Cholesterol 25-Hydroxylase Promotes Osteogenesis and Suppresses Adipogenesis of Bone Marrow Stromal Cells via mTOR Signaling Pathway

Increased adipogenesis and reduced osteogenesis of bone marrow stromal cells (BMSCs) are key features of glucocorticoid-induced osteoporosis (GIOP). However, the mechanism of controlling the differentiation balance of bone marrow stromal cells (BMSCs) is still unclear. Recent years, studies on the relationship between cholesterol metabolism and bone metabolism have been increasing. Various cholesterol metabolism-related molecules have been proved to be involved in BMSCs differentiation. Cholesterol-25-Hydroxylase (CH25H) is a multi-transmembrane endoplasmic reticulum related enzyme which participated in various metabolic process, including immunity, stem cell differentiation and inammation. Here we discovered and proved its role in regulating stem cell differentiation and explored the mechanism. signaling pathway. and suppresses adipogenesis via the mTOR signaling pathway. added rapamycin on the 14th day of osteogenic induction compared to CH25h knockdown cells. B-D. The expression of the biomarkers of osteogenesis including Runx2, Col1α1, OCN in control, shRNA and shRNA+Rapamcyin groups on the tenth day of adipogenic induction. E. The expression levels of mTOR, p-mTOR and p-P70S6K in control, shRNA and shRNA+Rapamcyin groups on the 0d and 14d of osteogenic induction.

Introduction Glucocorticoid-induced osteoporosis (GIOP) is one of the most severe complications of using glucocorticoids. But in diseases like chronic immune disease,the use of glucocorticoid is inevitable 1 . Due to the unclear pathogenesis of GIOP, bisphosphonates are always the preferred drug for inhibiting the process of osteoclast to prevent and treat GIOP for more than 20 years. However, inhibiting the process of osteoclast alone is not enough for the treatment of GIOP 2 . In the process of researching on GIPO, it's recognized that the abnormal differentiation of bone marrow stromal cells (BMSCs) is a main character.
As the common progenitor of various cells, including osteoblasts, adipocytes, chondrocytes, neurocytes, and myoblasts, BMSCs maintain a delicate balance of differentiation to keep an appropriate bone mass.
It's noteworthy that in the BMSCs of patients suffering from GIOP, there was a consistent trend of increased adipogenesis and reduced osteogenesis [3][4][5] . Therefore, seeking ways to promote osteogenesis as well as inhibit adipogenesis of BMSCs is important to control GIOP more effectively.
Recent years, successive clinical trials found that the metabolism disorder of cholesterol was associated with the decreased bone formation [6][7][8][9][10] . High level of serum cholesterol was negatively correlated with bone mineral density in patients 11 . Whereas the use of cholesterol-lowering drugs increased bone mass and reduced osteoporosis-associated fracture risks 12,13 . However, related basic research about cholesterol in the differentiation of BMSCs has contradictory results [14][15][16] . One explanation is that not only cholesterol itself but some certain molecules related to cholesterol metabolism play a part in the process of BMSCs differentiation. Cholesterol-25-Hydroxylase (CH25H) is a multi-transmembrane endoplasmic reticulum related enzyme that catalyze cholesterol to produce . Recent studies about CH25H suggest it may participate in controlling the adipo-osteogenic differentiation of BMSCs. For instance, the overexpression of CH25H could improve systemic insulin sensitivity in mouse, while the insulin resistance induced by glucocorticoid was considered as a potential mechanism to explain the accumulation of fat and the decreased formation of bone 17,18 . In addition, CH25H could promote the expression of collagen, which plays an important part in osteogenesis [19][20][21] .
But there is still no direct research on the function of CH25H in regulating BMSCs differentiation.
Combined with studies about CH25H, we presume that CH25H might participate in regulated adipoosteogenic differentiation of BMSCs and conducted an experimental demonstration in this study.
The mammalian target of rapamycin (mTOR) was discovered as a protein complex which is a member of protein kinase family related with Akt, integrating a variety of environmental cues to regulate organismal growth and homeostasis 22 . mTOR pathway contributed to maintain bone homeostasis and BMSCs lineage differentiation 23,24 . The activation of mTOR signaling pathway tends BMSCs to adipogenic differentiation and reduces osteogenic differentiation by regulating protein synthesis, adipose tissue morphogenesis and the activity of PPAR-γ [25][26][27][28] . The inhibition of mTOR signaling pathway could promote osteogenesis and suppress adipogenesis 29 .
In this study, to con rm CH25H participates in controlling the differentiation of BMSCs, we rstly detected the expression pattern of CH25H in the process of osteoblast and adipocyte differentiation. Then, through silencing or overexpressing CH25H in C3H10 C2C12 and 3T3-L1 cell lines, we con rmed the impact of expression levels of CH25H on osteogenesis and adipogenesis. The data of this study demonstrated CH25H could promote osteogenesis and inhibit adipogenesis by suppressing the mTOR signaling pathway, revealing a new function of CH25H in regulating BMSCs differentiation. These results may help furtherly explain the pathogenesis of GIOP and give a new potential therapeutic target for this disease.
Culture of cells and osteogenic/ adipogenic induction C3H10, C2C12, 3T3-L1 and 293 T cell lines were purchased from Cell Bank of the Chinese Academy of Science, Shanghai. All the four cell lines were cultured in fresh growth medium Which contains 90% high glucose DMEM (Gibco) and 10% FBS (Gibco). Cells were incubated in Cell incubator(ThermoFisher)at 37℃ and 5% CO 2 humid atmosphere. Osteogenic induction medium was made from fresh growth medium containing 100mM DXMS, 50 µM AA and 10 mM DXMS. Adipogenic induction medium was made from fresh growth medium containing with 500 mM IBMX, 200 mM ID, 1 uM DXMS and 10 uM insulin. The growth or induction medium was changed every two days.

