose dependence of Ps-GOS on proliferation, MTT assays were used to measure the metabolic activity of osteoblastic cells during proliferation. MC3T3-E1 cells were treated with 0.001-1000 µg/mL of Ps-GOS for 24-72 h ( Figure 1). Compared to control (cells without treatment), exposure to Ps-GOS for 48 h significantly increased cell proliferation at concentrations up to 10 µg/mL (p < 0.05) by 48 h, although proliferation was reduced at all concentrations after 72 h. Ps-GOS appeared to have a cytotoxic effect at higher concentrations (100 and 1000 µg/mL (p < 0.01)) after treatment for 72 h. Subsequently, only concentrations in the range 0.001 to 10 µg/mL were used in further experiments. This results indicated that low concentrations of Ps-GOS stimulate osteoblast proliferation without toxicity.
after treatment for 72 h. Subsequently, only concentrations in the range 0.001 to 10 μg/mL were used in further experiments. This results indicated that low concentrations of Ps-GOS stimulate osteoblast proliferation without toxicity.

Effect of Ps-GOS on Cell Cycle Distribution on MC3T3-E1 Cells
Flow cytometry was performed to investigate the effect of Ps-GOS on cell cycle progression. Cell cycle analysis was undertaken by measuring cellular DNA content corresponding to each of the three principle cell cycle stages; G0/G1, S, and G2/M. Actively proliferating cells were treated with indicated concentrations of Ps-GOS for 48 h. At higher concentrations (1 and 10 μg/mL), Ps-GOS significantly decreased the proportion of cells in G0/G1 phase while concomitantly increasing that in S phase when compared to control cells ( Figure 2). This study demonstrates that Ps-GOS can promote cell proliferation by accelerating progression into S phase from G1.

Effect of Ps-GOS on Cell Cycle Distribution on MC3T3-E1 Cells
Flow cytometry was performed to investigate the effect of Ps-GOS on cell cycle progression. Cell cycle analysis was undertaken by measuring cellular DNA content corresponding to each of the three principle cell cycle stages; G0/G1, S, and G2/M. Actively proliferating cells were treated with indicated concentrations of Ps-GOS for 48 h. At higher concentrations (1 and 10 µg/mL), Ps-GOS significantly decreased the proportion of cells in G0/G1 phase while concomitantly increasing that in S phase when compared to control cells ( Figure 2). This study demonstrates that Ps-GOS can promote cell proliferation by accelerating progression into S phase from G1.

Effects of Ps-GOS on Osteoblastic Differentiation of MC3T3-E1 Cells
Mineralization is the last stage of bone formation that results in the formation of calcified nodules that contribute to bone strength [38]. To examine the effect of Ps-GOS, MC3T3-E1 cells were treated with Ps-GOS for 14 and 21 days in an osteogenic induction medium to induce differentiation.
Calcium production in MC3T3-1 cells was determined by staining with Alizarin red S dye ( Figure   3A). Extraction and quantification of Alizarin red from treated cells showed that Ps-GOS significantly enhanced the calcium deposit formation at both day 14 and 21, with the former time-point showing the maximal increase (175%) when compared to control ( Figure 3B) at 1 and 10 μg/mL Ps-GOS. In addition, ALP a marker of mature osteoblastic differentiation [39] was

Effects of Ps-GOS on Osteoblastic Differentiation of MC3T3-E1 Cells
Mineralization is the last stage of bone formation that results in the formation of calcified nodules that contribute to bone strength [38]. To examine the effect of Ps-GOS, MC3T3-E1 cells were treated with Ps-GOS for 14 and 21 days in an osteogenic induction medium to induce differentiation. Calcium production in MC3T3-1 cells was determined by staining with Alizarin red S dye ( Figure 3A). Extraction and quantification of Alizarin red from treated cells showed that Ps-GOS significantly enhanced the calcium deposit formation at both day 14 and 21, with the former time-point showing the maximal increase (175%) when compared to control ( Figure 3B) at 1 and 10 µg/mL Ps-GOS. In addition, ALP a marker of mature osteoblastic differentiation [39] was determined at day 14. All Ps-GOS treatments markedly promoted ALP activity when compared to control ( Figure 3C). This result also correlated with the study of Yazid et al.: that the highest ALP level was produced from MC3T3-E1 cells at day 14 during osteoblast differentiation [40]. These results imply that Ps-GOS can promote osteoblast differentiation and mineralization in the process of bone formation.
Biomolecules 2020, 10, x 8 of 16 determined at day 14. All Ps-GOS treatments markedly promoted ALP activity when compared to control ( Figure 3C). This result also correlated with the study of Yazid et al.: that the highest ALP level was produced from MC3T3-E1 cells at day 14 during osteoblast differentiation [40]. These results imply that Ps-GOS can promote osteoblast differentiation and mineralization in the process of bone formation. .

