Cysteine Dioxygenase Type 1 Inhibits Osteogenesis by Regulating Wnt Signaling in Primary Mouse Bone Marrow Stromal Cells

Mesenchymal stem cells (MSCs) are multipotent cells, which can give rise to variety of cell types, including adipocytes and osteoblasts. Previously, we have shown that cysteine dioxygenase type 1 (Cdo1) promoted adipogenesis of primary mouse bone marrow stromal cells (BMSCs) and 3T3-L1 pre-adipocytes via interaction with Pparγ. However, the role of Cdo1 in osteogenesis remains unclear. Here, we demonstrated that expression of Cdo1 was elevated during osteoblastic differentiation of BMSCs in vitro. Interestingly, knockdown of Cdo1 by siRNA led to an increased expression of osteogenic related genes, elevated alkaline phosphatase (ALP) activity, and enhanced mineralization. Overexpression of Cdo1 in BMSCs inversely suppressed the osteogenesis. Furthermore, we found that overexpression of Cdo1 impaired Wnt signaling and restricted the Wnt3a induced expression of osteogenic transcriptional factors, such as Runx2 and Dlx5. Collectively, our findings indicate Cdo1 suppresses osteogenic differentiation of BMSCs, through a potential mechanism which involves in Wnt signaling reduction concomitantly.

Mesenchymal stem cells (MSCs) are heterogeneous cell populations with capacity for self-renewal and multipotency of differentiation, which can give rise to multiple cell types, such as adipocytes, chondrocytes, osteocytes, as well as other embryonic lineages 1,2 . To date, MSCs are found and isolated from various pre-natal and postnatal tissues, originated from bone marrow 3 , but also umbilical cord blood 4 , adipose tissue 5 , and dental tissues 6 . Further, MSCs are found to play a role in immune-modulation and anti-inflammation at injured sites 7,8 . Hence, MSCs have attracted much attention for stem cell-based bone repair 9,10 .
The process of osteogenic differentiation of MSCs can be categorized into commitment to osteoprogenitor cells, differentiation into pre-osteoblasts and maturation of osteoblasts 11,12 . The mature osteoblasts are capable of synthesizing the bone matrix that eventually becomes mineralized 12 . Mechanistically, the lineage specification of MSCs is a highly controlled process that involves several genetic and epigenetic mechanisms. One of the most extensively studied factors that is important in osteogenesis is runt-related gene 2 (RUNX2), a master transcription factor 13 . And other numerous factors are also required for osteogenesis, including growth factors, hormones, signaling molecules 14 . In addition to osteogenic differentiation, MSC can give rise to adipocytes under suitable conditions. Interestingly, a theoretical inverse relationship has been suggested between osteogenic differentiation and adipogenic differentiation of MSCs 15,16 . Several signaling pathways have been investigated to promote osteogenesis and inhibit adipogenesis, such as Wnt signaling, Hedgehog signaling, and NELL-1 signaling 11,17,18 .
Mammalian cysteine dioxygenase type 1 (Cdo1) is an essential enzyme for taurine biosynthesis by catalyzing the oxidation of cysteine to cysteine sulfinic acid 19 . In addition to the enzymatic activity of Cdo1, previous studies have also suggested that Cdo1 expression is upregulated during adipogenesis of human bone marrow-derived MSCs and adipose tissue-derived pre-adipocytes, and Cdo1 may serve as a marker of adipogenic differentiation of MSCs 20,21 . Furthermore, our group have demonstrated that Cdo1 promoted adipogenic differentiation via interaction with peroxisome proliferator-activated receptor gamma (Pparγ ) 21 . Given these findings, and an inverse relationship between osteogenesis and adipogenesis, we hypothesize that Cdo1 may inhibit osteoblastic differentiation of MSC. To address this hypothesis, in this study, we investigated the expression pattern of Cdo1 during osteogenic differentiation of BMSCs, and examined the effects of depletion of Cdo1 and overexpression of Cdo1 on this osteogenic process. Further, we observed overexpression of Cdo1 impaired Wnt signaling stimulated by Wnt3a in BMSCs. Our findings indicate Cdo1 suppresses osteogenesis via inhibition of Wnt signaling.

