- Email: [email protected]
Tigogenin inhibits adipocytic differentiation and induces osteoblastic differentiation in mouse bone marrow stromal cells
Molecular and Cellular Endocrinology 270 (2007) 17–22
Tigogenin inhibits adipocytic differentiation and induces osteoblastic differentiation in mouse bone marrow stromal cells Hua Zhou a,b , Xi Yang c , Naili Wang d , Yaou Zhang a,b , Guoping Cai a,b,∗ a
Life Science Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China c Department of Genetics and Developmental Biology, The Fourth Military Medical University, Xi’an 710032, PR China d Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 110015, PR China b
Received 27 October 2006; received in revised form 12 December 2006; accepted 29 January 2007
Abstract We investigated the effect of tigogenin on adipocytic and osteoblastic differentiation in mouse bone marrow stromal cells (BMSCs). Tigogenin enhanced the proliferation of BMSCs significantly. Tigogenin treatment reduced the adipogenic induction of lipid accumulation, visfatin secretion, and the expressions of peroxisome proliferation-activated receptor (PPAR)␥2 and adipocyte fatty acid-binding protein (ap)2. Moreover, tigogenin had no effect on the mitotic clonal expansion. On the other hand, tigogenin significantly elevated alkaline phosphatase (ALP) activity and the expressions of Cbfa1, collagen type I (COL I) and osteocalcin (OCN), as well as the content of matrix calcium in BMSCs. Further, SB-203580 antagonized the tigogenin-promoted osteogenesis. These observations suggested that tigogenin may modulate differentiation of BMSCs to cause a lineage shift away from the adipocytes and toward the osteoblasts, which is at least mediated by inhibition of PPAR␥ and via p38 MAPK pathway, and is a potential drug preventing the development of osteoporosis and the related disorders. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Tigogenin; Bone marrow stromal cells; Adipocyte; Osteoblast; Cell differentiation
1. Introduction Bone marrow stromal cells (BMSCs) are pluripotent cells that have the capacity to differentiate into adipocytes, osteoblasts, chondrocytes, myoblasts, or fibroblasts, and a reciprocal and inverse relationship exists between adipogenesis and osteogenesis in the cells (Ahdjoudj et al., 2004; Dominici et al., 2001; Li et al., 2003). The decrease in bone volume associated with osteoporosis and age-related osteopenia is accompanied by an increase in marrow adipose tissue at the expense of osteoblasts as seen following ovariectomy, immobilization, treatment with glucocorticoids, aging, and other conditions that lead to bone loss (Rosen and Bouxsein, 2006). It is possible, therefore, that the inhibition of marrow adipogenesis with a concomitant increase in osteogenesis could provide a therapeutic target with which to prevent further increase in adipocyte formation (Nuttall and Gimble, 2000).
Tigogenin is one of steroidal sapogenins which is widely used for synthesizing steroid drugs. It has been reported extensively that steroidal sapogenins from various plants have anti-obesity effect, but the underlying cellular and molecular mechanisms remain largely unknown. It is possible that some of these mechanisms involve alterations in differentiation of adipocytes. Our previous study have showed that tigogenin can inhibit the 3T3-L1 preadipocytes to differentiate into adipocytes (data unpublished). Thus it is likely that tigogenin might influence the differentiation of BMSCs. In the present study, to elucidate whether tigogenin can directly modulate the differentiation of BMSCs, we examined the effect of tigogenin on adipocytic and osteoblastic differentiation in BMSCs, and probed the possible mechanism. 2. Materials and methods 2.1. Isolation and culture of primary BMSCs
∗
Corresponding author at: Room 408 Building L, Tsinghua Campus, University Town, Shenzhen 518055, PR China. Tel.: +86 755 26036740. E-mail address: bjsz [email protected] (G. Cai). 0303-7207/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2007.01.017
Culture medium was Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, USA), supplemented with 10% calf serum (Gibco, USA), 100 U/ml penicillin and 100 g/ml streptomycin (Sangon, China). The BMSCs were
18
H. Zhou et al. / Molecular and Cellular Endocrinology 270 (2007) 17–22
obtained from BALB/c mice (4 weeks) as previously described (Verma et al., 2002) with minor modification. Briefly, mice were killed by cervical dislocation, metaphyses of femurs and tibias were cut aseptically, diaphysis cavities were repeatedly flushed with culture medium, and bone marrow cells were collected and cultured at 37 ◦ C in a 5% CO2 humidified incubator. Culture medium was replaced every 3 days and red blood cells and non-adherent cells were removed. For adipocyte-induced culture, cells were propagated to confluence, and were induced by adipogenic supplement containing 1 mol/L dexamethasone and 10 mg/L insulin (Sigma, USA).
2.2. Assessment of proliferation and mitotic clonal expansion of primary BMSCs BMSCs were plated in 96-well culture plates and cultured for 6 days. Tigogenin [(25R)-5␣-spirostane-3-ol] (gift from Dr. Naili Wang, Shenyang Pharmaceutical University, Shenyang, PR China) diluted with DMSO (Merck, Germany) to prepare the stock solution (10 mmol/L) was then added to the wells at final concentrations of 10, 30 or 90 mol/L. Cells were incubated for 72 h. Upon completion of the incubation, MTT dye solution (20 L, 5 g/L, Sangon, China) was added to each well. After 4 h incubation, the supernatant was removed and 100 L DMSO was added to solubilize the MTT. The optical density of each well was measured on a microplate spectrophotometer (TECAN, Switzerland) at a wavelength of 570 nm. For the assay of mitotic clonal expansion in adipogenesis, MTT test was conducted after BMSCs were treated with tigogenin (10, 30 or 90 mol/L) from day 0 to 3 of adipocytic differentiation.