Ps-GOS Up-Regulated Osteogenic-Related Gene Marker Expression in MC3T3-E1 Cells
During osteoblast differentiation, many osteogenic gene markers are expressed; these genes play important roles in matrix formation and calcium accumulation during bone formation. We determined the expression levels of OPN, ALP, COL1, OCN, RUNX2, and BMP-2, in MC3T3-E1 cells, after treating with Ps-GOS for 24 and 72 h using qRT-PCR. The results shown in Figure 4 indicate that the expression of ALP, COL1, OCN, and RUNX2 were up to two times higher than control levels

Ps-GOS Up-Regulated Osteogenic-Related Gene Marker Expression in MC3T3-E1 Cells
During osteoblast differentiation, many osteogenic gene markers are expressed; these genes play important roles in matrix formation and calcium accumulation during bone formation. We determined the expression levels of OPN, ALP, COL1, OCN, RUNX2, and BMP-2, in MC3T3-E1 cells, after treating with Ps-GOS for 24 and 72 h using qRT-PCR. The results shown in Figure 4 indicate that the expression of ALP, COL1, OCN, and RUNX2 were up to two times higher than control levels after 24 h exposure to Ps-GOS. Moreover, Ps-GOS at concentrations of 0.1 and 1.0 µg/mL sharply enhanced BMP-2 expression at 24 and 72 h, respectively. These results are consistent with the notion that Ps-GOS promotes osteoblastic differentiation by upregulating osteogenic-related gene expression via the BMP signaling pathway.

Ps-GOS Stimulates Osteoblast Differentiation via BMP Signaling Pathway
The BMP signaling pathway is responsible for osteoblast differentiation through up-regulation of the key proteins BMP-2 and Runx2. In the present study, we observed that Ps-GOS exposure resulted in an increase in BMP-2 and Runx2 mRNA expression (Figure 4). We thus examined the protein expression of BMP-2 and Runx2 to further validate the mechanism of action of Ps-GOS. Ps-GOS treatment at concentrations up to 10 μg/mL for 48 h resulted in elevated levels of BMP-2, as judged by both indirect immunofluorescence (IF) microscopy ( Figure 5A), as well as by Western blotting ( Figure 5D). Quantitative analysis of BMP-2 staining ( Figure 5A), following treatment with 0.01, 0.1, and 10 μg/mL Ps-GOS for two days, indicated a significant increase in BMP-2 positive staining cells compared to the control ( Figure 5B). Similarly, western blotting analysis showed that all Ps-GOS treatments significantly enhanced protein expression level of BMP-2 at day 2, with maximal increase at concentrations >0.1 μg/mL ( Figure 5C). Consistent with the notion that Runx

Ps-GOS Stimulates Osteoblast Differentiation via BMP Signaling Pathway
The BMP signaling pathway is responsible for osteoblast differentiation through up-regulation of the key proteins BMP-2 and Runx2. In the present study, we observed that Ps-GOS exposure resulted in an increase in BMP-2 and Runx2 mRNA expression (Figure 4). We thus examined the protein expression of BMP-2 and Runx2 to further validate the mechanism of action of Ps-GOS. Ps-GOS treatment at concentrations up to 10 µg/mL for 48 h resulted in elevated levels of BMP-2, as judged by both indirect immunofluorescence (IF) microscopy ( Figure 5A), as well as by Western blotting ( Figure 5D). Quantitative analysis of BMP-2 staining ( Figure 5A), following treatment with 0.01, 0.1, and 10 µg/mL Ps-GOS for two days, indicated a significant increase in BMP-2 positive staining cells compared to the control ( Figure 5B). Similarly, western blotting analysis showed that all Ps-GOS treatments significantly enhanced protein expression level of BMP-2 at day 2, with maximal increase at concentrations >0.1 µg/mL ( Figure 5C). Consistent with the notion that Runx operates downstream of BMP-2, Western blotting of Runx l showed that levels of this protein were significantly elevated after exposure for 10 days to Ps-GOS concentrations > 0.1 µg/mL ( Figure 5D).
Biomolecules 2020, 10, x 10 of 16 operates downstream of BMP-2, Western blotting of Runx l showed that levels of this protein were significantly elevated after exposure for 10 days to Ps-GOS concentrations > 0.1 μg/mL ( Figure 5D). . The data were obtained from four replicates as mean ± SEM. The data in columns with different letters in each group were significantly different at p < 0.05.