Plasmids and viral infection
Three plasmids including pLKO.1-EGFP-puromycin, psPAX2, and pMD2.G (GeneChem, CHN) were involved in the lentivirus packing system for short hairpin RNA (shRNA) expression. Three CH25H RNAi sequences (Table 1) were chosen to knock down the expression of CH25H, which were annealed and inserted into the pLKO.1-EGFP-puromycin vector. Lentiviruses were packed in 293T cell line and infected targeted cells which seeded into 6 cm culture dishes at the density of around 60%. Stably transfected cells lines were screened out by puromycin (Sigma) for at least two weeks. method was applied to analyze the results. β-actin was set as the internal reference to normalize the gene expression of samples. The primers used in qRT-PCR were searched from PrimerBank (https://pga.mgh.harvard.edu/primerbank/) and listed in Table 2. Table 2 Gene Forward Reverse

Western blot
Western blotting was performed to compare the relative expression of genes at protein level. The medium of cells was removed and the adherent cells was washed with cold PBS twice. 60 µl RIPA(p0013b, Byotime) buffer with 1% PMSF(st506, Beyotime) was added to the 60mm dishes and the cells were scraped into EP tubes. Lysates were vibrated 10s every 5 min for 30 min at 0°C, and centrifuged at 12000 rpm at 4°C for 10 min to harvest the supernatant. The BCA kit(P0010, Beyotime)was used to determine the concentration of total protein. The protein sample was subjected to gel electrophoresis and transferred to PVDF membranes (Milipore, USA), the later were blocked using 5% non-fat milk for 2 h at room temperature. The PVDF membrane then incubated by primary antibodies at 4°C overnight, the membranes were rinsed by TBST three times for 10 min, and then probed with goat anti-rabbit or mouse secondary antibodies (7074, 4410, CST, USA) at room temperature for 2 h, and detection with an ECL kit (Share-bio, CHN). Images were read by Fluor Chem E system (Proteinsimple, USA).
In vivo ectopic bone formation assay 3 * 10 6 control or CH25H-overexpressed C3H10 cells was injected subcutaneously to skin under the right front leg of male BALB/c nude mice aged 4 weeks. 6 replicates were set in each group. Implants were harvested and xed in 10% formaldehyde for Oil Red O, HE, Masson's staining and Immunohistochemistry (IHC) after six weeks' observation. All animal care and experimental protocols were approved by the Animal Care and Use Committee of Fudan University and complied with the Animal Management Guidelines of China.