2.3. Oil red O staining and measurement BMSCs were cultured in the presence of adipogenic inducers and tigogenin at the concentration of 10, 30 or 90 mol/L for 18 days. Fat droplets within differentiated adipocytes from BMSCs were observed using the oil red O staining method. Cell monolayers were fixed in 4% formaldehyde for 20 min, washed in water and stained with a 0.6% (w/v) oil red O solution (60% isopropanol, 40% water) for 45 min at 37 ◦ C. For quantification, cell monolayers were then washed extensively with water to remove unbound dye, then 1 ml of isopropyl alcohol was added to the stained culture plate. After 5 min, the absorbance of the extract was assayed by a spectrophotometer at 510 nm. Quantification of oil red O staining is normalized to cell number.
2.4. ELISA for secreted visfatin protein After being treated with adipogenic supplement and tigogenin (10, 30 or 90 mol/L) for 18 days, the culture medium of BMSCs was collected. The levels of extracellular visfatin protein in culture medium were assayed by using EIA kit (Phoenix Biotech, USA) according to the user’s manual of the kit.
according to the manufacturer’s directions. PCR primers used for PPAR␥2, ap2, Cbfa1, COL I, OCN and -actin mRNA detection are as follows: PPAR␥2 (sense: 5 -G G G T C A G C T C T T G T G A A T G G-3 , antisense: 5 -C T G A T G C A C T GCCTATGAGC-3 ; GenBank accession no.: NM 011146), ap2 (sense: 5 -T C T C A C C T G G A A G A C A G C TCCTCCTCG-3 , antisense: 5 -T T C C A T CCAGGCCTCTTCCTTTGGCTC-3 ; GenBank accession no.: K02109), Cbfa1 (sense: 5 -CCG CACGACAACCGCACCAT-3 , antisense: 5 -CG CTCC GGCCC ACAAAT CTC-3 ; GenBank accession no.: AF010284), COL I (sense: 5 -CT GCC TGCT TC GTGTAAA-3 , antisense: 5 -AC GTTC AGTTG GT CAAAGGTA-3 ; GenBank accession no.: NM 007742), OCN (sense: 5 TCGGT TC TGGTGGCGC T T TGCTAC-3 , antisense: 5 -AAG TTC CC CGC T G GTG T CCTATGT-3 ; GenBank accession no.: L24431) and -actin (sense: 5 -T TCGTGGATGCCACAGGACT-3 , antisense: 5 -TCACCAACTG G GA CG AC ATG-3 ; GenBank accession no.: NM 009606). The gene expression levels were normalized by house keeping gene -actin.
2.7. Western blotting analysis BMSCs treated with tigogenin (30 mol/L) were lysed with NETN buffer (20 mM Tris–HCl, pH 7.8, 1 mM EDTA, 50 mM sodium chloride, and 0.5% NP-40) at day 5 of adipogenic induction and lysates were centrifuged at 12,000 rpm at 4 ◦ C for 10 min. Supernatants were collected and protein concentration was determined by BCA assay kit (Nanjing Jiancheng Biotech, China). Western blotting analysis was carried out according to the manufactuers’ protocol. Antibodies against PPAR␥, COL I, -actin (Biolegend; 1:500), phospho-p38, and non-phospho-p38 (Cell signaling, USA; 1:1000) were used. Protein expression was visualized with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, USA; 1:2000) and enhanced chemiluminescence (KPL, USA).
2.8. Quantification of matrix calcium deposition BMSCs were treated with varied concentrations of tigogenin, and the culture continued up to 18 days. Calcification was assessed by the method previously described (Wada et al., 1999) with modification. Briefly, BMSCs were decalcified for 24 h with 0.6 mol/L HCl and the calcium content in supernatant was determined with the use of the calcium C-test Wako Kit (Wako Chemicals, Japan). After decalcification, the cells were washed three times with PBS and solubilized with 0.1 mol/L NaOH/0.1% SDS. The total protein concentration was determined by BCA assay kit. The calcium content of the cells was normalized to protein content.
2.9. Statistical analysis Data were presented as mean ± S.E. and statistically analyzed using Student’s t-test. Differences were considered significant when P < 0.05.
2.5. Alkaline phosphatase (ALP) activity assay BMSCs were propagated to confluence, and 2 days later, cells were treated with 10, 30 and 90 mol/L tigogenin respectively for 5 days, and then the cells were washed twice with ice-cold PBS and lysed by two cycles of freezing and thaw. Aliquots of supernatants were subjected to ALP activity and protein content measurement using an ALP assay kit and a micro-Bradford assay kit (Nanjing Jiancheng Biotech, China) respectively. In the experiment for probing the signaling pathway, cells were pretreated with 50 mol/L ERK1/2 pathway inhibitor PD-98059 or 10 mol/L p38 MAPK pathway inhibitor SB-203580 (Sigma, USA) for 60 min, and then stimulated with 30 mol/L tigogenin for 5 days. Cells were collected and ALP activity was determined.
3. Results 3.1. Effect of tigogenin on the BMSCs proliferation To determine whether tigogenin can affect the proliferation activity of BMSCs, the cells were treated with 10, 30 and 90 mol/L tigogenin respectively for 3 days. MTT tests showed that tigogenin significantly promoted BMSCs proliferation in a dose-dependent manner (Fig. 1).
2.6. Semi-quantitative RT-PCR
3.2. Effect of tigogenin on adipogenesis of BMSCs
BMSCs were propagated to confluence, and 2 days later, cells were treated with 10, 30 and 90 mol/L tigogenin respectively for 5 days and harvested. In another experiment, cells were collected at day 5 and 18 after adipogenic inducers and tigogenin treatment. Total RNA was extracted from cells and single strand cDNA synthesis was performed by using ReverTra Ace ␣ kit (TOYOBO, Japan)
To test whether tigogenin inhibits adipocytic differentiation of BMSCs, tigogenin at the concentrations of 10, 30 or 90 mol/L was added to the medium from day 0 to 18 of differentiation and adipocytes were stained by oil red O at day
H. Zhou et al. / Molecular and Cellular Endocrinology 270 (2007) 17–22
19
Fig. 1. Effect of tigogenin on the proliferation of BMSCs. BMSCs were treated with 10, 30 or 90 mol/L tigogenin for 3 days, and the proliferation rate was assessed by MTT test. Values are mean ± S.E. for triplicate cultures and * P < 0.05, ** P < 0.01 vs. control (the values in cells not receiving tigogenin were set at 100%).