Stimulating effect on Osteoblastic Differentiation by Ps-GOS could Affect through MAPK and Wnt/β-Catenin Signaling Pathways
While BMP signaling plays a critical role in osteoblast differentiation and bone formation, additional pathways, participating in a range of cross-talk signaling, are also involved. The expression of a range of MAPK family members was investigated by qRT-PCR. As shown in Figure  6, Ps-GOS significantly promoted the expression of ERK1, ERK2, JNK1, and JNK2 following 12 and 24 h exposure, compared to control, and in all cases, expression levels reverted to control levels or below after 48 h. A similar pattern was observed with p38α, although Ps-GOS-enhanced expression was reduced after 48 h exposure and did not revert to control levels. We also investigated expression of genes involved in Wnt/β-catenin signaling (Figure 7). Wnt5a, Fzd4, β-catenin, and LRP5 were significantly up-regulated with Ps-GOS after 12 and 24 h exposure, and expression levels then slightly decreased at 48 h (the same time observed with MAPK). These outcomes suggest that the stimulatory effect of Ps-GOS on osteoblastic differentiation additionally involves stimulation via MAPK and Wnt/β-catenin signaling pathways.

Stimulating Effect on Osteoblastic Differentiation by Ps-GOS Could Affect through MAPK and Wnt/β-Catenin Signaling Pathways
While BMP signaling plays a critical role in osteoblast differentiation and bone formation, additional pathways, participating in a range of cross-talk signaling, are also involved. The expression of a range of MAPK family members was investigated by qRT-PCR. As shown in Figure 6, Ps-GOS significantly promoted the expression of ERK1, ERK2, JNK1, and JNK2 following 12 and 24 h exposure, compared to control, and in all cases, expression levels reverted to control levels or below after 48 h. A similar pattern was observed with p38α, although Ps-GOS-enhanced expression was reduced after 48 h exposure and did not revert to control levels. We also investigated expression of genes involved in Wnt/β-catenin signaling (Figure 7). Wnt5a, Fzd4, β-catenin, and LRP5 were significantly up-regulated with Ps-GOS after 12 and 24 h exposure, and expression levels then slightly decreased at 48 h (the same time observed with MAPK). These outcomes suggest that the stimulatory effect of Ps-GOS on osteoblastic differentiation additionally involves stimulation via MAPK and Wnt/β-catenin signaling pathways.