Statistical analyzes
All the experiments were performed independently for three times at least. The results were analyzed with the Statistical Package for Social Sciences (SPSS) 20.0 software for Windows which was provided by Fudan university. The results were presented as the mean ± SD. Statistical analysis of the differences between the groups was performed by analysis of variance (oneway ANOVA). P < 0.05 indicates that the difference was statistically signi cant. The value of *P < 0.05, **P < 0.01 and ***P < 0.001 was considered statistically signi cant. The graphs were made by ggplot2 R package in the environment of R 4.0.2 for windows.

Results
3.1. CH25H expression was upregulated during osteogenesis and downregulated during adipogenesis.
We chose mouse mesenchymal stem cells C3H10 and mouse myoblast precursor cells C 2C12 for osteoblasts induction, C3H10 and mouse adipose progenitor cells 3T3-L1 for adipocytes induction. All these three cell lines had been proved having osteogenic or adipogenic differentiation potential 30,31 .
Total protein and mRNA of the cells was collected after osteogenic induction for 0d, 7d and 14d, adipogenic induction for 0d, 5d, and 10d. As shown in Fig. 1A with the time for osteogenic induction extends, osteogenesis and mineralization degree enhanced as the ALP and AR-S staining deepen. The biomarkers Runx2, OCN and Col1α1 increased (Fig. 1C, D). Meanwhile, the mRNA and protein levels of CH25H were promoted in a gradual manner along with the osteogenic process ( Fig. 1B-D). While during the process of adipogenic induction, fat droplets stained by Oil Red O in C3H10 and 3T3-L1 cell lines increased as shown in Fig. 1E, and the classic biomarkers CEBPα, PPARγ and SREBP1 were increased as well (Fig. 1G-I). But the protein and mRNA levels of CH25H were decreased during this period (Fig. 1F). All of these ndings suggested that CH25H might play an important role in the process of adipo-osteogenic differentiation.

Establishment of CH25H knocking down cell lines
Lentivirus transfection system were used to knockdown CH25H in C3H10, C2C12 and 3T3-L1 cell lines to deeply assess the effect of CH25H in adipo-osteogenic differentiation. To ensure the effect of knockdown, we designed three shRNA sequences and evaluated the e ciency of knockdown in both protein and mRNA levels respectively. Western blotting and qRT-PCR result as shown in Fig. 2. A and B suggested that CH25H expression was most signi cantly silenced in the CH25H-shRNA3 group, consistent in the three cell lines.

CH25H knockdown inhibited osteogenesis and promoted adipogenesis
We next examined the effect of CH25H in the process of adipo-osteogenic differentiation. The control and shRNA treated C3H10 and C2C12 cells were cultured in the osteogenic differentiation medium. We evaluated the difference of their osteogenesis ability by the means of ALP, AR-S staining and the biomarkers of osteogenesis (Fig. 2C, E-G). All the results proved that the ability of osteogenesis in CH25H knockdown cell lines was signi cantly weakened. In the aspect of regulating adipogenic differentiation, we cultured control or shRNA treated C3H10 and 3T3-L1 cell lines in adipogenic medium and used Oil Red O staining to show the degree of lipid droplet formation at 0d, 5d and 10d. As shown in Fig. 2D, CH25H-knockdown cell lines had greater fat droplets formation capability, and the classic biomarkers of adipogenesis expressed more than control cells (Fig. 2H-J). Data above proved that CH25H knockdown inhibits osteogenesis and promotes adipogenesis.