Materials and Methods
Cell Culture. Primary mouse bone marrow stromal cells (BMSCs) were isolated and cultured as described previously 22 . The derived cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-Glutamine, plus 100 U/ml of K-Penicillin G and 100mg/ml of Streptomycin sulfate (all from Gibco) at 37 °C with a humidified atmosphere of 5% CO2. All animal procedures were conducted in accordance with The Guidelines for the Care and Use of Laboratory Animals of State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University. To induce osteogenic differentiation, BMSCs were seeded at 5 × 10 3 cells per well in 24-well-plates, and cultured with osteogenic medium (OS). Osteogenic medium was comprised of 90% α -MEM (Gibco), 10% FBS (Gibco), 100 μ M ascorbic acid, 10 mM β -glycerophosphate, and 10nM dexamethasone (all from Sigma).
Characterization of osteoblastic phenotypes. After several days of osteogenic induction, the cells were fixed in 70% ethanol (Fisher), and alkaline phosphatase (ALP) staining was performed according to the manufacturer's instructions (System Biosciences). For quantitative determination of ALP activity, 20 μ L cell protein solution was incubated with 50 μ L ALP stabilizing buffer (Sigma) and 50 μ L ALP yellow (pNPP) liquid substrate (sigma) for 20 min at 37 °C . The absorbance was then read on a microplate reader (Bio-Rad) at OD405 nm. Alizarin Red S (ARS) staining was performed to assess the mineralization of extra cellular matrix, after 14 days of osteogenic induction. Briefly, the cells were fixed with 70% ethanol for 1 hour, and stained with 40 mM Alizarin red for 10 min. The stained cultures were destained by 10% CPC, and absorbance of the solution was read at 562 nM.
Luciferase Reporter Assay. One day before transfection, BMSCs were seeded per well into 12-well plate at 10 5 cells per well. After overnight incubation, the cells were transiently transfected with 1 μ g DNA of reporter constructs (TOPflash, Millipore) using 2 μ L Lipofectamine 2000 TM (Invitrogen) in 50 μ L OptiMEM I (Gibco) reduced serum media. Thereafter, the test cells were stimulated with human recombinant Wnt3a (100 ng/ml, System Biosciences); and control cells were treated with phosphate buffered saline (PBS). After 24 hours, cells were lysed and firefly luciferase activity was measured in triplicate according to the manufacturer's protocol (Promega).The firefly luciferase activity was normalized to protein concentrations. to assess the statistical inference on difference among each pair of data sets, respectively. A p value < 0.05 was considered to be statistically significant.
All experimental protocols and procedures were approved by State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University.

Results
Cdo1 is upregulated during osteogenic differentiation of BMSCs. To explore the role of Cdo1 in osteogenesis, we first examined the expression level of Cdo1 during osteogenic differentiation of primary BMSCs. As shown in Fig. 1, the mRNA expression of Cdo1 was elevated in response to osteogenic stimulation. However, the protein expression of Cdo1 was detected during osteogenic differentiation of primary BMSCs by Western blot (data not shown).
Depletion of Cdo1 enhances osteogenic differentiation of BMSCs. Next, we used two specific siR-NAs to knockdown the expression of Cdo1 in BMSCs, and the knockdown efficiency in the presence or absence of osteogenic stimulus was assessed by RT-PCR ( Fig. 2A). After osteogenic induction, we found siRNA-mediated depletion of Cdo1 significantly promoted expression of osteogenic-related genes, such as Col1a1 (Collagen, type I, alpha 1), and Ibsp (Integrin binding sialoprotein) (Fig. 2B). Consistently, knockdown of Cdo1 enhanced ALP activity, an early marker of osteoblastic differentiation (Fig. 2C,D). Furthermore, we assessed the extracellular matrix (ECM) mineralization by ARS staining. As shown in Fig. 2E,F, the ECM mineralization was significantly enhanced by depletion of Cdo1.

Overexpression of Cdo1 inhibits osteogenic differentiation of BMSCs.
To investigate the effects of ectopic overexpression of Cdo1 on osteogenic differentiation, BMSCs cells were stably transduced with retroviruses expressing Cdo1 (Fig. 3A,B). As expected, the expression of Col1a1 and Ibsp was downregulated by overexpression of Cdo1 after osteogenic induction (Fig. 3C). In addition, The ALP activity and ECM mineralization were also impaired by overexpression of Cdo1 in BMSCs (Fig. 3D-G).
Overexpression of Cdo1 impairs Wnt signaling. Wnt signaling plays an essential role in regulation osteogenic and adipogenic differentiation of MSCs 17 . To investigate whether the inhibitory effect of Cdo1 on osteogenesis was mediated by Wnt signaling, BMSCs were transfected with TOPflash reporter plasmids. We found that overexpression of Cdo1 significantly reduced the luciferase activity stimulated by recombinant Wnt3a protein (Fig. 4A). Consistently, the expression of downstream genes, Axin2 and Dkk1, was also downregulated by overexpression of Cdo1 in response to Wnt3a treatment (Fig. 4B). Runx2 and Dlx5 are important transcription factors in osteogenic differentiation of MSCs, and both of them are target genes of Wnt signaling 23 . Further, we found the expression of Runx2 and Dlx5 are also inhibited by overexpression of Cdo1 when treated with Wnt3a. Taken together, Cdo1 suppresses osteogenic differentiation of BMSCs, through a potential mechanism which involves in Wnt signaling reduction concomitantly.