Discussion
It is well known that osteoblasts are critical players in the regulation of bone formation and bone remodeling. The reduction of osteoblast activity results in low bone mass and osteoporosis. Recently, it has been shown that β-glucan has a therapeutic effect on osteoporosis by promoting the function of osteoblasts. Lee at al. have suggested that β-(1-3),(1-6)-glucan has a stimulatory effect on BMP-7 promoting bone formation by inducing the production of ALP and calcium accumulation on mineralization [26]. However, the mechanism by which small molecular mass β-glucans, such as Ps-GOS, promote bone formation has not yet been determined.
This study has demonstrated that Ps-GOS has the potential to prevent osteoporosis. At low concentrations (<10 µg/mL), Ps-GOS induced osteoblastic proliferation in MC3T3-E1 cells without toxicity for up to 48 h of treatment. The elevated rate of proliferation correlated with an increase in the proportion of cells in S-phase, suggesting that Ps-GOS promotes proliferation by stimulating the G1-S transition within the cell cycle.
The production of ALP and calcium accumulation are essential steps in the process of mineralization leading to bone formation. ALP, a marker of early-stage osteoblastic differentiation, can be detected on the cell surface and in matrix vesicles of mature osteoblasts [41]. High level expression of ALP initiates and plays a fundamental role during the process of mineralization process [42], where it has been suggested that it hydrolyses pyrophosphate, a natural inhibitor of mineralization, concomitantly increasing local phosphate concentration [43,44]. We found that Ps-GOS stimulated the expression of ALP and promoted the accumulation of calcium, consistent with a potential role for Ps-GOS in promoting bone formation.
In normal development, mesenchymal stem cells function as progenitors of osteoblasts and osteocytes, as well as adipocytes and myocytes [45]. Differentiation of stem cells into an osteoblast lineage requires specific osteogenic signaling proteins and transcription factors. BMP-2, a member of the transforming growth factor-beta (TGF-β) superfamily, is synthesized by osteoblasts and is a critical inducer regulating osteoblastic differentiation and bone formation [46]. BMP-2-induced osteogenesis is absolutely dependent on the formation and transcriptional activity of the downstream osteogenic complex Runx2-SMAD. Runx2 has been reported to mediate the expression of many osteogenic gene markers, including ALP, COL1, OCN, and OPN [47]. Mutations on one allele of human Runx2 gene cause cleidocranial dysplasia [48], while deletion of Runx2 in mice leads to a complete failure of bone formation and ossification [49]. We found that Ps-GOS promoted bone formation by upregulating both gene and protein expression levels of BMP-2 and Runx2. As a consequence of Runx2 up-regulation, Ps-GOS also promoted expression of ALP, COL1, and OCN. Surprisingly, Ps-GOS had no effect on OPN expression. The significance of this observation is not yet clear.
The Wnt/β-catenin signaling pathway works cooperatively with BMP signaling to regulate osteoblast differentiation and bone formation. Crosstalk between Wnt/β-catenin and BMP signaling pathways results in an elevation in BMP-2 and Runx2 expression; the target of this signaling in osteoblasts [50]. Wnt/β-catenin signaling is activated when Wnt ligand binds to the receptors Frizzled (Fzd) and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), which in turn inhibits β-catenin phosphorylation. The translocation of β-catenin into nucleus leads to the activation of members of the TCF/LEF transcription factor family in order to promote osteogenic gene expression [51]. Mutation of LRP5 can cause osteoporosis pseudoglioma syndrome (OPPG) in humans [52] and decrease osteoblast numbers in mice [53]. In addition, Wnt5a is an important component in the regulation of Wnt/β-catenin signaling in osteoblast linear cells, as lack of Wnt5a leads to a reduction in LRP5 and LRP6 expression [54]. To identify additional molecular targets of Ps-GOS that might be associated with the up-regulation of BMP-2 and Runx2, we examined expression levels of Wnt/β-catenin-related genes. Our data reveled that Ps-GOS significantly up-regulates the expression of Wnt5a, LRP5, β-catenin, and Fzd4 mRNA, in MC3T3-E1 cells, suggesting that Ps-GOS may prevent bone loss in osteoporosis through Wnt/β-catenin together with BMP-2 signaling pathways.
The JNK-ERK-p38 MAPK signaling pathways also play an essential role in the control of bone formation and bone homeostasis during osteoblast differentiation [55]. Specifically, JNK is important for late-stage osteoblast differentiation, as inactivation of JNK by a specific small molecule inhibitor causes a lack of mineralization in MC3T3-E1 cells [56], while deletion of JNK1 and JNK2 in mice (Jnk1-2osx) results in severe osteopenia arising from bone loss [57]. Phosphorylation and consequent activation of Runx2 in osteoblasts is dependent on the expression of both ERK1 and ERK2 [58], while inactivation of ERK1 and ERK2 reduces β-catenin expression [59]. In addition, p38 is required for early-stage of osteoblast differentiation by promoting osteogenic differentiation markers, including ALP, osteocalcin, and mineralization [60].
In summary, this study demonstrated for the first time that the short-chain-length, lower-molecular-weight, higher-water-solubility Pleurotus sajor-caju glucanoligosaccharide (Ps-GOS) promotes osteoblast differentiation and mineralization, which are accompanied by the increase of ALP, COL1, and OCN expression through the induction of BMP-2 and Runx2. Furthermore, we have found that Ps-GOS also promotes bone formation via Wnt/β-catenin and MAPK signaling pathways. Thus, our findings show that Ps-GOS could be developed as a candidate supplement promoting bone formation. As such, this represents an example of both value creation as well as value addition in the biotechnological exploitation and use of natural rubber latex serum. Future work will be directed at determining the precise mechanism of action of Ps-GOS for comparison with other known modulators of osteoblastogenesis. Given the important role of the RANKL/RANL/OPG system in regulating osteoclast function, it will be iintersting to establish whether Ps-GOS can impact this system also.

Conclusions
In the present study, we report for the first time that small size Pleurotus sajor-caju glucanoligosaccharide (Ps-GOS) has a stimulatory effect, promoting bone formation by stimulating osteoblast proliferation, differentiation, and mineralization possibility through BMP-2, Wnt/β-catenin, and MAPK signaling pathways.
Ps-GOS has potential as a useful therapeutic drug to prevent osteoporosis.