Fig. 3. Effect of tigogenin on the visfatin secretion of adipocytes induced from BMSCs for 18 days. BMSCs were induced for 18 days, then content of visfatin protein in culture medium was measured. Values are mean ± S.E. for triplicate cultures and * P < 0.05, ** P < 0.01 vs. control (the values in cells not receiving tigogenin were set at 100%).
18. Control cells displayed significant lipid accumulation when induced for 18 days compared with cells uninduced (Fig. 2A and B). In tigogenin-treated cells, the amount of stained lipid was less than that of control (Fig. 2C). We then extracted stained lipid and measured its absorbance to compare lipid accumulation between control and tigogenin-treated cells. As shown in Fig. 2D, a significant dose-dependently reduction
of absorbance was observed in cells exposed to tigogenin. At 10 mol/L, tigogenin reduced adipocytic differentiation rate by about 10%, and at 30–90 mol/L it blocked the differentiation by about 40–60%. Next, the levels of visfatin secreted from adipocytes induced from BMSCs for 18 days were measured and found to be decreased significantly by tigogenin also in a dosedependent manner (Fig. 3). To investigate whether tigogenin
Fig. 2. Effect of tigogenin on adipocytic differentiation of BMSCs induced by dexamethasone and insulin. The cells induced for 18 days were stained with oil red O and photographed (100×). (A) Cells untreated. (B) Control cells treated with adipogenic inducers. (C) Cells treated with adipogenic inducers and 90 mol/L tigogenin. After staining with oil red O, stained lipid was extracted and the absorbance at 510 nm was measured (D). Values are mean ± S.E. for triplicate cultures and * P < 0.05, ** P < 0.01 vs. control (the values in cells not receiving tigogenin were set at 100%). The experiment was repeated for three times.
20
H. Zhou et al. / Molecular and Cellular Endocrinology 270 (2007) 17–22
Fig. 5. Effect of tigogenin on the mitotic clonal expansion of BMSCs during adipogenesis. BMSCs were treated with tigogenin (10, 30 or 90 mol/L) during early differentiation phrase (from day 0 to 3) and MTT test was employed to quantify the changes in proliferation. Values are mean ± S.E. for triplicate cultures (the values in normal control cells were set at 100%).
3.4. Effect of tigogenin on osteogenesis of BMSCs
Fig. 4. Effect of tigogenin on PPAR␥2, ap2 and OCN mRNA expression in BMSCs. Expression of PPAR␥2, ap2, Cbfa1, COL I and OCN mRNAs was analyzed by semi-quantitative RT-PCR after cells were treated with tigogenin (10, 30 or 90 mol/L) for 5 or 18 days with (A) or without (B) adipogenic inducers. PPAR␥2 and COL I protein levels were analyzed by Western blot (C). The experiment was repeated for four times.
suppresses adipogenesis through a PPAR␥ pathway, the expression of PPAR␥ was observed. It was found that PPAR␥ mRNA was strongly inhibited by tigogenin, and PPAR␥ protein showed a similar change (Fig. 4A and C). Additionally, mRNA levels of ap2, one of the late target maker genes of PPAR␥ in adipocytic differentiation, was down-regulated by tigogenin (Fig. 4A). The inhibitory effects of tigogenin on expression of PPAR␥ and ap2 are concentration dependent. 3.3. Effect of tigogenin on the mitotic clonal expansion of BMSCs during adipogenesis To further determine whether tigogenin regulates upstream signaling of PPAR␥, we observed the mitotic clonal expansion in adipocytic differentiation of BMSCs and MTT test was used to quantitate the proliferation. As an early critical event of differentiation, suppression of clonal expansion appears to abort adipocyte differentiation. Fig. 5 showed that the proliferation rate of BMSCs increased almost twofold when the cells were induced for 3 days by adipogenic inducers and no substantial difference in clonal expansion was observed between cells treated with or without tigogenin in the concentrations of 10–90 mol/L.
To determine whether tigogenin might have an corresponding ability to promote osteogenesis of BMSCs, the markers characteristic of osteoblastic differentiation such as ALP activity, Cbfa1, COL I and OCN, as well as the content of matrix calcium in BMSCs were measured. After BMSCs were treated with tigogenin for 5 days, expression levels of mRNA for Cbfa1, COL I and OCN were promoted in a dose-dependent manner (Fig. 4B), and COL I protein got the same elevation (Fig. 4C). Also, BMSCs exhibited increased ALP activity in response to tigogenin and ALP activity was elevated almost 1.2–1.5-fold above basal levels in the presence of 10–90 mol/L tigogenin (Fig. 6A). Moreover, we measured the amount of matrix calcium deposition in BMSCs. Calcium deposition was found to be undetectable in control BMSCs and increased slightly but significantly in cells treated with 30–90 mol/L tigogenin for 18 days (Fig. 6B). 3.5. Effect of MAPK pathway inhibitors on the tigogenin-promoted osteogenesis in BMSCs Whether tigogenin-promoted osteogenesis in BMSCs depended on the MAPK pathway was assessed by using specific inhibitors of ERK1/2 and p38 MAPK pathway. BMSCs were pretreated with either the ERK1/2 pathway inhibitor PD-98059 or the p38 MAPK pathway inhibitor SB-203580, then treated with 30 mol/L tigogenin for 5 days. As shown in Fig. 6C, ALP activity in BMSCs treated with ERK1/2 pathway inhibitor PD-98059 or p38 MAPK pathway inhibitor SB-203580 alone barely changed compared with control. PD-98059 exerted no significant influences on the stimulatory effect of tigogenin on the osteogenesis of BMSCs, whereas, SB-203580 significantly impaired this upregulation. Furthermore, Western blotting analysis showed that exposure of BMSCs to 30 mol/L tigogenin activated the phosphorylation of p38 kinase. This activation occurred within 5 min of tigogenin application and was still active after 40 min (Fig. 6D).