Discussion
MSCs have generated a great deal of enthusiasm over the past decade for tissue engineering and regenerative medicine 24,25 . Understanding the mechanisms of MSC lineage specification and directing its differentiation in a determined manner are critical for the fundamental and clinical applications 26,27 . In the present study, we have found that the expression of Cdo1 was up-regulated during osteogenic differentiation of BMSCs in vitro. While siRNA mediated knockdown of Cdo1 promoted osteogenic differentiation of BMSCs, ectopic overexpression of Cdo1 significantly reduced the expression of osteogenic related genes, ALP activity, and ECM mineralization. However, we also noticed that depletion of Cdo1 did not upregulate ALP activity and ECM mineralization   without additional osteogenic stimulus, as shown in Fig. 2c-f. It is possible that depletion of Cdo1is not sufficient to initial the osteoblastic commitment of MSCs. To explore the mechanism by which Cdo1 regulates osteogenesis, we further performed luciferase assay after transfection with TOPflash reporter. And we found that overexpression of Cdo1 inhibited Wnt signaling, and suppressed expression of Wnt target genes in BMSCs.
Previous studies have suggested that osteogenesis and adipogenesis have an inverse correlation 15,28 . We had reported that Cdo1 promoted adipogenesis, and we further found Cdo1 inhibited osteogenic differentiation of BMSCs in this study. Although Cdo1 expression was upregulated in both adipogenesis and osteogenesis, the increased fold of Cdo1 in adipogenesis was much greater compared to its upregulation in osteogenesis. And the upregulation of Cdo1 in osteogenesis was observed at medium and late stages of osteogenesis. In contrast, the upregulation of Cdo1 took place earlier in adipogenesis of mBMSCs 21 . It is possible that a relative higher expression level of Cdo1 is required to exert its inhibitory effects on osteogenesis. Under physiological conditions, the osteogenic differentiation and adipogenic differentiation of MSCs are well balanced. However, disruption of this homeostasis may lead to bone dysregulations, such as osteoporosis, which is characterized by excessive accumulation of adipocytes and decreased bone mass 29,30 . Our results indicate that Cdo1 may contribute to the development of osteoporosis. While osteoporosis occurs more commonly in aging population, several changes in MSC take place with age, including loss of proliferation potential, decrease in capacity to differentiate into osteoblasts, and increase in capacity to differentiate into adipocytes 31 . It would be interesting to investigate whether Cdo1 is involved in the age-related changes in MSCs. In addition, our results showed Cdo1 suppressed the differentiation from MSCs to osteoblasts, but if Cdo1 affects maturation of osteoblasts still need further investigations.
We also found that overexpression of Cdo1 impaired Wnt signaling, and inhibited expression of Wnt target genes, such as Axin2, Dkk1, Runx2, and Dlx5. Runx2 and Dlx5 play an important role in initiation of osteogenesis. Thus, our results indicate that such inhibitory effects of Cdo1 on osteogenesis may be mediated by Wnt signaling. While activation of Wnt signaling promotes osteogenic differentiation of MSCs, it also strongly inhibits adipogenesis, through both-catenin dependent and beta-catenin independent mechanisms 32,33 . Further, Song et al. reported that loss of Wnt signaling results in a cell-fate shift of preosteoblasts from osteoblasts to adipocytes 34 . It is likely that Cdo1 is a key factor in lineage specification by regulating Wnt signaling. However, the mechanism by which Cdo1 regulate wnt signaling still need further investigation. In addition, we have reported that Cdo1 interacted with Pparγ 21 . Since Pparγ can suppress osteogenesis 35,36 , activation of Pparγ might be an alternative mechanism that Cdo1 inhibits osteogenesis.
Collectively, we have found that Cdo1 inhibits osteogenic differentiation by regulating Wnt signaling in primary BMSCs. Together with previous studies, our results indicate that Cdo1 may play an important role in regulation of the balance between osteogenesis and adipogenesis during MSC differentiation, and upregulation of Cdo1 may be correlated to the bone-related diseases.