CH25H knockdown activated the mTOR signaling pathway
For the purpose of exploring the potential mechanism involved,we then identi ed differences of key molecules in common osteogenesis related signaling pathways between the two groups. We found that mTOR signaling pathway, including Akt, p-Akt, mTOR, p-mTOR and p-P70S6K were upregulated in CH25H knockdown cell lines (Fig. 3A). what's more, we collected the protein samples in 0d and 14d during the process of osteogenic induction, 0d and 10d during the process of adipogenic process to test the key molecules of mTOR signaling pathway expression trends. As Fig. 3B and C shown, the mTOR signaling pathway was not only re ected different expression trends in the process of osteogenic or adipogenic differentiation but also highly expressed in CH25H knockdown cell lines in both three cell lines. As an important signaling pathway for the regulation of cell differentiation and development, there have been researches proved that mTOR pathway could regulate lineage differentiation between osteoblasts and adipocytes 23,32 . We hypothesized that mTOR signaling play a role in CH25H-induced change in MSCs differentiation.

Rapamycin rescued the shift of adipo-osteogenic balance in MSCs caused by CH25H knockdown
To certify the function of mTOR signaling pathway in CH25H induced change of differentiation direction, we used rapamycin, a speci c inhibitor of mTOR, nding that with rapamycin treatment, the osteogenesis inhibition effect caused by CH25H knockdown was rescued (Fig. 4A-D) and the expression levels of mTOR, p-mTOR, p-P70S6K were suppressed as well (Fig. 4E). In the process of adipogenic differentiation, the using of rapamycin also rescued the adipogenesis enhancement caused by CH25H knockdown (Fig. 5A-D), and the proteins of mTOR, p-mTOR, p-P70S6K were suppressed before and after adipogenic induction (Fig. 5E).
3.6. Overexpression of CH25H promotes osteogenesis and suppresses adipogenesis through inhibiting the mTOR signaling pathway.
In order to validate the results above, we established CH25H overexpression C3H10 cell lines (C3H10-cDNA), and test expression effect of CH25H in both protein and mRNA levels( Fig. 6A-B). As Fig. 6C showed, contrary to C3H10-blank cells, the mTOR signaling pathway is inhibited in C3H10-cDNA cell lines. ALP and AR-S and Oil Red O staining shows that in C3H10-cDNA cells, the differentiation of osteoblasts was upregulated, while adipogenesis was suppressed ( Fig. 6D and E). The express changes of biomarker in mRNA levels was consistent with the results of staining (Fig. 6F-K).
3.7. The function of CH25H in regulating osteo-adipogenic differentiation in vivo To con rm the results in vitro, we performed animal assays with CH25H coexpression C3H10 cell lines and blank C3H10 cell lines. 4*10 6 of these two kinds of cells were injected into nude mice aged four weeks respectively (6 independent repeats in each group). After six weeks feeding, implants were taken out. HE, Masson, Oil Red O staining and IHC were used to con rm the degree of osteogenic or adipogenic differentiation. As shown in Fig. 7A, CH25H-overexpressed C3H10 cells had an increased tendency of osteogenesis and a decreased tendency of adipogenesis. The results of IHC (Fig. 7B)