H. Zhou et al. / Molecular and Cellular Endocrinology 270 (2007) 17–22
21
Fig. 6. Effect of tigogenin on ALP activity of BMSCs and the involved signaling pathways. (A) effect of tigogenin on ALP activity. After Cells were treated with tigogenin (10, 30 or 90 mol/L) for 5 days, ALP activity was assayed. (B) Effect of tigogenin on calcium deposition. BMSCs were treated with varied concentrations of tigogenin, and calcium deposition was measured at day 18 of culture. (C) Effect of PD-98059 and SB-203580 on the tigogenin-promoted osteogenesis in BMSCs. BMSCs were pretreated with or without 50 mol/L PD-98059 or 10 mol/L SB-203580 for 60 min and then stimulated with tigogenin (30 mol/L) for 5 days, and ALP activity was determined. (D) Tigogenin induced p38 phosphorylation. Tigogenin was treated at indicated time periods. Total protein extracts were prepared and Western blotting analysis was performed. Antibodies against phospho-p38, p38 and -actin were used. Values are mean ± S.E. for triplicate cultures and * P < 0.05, ** P < 0.01 vs. control (the values in cells not receiving tigogenin were set at 100%).
4. Discussion Studies have demonstrated that BMSCs can differentiate into multiple cell types, including osteoblasts, myoblasts, chondrocytes, and adipocytes (Dennis et al., 1999). Adipocytic and osteoblastic cells derived from multipotential stromal cells are reciprocal cell types that are dominant in marrow (Beresford et al., 1992). It is thought that changes in the ratios of these cells are involved in bone volume decreases associated with osteoporosis and age-related osteopenia. One mechanism that may account for the reciprocal relationship between decreased bone density and increased fat formation is an imbalance in the production of osteogenesis and adipogenesis cells in the bone marrow cavity, and an increase in the number of adipocytes occurs at the expense of osteoblasts in osteopenic disorders. Data have suggested that medullary adipocytes are secretory cells that may influence osteogenesis by impairing osteoblast proliferation, differentiation and mineralization, and promoting osteoclasts formation and activation (Gimble, 1990; Mundy, 1996; Sakaguchi et al., 2000). Therefore, inhibition of marrow adipocyte differentiation and a concomitant enhancement
of osteoblastogenesis may provide a strategy for the treatment of osteoporosis. Agents, which inhibit adipogenic and promote osteogenic differentiation of BMSCs, might be helpful in preventing the development of osteoporosis. In the present study, we have examined the effect of tigogenin on adipogenic and osteogenic differentiation of isolated mouse primary BMSCs. To determine whether tigogenin has an ability to decrease adipocyte formation of BMSCs, we detected the adipogenic differentiation using oil red O staining and observed the changes of several differentiation markers. It is found that tigogenin markedly inhibited BMSCs to differentiate into adipocyte and decreased the visfatin secretion and expression levels of PPAR␥ and ap2. Since tigogenin can inhibit the expression of PPAR␥, an early key transcription factor in the signaling cascade during adipogenesis (Gregoire et al., 1998), it possibly prevents adipogenesis of BMSCs in the early phase. Thus, we measured whether tigogenin affected mitotic clonal expansion of adipocyte differentiation which initiates the process of differentiation in response to adipogenic inducers (Tang et al., 2003), and found that tigogenin did not affect this re-entry into the cell cycle. Our previous study also showed that tigogenin treatment
22
H. Zhou et al. / Molecular and Cellular Endocrinology 270 (2007) 17–22
did not alter the expression of C/EBP, the upstream regulator of clonal expansion and PPAR␥ (data not shown). Thus, we deduced from the results that tigogenin could directly act upon the expression of PPAR␥, which might lead to the inhibition of adipocytic differentiation of BMSCs and is at least one major mechanism involved in the inhibition of adipocytic differentiation by tigogenin. To explore whether tigogenin exerted the corresponding influences on osteogenesis of BMSCs, ALP activity, expression levels of Cbfa1, COL I and OCN, and the content of matrix calcium in BMSCs exposed to tigogenin were assayed. We have ever compared the capacity for osteogenesis in BMSCs induced by osteogenic supplements with or without tigogenin in previous studies, and found that tigogenin treatment did not significantly promoted the osteogenesis of BMSCs induced by osteogenic supplements, whereas the osteogenesis could be slightly but significantly elevated when BMSCs were treated with tigogenin only. We speculated that the strong effect of osteogenic inducers on the osteogenesis of BMSCs might make it inapparent the weak effect of tigogenin when both were added to the cells. Thus, in the current study, we employed BMSCs treated without osteogenic inducers. It was found that ALP activity was significantly elevated in the presence of tigogenin. Similarly, the mRNA expression of other osteoblastic phenotypes, such as Cbfa1, COL I and OCN, also increased following the addition of tigogenin. These results in osteoblastic differentiation suggest that tigogenin is able to differentiate BMSCs into an osteoblastic lineage without adding any osteogenic factors, although the effect appears modest. This conclusion is further strengthened by the evidence that tigogenin increased the matrix calcium deposition in BMSCs slightly but significantly. To make it clear the mechanism involved in this stimulatory effect of tigogenin, the role of MAPK in mediating the cell response to tigogenin was assessed. It has been reported that two key MAPK pathways, ERK1/2 and p38, are essential for osteoblastic differentiation of bone cells (Lai et al., 2001; Lai and Cheng, 2002; Chen et al., 2004). In the current study, SB-203580, a specific inhibitor of p38 pathway, remarkably blocked the stimulatory effect of tigogenin on ALP activity of BMSCs, whereas, PD-98059, inhibitor of ERK1/2, barely changed this promotion. Furthermore, we found that the amount of phospo-p38 protein was increased in BMSCs treated with tigogenin. Thus, it is suggested that this promotion of osteoblastic differentiation by tigogenin is at least partially mediated via p38 MAPK pathway. The above results indicate that tigogenin exerts dual influences on BMSCs by inhibiting adipocytic differentiation and promoting osteoblastic differentiation. These results got further substantiated by the fact that tigogenin promoted the proliferation of BMSCs.
Taken together, we deduced from our results that tigogenin might have protective effect on bone and be helpful in preventing the development of osteoporosis by inhibiting adipocyte formation and stimulating osteoblast formation from BMSCs. References Ahdjoudj, S., Fromigue, O., Marie, P.J., 2004. Plasticity and regulation of human bone marrow stromal osteoprogenitor cells: potential implication in the treatment of age-related bone loss. Histopathology 19, 151–157. Beresford, J.N., Bennett, J.H., Devlin, C., Leboy, P., Owen, M.E., 1992. Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J. Cell. Sci. 102, 341–351. Chen, Y.J., Kuo, Y.R., Yang, K.D., Wang, C.J., Chen, S.M., Huang, H.C., Yang, Y.J., 2004. Activation of extracellular signal-regulated kinase (ERK) and p38 kinase in shock wave-promoted bone formation of segmental defect in rats. Bone 34 (3), 466–477. Dennis, J.E., Merriam, A., Awadallah, A., Yoo, J.U., Johnstone, B., Caplan, A.I., 1999. A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J. Bone Miner. Res. 14, 700–709. Dominici, M., Hofmann, T.J., Horwitz, E.M., 2001. Bone marrow mesenchymal cells: biological properties and clinical applications. J. Biol. Regul. Homeost. Agents 15, 28–37. Gimble, J.M., 1990. The function of adipocytes in the bone marrow stroma. New Biol. 2, 304–312. Gregoire, F.M., Smas, C.M., Sul, H.S., 1998. Understanding adipocyte differentiation. Physiol. Rev. 78, 783–809. Lai, C.F., Cheng, S.L., 2002. Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor- in normal human osteoblastic cells. J. Biol. Chem. 277, 15514–15522. Lai, C.F., Chaudhary, L., Fausto, A., Halstead, L.R., Ory, D.S., Avioli, L.V., Cheng, S.L, 2001. Erk is essential for growth, differentiation, integrin expression, and cell function in human osteoblastic cells. J. Biol. Chem. 276, 14443–144509. Li, X., Cui, Q., Kao, C., Wang, G.J., Balian, G., 2003. Lovastatin inhibits adipogenic and stimulates osteogenic differentiation by suppressing PPAR␣2 and increasing Cbfa1/Runx2 expression in bone marrow mesenchymal cell cultures. Bone. 33, 652–659. Mundy, G.R., 1996. Cytokines and bone remodeling. In: Marcus, R., Feldman, D., Kelsey, J. (Eds.), Osteoporosis. Academic Press, San Diego, p. 301. Nuttall, M.E., Gimble, J.M., 2000. Is there a therapeutic opportunity to either prevent or treat osteopenic disorders by inhibiting marrow adipogenesis? Bone 27, 177–184. Rosen, C.J., Bouxsein, M.L., 2006. Mechanism of disease: is osteoporosis the obesity of bone. Nat. Clin. Pract. Rheumatol. 2 (1), 35–43. Sakaguchi, K., Morita, I., Murota, S., 2000. Relationship between the ability to support differentiation of osteoclast-like cells and adipogenesis in murine stromal cells derived from bone marrow. Prostag. Leukotr. Ess. 62, 319–327. Tang, Q.Q., Otto, T.C., Lane, M.D., 2003. Mitotic clonal expansion: a synchronous process required for adipogenesis. Proc. Natl. Acad. Sci. U.S.A. 100, 44–49. Verma, S., Rajaratnam, J.H., Denton, J., Hoyland, J.A., Byer, R.J., 2002. Adipocytic proportion of bone marrow is inversely related to bone formation in osteoporosis. J. Clin. Pathol. 55, 693–698. Wada, T., McKee, M.D., Steitz, S., Giachelli, C.M., 1999. Calcification of vascular smooth muscle cell cultures inhibited by osteopontin. Circ. Res. 84, 166–178.
Tigogenin inhibits adipocytic differentiation and induces osteoblastic differentiation in mouse bone marrow stromal cells Hua Zhou a,b , Xi Yang c , Naili Wang d , Yaou Zhang a,b , Guoping Cai a,b,∗ a
Life Science Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China c Department of Genetics and Developmental Biology, The Fourth Military Medical University, Xi’an 710032, PR China d Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 110015, PR China b
Received 27 October 2006; received in revised form 12 December 2006; accepted 29 January 2007
Abstract We investigated the effect of tigogenin on adipocytic and osteoblastic differentiation in mouse bone marrow stromal cells (BMSCs). Tigogenin enhanced the proliferation of BMSCs significantly. Tigogenin treatment reduced the adipogenic induction of lipid accumulation, visfatin secretion, and the expressions of peroxisome proliferation-activated receptor (PPAR)␥2 and adipocyte fatty acid-binding protein (ap)2. Moreover, tigogenin had no effect on the mitotic clonal expansion. On the other hand, tigogenin significantly elevated alkaline phosphatase (ALP) activity and the expressions of Cbfa1, collagen type I (COL I) and osteocalcin (OCN), as well as the content of matrix calcium in BMSCs. Further, SB-203580 antagonized the tigogenin-promoted osteogenesis. These observations suggested that tigogenin may modulate differentiation of BMSCs to cause a lineage shift away from the adipocytes and toward the osteoblasts, which is at least mediated by inhibition of PPAR␥ and via p38 MAPK pathway, and is a potential drug preventing the development of osteoporosis and the related disorders. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Tigogenin; Bone marrow stromal cells; Adipocyte; Osteoblast; Cell differentiation
1. Introduction Bone marrow stromal cells (BMSCs) are pluripotent cells that have the capacity to differentiate into adipocytes, osteoblasts, chondrocytes, myoblasts, or fibroblasts, and a reciprocal and inverse relationship exists between adipogenesis and osteogenesis in the cells (Ahdjoudj et al., 2004; Dominici et al., 2001; Li et al., 2003). The decrease in bone volume associated with osteoporosis and age-related osteopenia is accompanied by an increase in marrow adipose tissue at the expense of osteoblasts as seen following ovariectomy, immobilization, treatment with glucocorticoids, aging, and other conditions that lead to bone loss (Rosen and Bouxsein, 2006). It is possible, therefore, that the inhibition of marrow adipogenesis with a concomitant increase in osteogenesis could provide a therapeutic target with which to prevent further increase in adipocyte formation (Nuttall and Gimble, 2000).