Discussion
Glucocorticoid-induced osteoporosis (GIOP), which is a common form of secondary osteoporosis, charactered as abnormal decreased osteogenic differentiation and increased adipogenic differentiation, has been relatively well established 33,34 . However, the underlying mechanisms are not fully understood.
Bone marrow surrounds the trabecular elements of the skeleton and is made up of bone marrow mesenchymal stem cells (BMSCs). As a type of non-hematopoietic stem cell with strong potential for multipotent differentiation, BMSCs differentiate under the regulation of multiple pathways. These signaling pathways eventually effect on Runx2, which controls osteogenic differentiation, or nuclear receptor PPARγ, which controls adipogenic differentiation, and eventually determine how stem cells mature. For the osteoblasts and adipocytes have a common origin, a switching mechanism in BMSCs might help to explain previous observations that enhanced bone marrow adipogenesis seemed to be at the cost of osteoblast differentiation 35,36 . For example, the BMP pathway could regulate the commitment of BMSCs to the osteoblast lineage. While Akt pathway could enhance adipogenesis by activation CEBPs. However, BMSCs lineage differentiation could be regulated at various molecular levels, when they are stimulated by multiple micro-environmental factors. The lineage switch between osteoblasts and adipocytes might be regulated by multiple molecular pathways, such as mTOR and Wnt/β-Catenin signaling.
The mTOR, a conserved serine-threonine protein kinase, belongs to the Akt related kinase family. Recent evidence has indicated that mTOR could modulate cell proliferation and differentiation in various of cell types. Studies in vitro and in vivo have demonstrated that the expression of of mTOR is induced in several types of cells, including adipocytes [37][38][39] . It is reported that mTOR signaling plays a crucial role in both initiating and maintaining adipogenesis by promoting the expression of adipogenesis related gene PPARγ, which is crucial for the feedback of CEBPα and other adipogenesis related gene expression program 37,39 . The use of rapamycin could inhibit mTOR-activated adipogenesis 40 . In osteogenesis, the use of rapamycin could rescue the reduced capacity to form new bone caused by mTOR activation 29 .
Based on the results above, it is reasonable to speculate that mTOR signaling pathway is a key switch to determine the linage differentiation of BMSCs.
As a kind of hydroxylase, the main function of CH25H is to oxidize cholesterol to be 25hydroxycholerterol(25HC) 41 . In regulating cholesterol, CH25H are traditionally regarded as an important regulator that maintain cholesterol homeostasis by inhibiting sterol regulator-binding protein (SREBP) and activating liver X receptor (LXR), which are two key transcriptional regulators 42,43 . These years, furtherly studies about CH25H had revealed that CH25H is not only an enzyme involved in cholesterol metabolism but has many other biological functions like immunomodulatory and participating in in ammation 44,45 . But there has no report whether CH25H could regulate the differentiation of BMSCs. In this study, to strengthen the reliability of our data, three cell lines including C3H10, C2C12 and 3T3-L1 were used Among them, C3H10 was used in both adipogenic and osteogenic induction, C2C12 and 3T3-L1 cell lines were respectively used for osteogenic and adipogenic induction. The corresponding inductivity of each cell line has been widely used by researchers [46][47][48][49] . Our design insured that there was data of two kinds of cells in each experiment. We demonstrated that CH25H could promote osteogenesis and inhibit adipogenesis through suppressing the mTOR signaling pathway. For the rst time, to our knowledge, we revealed the function of CH25H in regulating BMSCs differentiation. The data we provide may help furtherly understand the adipo-osteogenic differentiation regulation, and give a potential therapeutic target in treating decease caused by disordered differentiation of BMSCs like GIOP.

Conclusion
In this study, we demonstrated that CH25H could increase osteogenesis and suppress adipogenesis for the rst time. Besides, the mTOR signaling pathway was found to be the downstream signaling pathway of CH25H in regulating osteo-adipogenic differentiation of MSCs. Taken together, our results indicate that CH25H promotes osteogenesis and suppresses adipogenesis via the mTOR signaling pathway. MH Wang: Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Abbreviations
All authors have read and approved the manuscript. Knockdown upregulated the mTOR signaling pathway. A. Akt, p-Akt, mTOR, p-mTOR and p-P70S6K were promoted after CH25H knockdown in C3H10, C2C12 and 3T3-L1 cell lines. B and C. The expression levels of Akt, p-Akt, mTOR, p-mTOR and p-P70S6K at the 0d, 7d and 14d in CH25H knockdown and control cell lines during the process of osteogenic induction. C. The expression levels of Akt, p-Akt, mTOR, p-mTOR and p-P70S6K at the 0d, 5d and 10d in CH25H knockdown and control cell lines during the process of adipogenic induction   The overexpression of CH25H in C3H10 suppressed the mTOR signaling pathway, promoted osteogenesis and suppressed adipogenesis. A and B. the protein and mRNA levels of CH25H in C3H10-Blank and C3H10-cDNA groups. C. the key moleculars of mTOR signaling pathways were suppressed in CH25H overexpressed cells. D. AR-S and ALP staining showed the osteogenesis and extracellular mineralization were enhanced in CH25H overexpress cells. E. The Oil Red O staining indicated the adipogenic differentiation was suppressed in CH25H overexpress cells. G-K. the relative mRNA levels of osteogenesis and adipogenesis biomarkers during the process of osteogenic and adipogenic induction between C3H10-Blank and C3H10-cDNA groups.