Tigogenin is one of steroidal sapogenins which is widely used for synthesizing steroid drugs. It has been reported extensively that steroidal sapogenins from various plants have anti-obesity effect, but the underlying cellular and molecular mechanisms remain largely unknown. It is possible that some of these mechanisms involve alterations in differentiation of adipocytes. Our previous study have showed that tigogenin can inhibit the 3T3-L1 preadipocytes to differentiate into adipocytes (data unpublished). Thus it is likely that tigogenin might influence the differentiation of BMSCs. In the present study, to elucidate whether tigogenin can directly modulate the differentiation of BMSCs, we examined the effect of tigogenin on adipocytic and osteoblastic differentiation in BMSCs, and probed the possible mechanism. 2. Materials and methods 2.1. Isolation and culture of primary BMSCs
∗
Corresponding author at: Room 408 Building L, Tsinghua Campus, University Town, Shenzhen 518055, PR China. Tel.: +86 755 26036740. E-mail address: bjsz [email protected] (G. Cai). 0303-7207/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2007.01.017
Culture medium was Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, USA), supplemented with 10% calf serum (Gibco, USA), 100 U/ml penicillin and 100 g/ml streptomycin (Sangon, China). The BMSCs were
18
H. Zhou et al. / Molecular and Cellular Endocrinology 270 (2007) 17–22
obtained from BALB/c mice (4 weeks) as previously described (Verma et al., 2002) with minor modification. Briefly, mice were killed by cervical dislocation, metaphyses of femurs and tibias were cut aseptically, diaphysis cavities were repeatedly flushed with culture medium, and bone marrow cells were collected and cultured at 37 ◦ C in a 5% CO2 humidified incubator. Culture medium was replaced every 3 days and red blood cells and non-adherent cells were removed. For adipocyte-induced culture, cells were propagated to confluence, and were induced by adipogenic supplement containing 1 mol/L dexamethasone and 10 mg/L insulin (Sigma, USA).
2.2. Assessment of proliferation and mitotic clonal expansion of primary BMSCs BMSCs were plated in 96-well culture plates and cultured for 6 days. Tigogenin [(25R)-5␣-spirostane-3-ol] (gift from Dr. Naili Wang, Shenyang Pharmaceutical University, Shenyang, PR China) diluted with DMSO (Merck, Germany) to prepare the stock solution (10 mmol/L) was then added to the wells at final concentrations of 10, 30 or 90 mol/L. Cells were incubated for 72 h. Upon completion of the incubation, MTT dye solution (20 L, 5 g/L, Sangon, China) was added to each well. After 4 h incubation, the supernatant was removed and 100 L DMSO was added to solubilize the MTT. The optical density of each well was measured on a microplate spectrophotometer (TECAN, Switzerland) at a wavelength of 570 nm. For the assay of mitotic clonal expansion in adipogenesis, MTT test was conducted after BMSCs were treated with tigogenin (10, 30 or 90 mol/L) from day 0 to 3 of adipocytic differentiation.
2.3. Oil red O staining and measurement BMSCs were cultured in the presence of adipogenic inducers and tigogenin at the concentration of 10, 30 or 90 mol/L for 18 days. Fat droplets within differentiated adipocytes from BMSCs were observed using the oil red O staining method. Cell monolayers were fixed in 4% formaldehyde for 20 min, washed in water and stained with a 0.6% (w/v) oil red O solution (60% isopropanol, 40% water) for 45 min at 37 ◦ C. For quantification, cell monolayers were then washed extensively with water to remove unbound dye, then 1 ml of isopropyl alcohol was added to the stained culture plate. After 5 min, the absorbance of the extract was assayed by a spectrophotometer at 510 nm. Quantification of oil red O staining is normalized to cell number.
2.4. ELISA for secreted visfatin protein After being treated with adipogenic supplement and tigogenin (10, 30 or 90 mol/L) for 18 days, the culture medium of BMSCs was collected. The levels of extracellular visfatin protein in culture medium were assayed by using EIA kit (Phoenix Biotech, USA) according to the user’s manual of the kit.
according to the manufacturer’s directions. PCR primers used for PPAR␥2, ap2, Cbfa1, COL I, OCN and -actin mRNA detection are as follows: PPAR␥2 (sense: 5 -G G G T C A G C T C T T G T G A A T G G-3 , antisense: 5 -C T G A T G C A C T GCCTATGAGC-3 ; GenBank accession no.: NM 011146), ap2 (sense: 5 -T C T C A C C T G G A A G A C A G C TCCTCCTCG-3 , antisense: 5 -T T C C A T CCAGGCCTCTTCCTTTGGCTC-3 ; GenBank accession no.: K02109), Cbfa1 (sense: 5 -CCG CACGACAACCGCACCAT-3 , antisense: 5 -CG CTCC GGCCC ACAAAT CTC-3 ; GenBank accession no.: AF010284), COL I (sense: 5 -CT GCC TGCT TC GTGTAAA-3 , antisense: 5 -AC GTTC AGTTG GT CAAAGGTA-3 ; GenBank accession no.: NM 007742), OCN (sense: 5 TCGGT TC TGGTGGCGC T T TGCTAC-3 , antisense: 5 -AAG TTC CC CGC T G GTG T CCTATGT-3 ; GenBank accession no.: L24431) and -actin (sense: 5 -T TCGTGGATGCCACAGGACT-3 , antisense: 5 -TCACCAACTG G GA CG AC ATG-3 ; GenBank accession no.: NM 009606). The gene expression levels were normalized by house keeping gene -actin.
2.7. Western blotting analysis BMSCs treated with tigogenin (30 mol/L) were lysed with NETN buffer (20 mM Tris–HCl, pH 7.8, 1 mM EDTA, 50 mM sodium chloride, and 0.5% NP-40) at day 5 of adipogenic induction and lysates were centrifuged at 12,000 rpm at 4 ◦ C for 10 min. Supernatants were collected and protein concentration was determined by BCA assay kit (Nanjing Jiancheng Biotech, China). Western blotting analysis was carried out according to the manufactuers’ protocol. Antibodies against PPAR␥, COL I, -actin (Biolegend; 1:500), phospho-p38, and non-phospho-p38 (Cell signaling, USA; 1:1000) were used. Protein expression was visualized with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, USA; 1:2000) and enhanced chemiluminescence (KPL, USA).
2.8. Quantification of matrix calcium deposition BMSCs were treated with varied concentrations of tigogenin, and the culture continued up to 18 days. Calcification was assessed by the method previously described (Wada et al., 1999) with modification. Briefly, BMSCs were decalcified for 24 h with 0.6 mol/L HCl and the calcium content in supernatant was determined with the use of the calcium C-test Wako Kit (Wako Chemicals, Japan). After decalcification, the cells were washed three times with PBS and solubilized with 0.1 mol/L NaOH/0.1% SDS. The total protein concentration was determined by BCA assay kit. The calcium content of the cells was normalized to protein content.
2.9. Statistical analysis Data were presented as mean ± S.E. and statistically analyzed using Student’s t-test. Differences were considered significant when P < 0.05.
2.5. Alkaline phosphatase (ALP) activity assay BMSCs were propagated to confluence, and 2 days later, cells were treated with 10, 30 and 90 mol/L tigogenin respectively for 5 days, and then the cells were washed twice with ice-cold PBS and lysed by two cycles of freezing and thaw. Aliquots of supernatants were subjected to ALP activity and protein content measurement using an ALP assay kit and a micro-Bradford assay kit (Nanjing Jiancheng Biotech, China) respectively. In the experiment for probing the signaling pathway, cells were pretreated with 50 mol/L ERK1/2 pathway inhibitor PD-98059 or 10 mol/L p38 MAPK pathway inhibitor SB-203580 (Sigma, USA) for 60 min, and then stimulated with 30 mol/L tigogenin for 5 days. Cells were collected and ALP activity was determined.
3. Results 3.1. Effect of tigogenin on the BMSCs proliferation To determine whether tigogenin can affect the proliferation activity of BMSCs, the cells were treated with 10, 30 and 90 mol/L tigogenin respectively for 3 days. MTT tests showed that tigogenin significantly promoted BMSCs proliferation in a dose-dependent manner (Fig. 1).
2.6. Semi-quantitative RT-PCR
3.2. Effect of tigogenin on adipogenesis of BMSCs
BMSCs were propagated to confluence, and 2 days later, cells were treated with 10, 30 and 90 mol/L tigogenin respectively for 5 days and harvested. In another experiment, cells were collected at day 5 and 18 after adipogenic inducers and tigogenin treatment. Total RNA was extracted from cells and single strand cDNA synthesis was performed by using ReverTra Ace ␣ kit (TOYOBO, Japan)
To test whether tigogenin inhibits adipocytic differentiation of BMSCs, tigogenin at the concentrations of 10, 30 or 90 mol/L was added to the medium from day 0 to 18 of differentiation and adipocytes were stained by oil red O at day
H. Zhou et al. / Molecular and Cellular Endocrinology 270 (2007) 17–22
19
Fig. 1. Effect of tigogenin on the proliferation of BMSCs. BMSCs were treated with 10, 30 or 90 mol/L tigogenin for 3 days, and the proliferation rate was assessed by MTT test. Values are mean ± S.E. for triplicate cultures and * P < 0.05, ** P < 0.01 vs. control (the values in cells not receiving tigogenin were set at 100%).
Fig. 3. Effect of tigogenin on the visfatin secretion of adipocytes induced from BMSCs for 18 days. BMSCs were induced for 18 days, then content of visfatin protein in culture medium was measured. Values are mean ± S.E. for triplicate cultures and * P < 0.05, ** P < 0.01 vs. control (the values in cells not receiving tigogenin were set at 100%).
18. Control cells displayed significant lipid accumulation when induced for 18 days compared with cells uninduced (Fig. 2A and B). In tigogenin-treated cells, the amount of stained lipid was less than that of control (Fig. 2C). We then extracted stained lipid and measured its absorbance to compare lipid accumulation between control and tigogenin-treated cells. As shown in Fig. 2D, a significant dose-dependently reduction
of absorbance was observed in cells exposed to tigogenin. At 10 mol/L, tigogenin reduced adipocytic differentiation rate by about 10%, and at 30–90 mol/L it blocked the differentiation by about 40–60%. Next, the levels of visfatin secreted from adipocytes induced from BMSCs for 18 days were measured and found to be decreased significantly by tigogenin also in a dosedependent manner (Fig. 3). To investigate whether tigogenin
Fig. 2. Effect of tigogenin on adipocytic differentiation of BMSCs induced by dexamethasone and insulin. The cells induced for 18 days were stained with oil red O and photographed (100×). (A) Cells untreated. (B) Control cells treated with adipogenic inducers. (C) Cells treated with adipogenic inducers and 90 mol/L tigogenin. After staining with oil red O, stained lipid was extracted and the absorbance at 510 nm was measured (D). Values are mean ± S.E. for triplicate cultures and * P < 0.05, ** P < 0.01 vs. control (the values in cells not receiving tigogenin were set at 100%). The experiment was repeated for three times.
20
H. Zhou et al. / Molecular and Cellular Endocrinology 270 (2007) 17–22
Fig. 5. Effect of tigogenin on the mitotic clonal expansion of BMSCs during adipogenesis. BMSCs were treated with tigogenin (10, 30 or 90 mol/L) during early differentiation phrase (from day 0 to 3) and MTT test was employed to quantify the changes in proliferation. Values are mean ± S.E. for triplicate cultures (the values in normal control cells were set at 100%).
3.4. Effect of tigogenin on osteogenesis of BMSCs
Fig. 4. Effect of tigogenin on PPAR␥2, ap2 and OCN mRNA expression in BMSCs. Expression of PPAR␥2, ap2, Cbfa1, COL I and OCN mRNAs was analyzed by semi-quantitative RT-PCR after cells were treated with tigogenin (10, 30 or 90 mol/L) for 5 or 18 days with (A) or without (B) adipogenic inducers. PPAR␥2 and COL I protein levels were analyzed by Western blot (C). The experiment was repeated for four times.
suppresses adipogenesis through a PPAR␥ pathway, the expression of PPAR␥ was observed. It was found that PPAR␥ mRNA was strongly inhibited by tigogenin, and PPAR␥ protein showed a similar change (Fig. 4A and C). Additionally, mRNA levels of ap2, one of the late target maker genes of PPAR␥ in adipocytic differentiation, was down-regulated by tigogenin (Fig. 4A). The inhibitory effects of tigogenin on expression of PPAR␥ and ap2 are concentration dependent. 3.3. Effect of tigogenin on the mitotic clonal expansion of BMSCs during adipogenesis To further determine whether tigogenin regulates upstream signaling of PPAR␥, we observed the mitotic clonal expansion in adipocytic differentiation of BMSCs and MTT test was used to quantitate the proliferation. As an early critical event of differentiation, suppression of clonal expansion appears to abort adipocyte differentiation. Fig. 5 showed that the proliferation rate of BMSCs increased almost twofold when the cells were induced for 3 days by adipogenic inducers and no substantial difference in clonal expansion was observed between cells treated with or without tigogenin in the concentrations of 10–90 mol/L.
To determine whether tigogenin might have an corresponding ability to promote osteogenesis of BMSCs, the markers characteristic of osteoblastic differentiation such as ALP activity, Cbfa1, COL I and OCN, as well as the content of matrix calcium in BMSCs were measured. After BMSCs were treated with tigogenin for 5 days, expression levels of mRNA for Cbfa1, COL I and OCN were promoted in a dose-dependent manner (Fig. 4B), and COL I protein got the same elevation (Fig. 4C). Also, BMSCs exhibited increased ALP activity in response to tigogenin and ALP activity was elevated almost 1.2–1.5-fold above basal levels in the presence of 10–90 mol/L tigogenin (Fig. 6A). Moreover, we measured the amount of matrix calcium deposition in BMSCs. Calcium deposition was found to be undetectable in control BMSCs and increased slightly but significantly in cells treated with 30–90 mol/L tigogenin for 18 days (Fig. 6B). 3.5. Effect of MAPK pathway inhibitors on the tigogenin-promoted osteogenesis in BMSCs Whether tigogenin-promoted osteogenesis in BMSCs depended on the MAPK pathway was assessed by using specific inhibitors of ERK1/2 and p38 MAPK pathway. BMSCs were pretreated with either the ERK1/2 pathway inhibitor PD-98059 or the p38 MAPK pathway inhibitor SB-203580, then treated with 30 mol/L tigogenin for 5 days. As shown in Fig. 6C, ALP activity in BMSCs treated with ERK1/2 pathway inhibitor PD-98059 or p38 MAPK pathway inhibitor SB-203580 alone barely changed compared with control. PD-98059 exerted no significant influences on the stimulatory effect of tigogenin on the osteogenesis of BMSCs, whereas, SB-203580 significantly impaired this upregulation. Furthermore, Western blotting analysis showed that exposure of BMSCs to 30 mol/L tigogenin activated the phosphorylation of p38 kinase. This activation occurred within 5 min of tigogenin application and was still active after 40 min (Fig. 6D).
H. Zhou et al. / Molecular and Cellular Endocrinology 270 (2007) 17–22
21
Fig. 6. Effect of tigogenin on ALP activity of BMSCs and the involved signaling pathways. (A) effect of tigogenin on ALP activity. After Cells were treated with tigogenin (10, 30 or 90 mol/L) for 5 days, ALP activity was assayed. (B) Effect of tigogenin on calcium deposition. BMSCs were treated with varied concentrations of tigogenin, and calcium deposition was measured at day 18 of culture. (C) Effect of PD-98059 and SB-203580 on the tigogenin-promoted osteogenesis in BMSCs. BMSCs were pretreated with or without 50 mol/L PD-98059 or 10 mol/L SB-203580 for 60 min and then stimulated with tigogenin (30 mol/L) for 5 days, and ALP activity was determined. (D) Tigogenin induced p38 phosphorylation. Tigogenin was treated at indicated time periods. Total protein extracts were prepared and Western blotting analysis was performed. Antibodies against phospho-p38, p38 and -actin were used. Values are mean ± S.E. for triplicate cultures and * P < 0.05, ** P < 0.01 vs. control (the values in cells not receiving tigogenin were set at 100%).
4. Discussion Studies have demonstrated that BMSCs can differentiate into multiple cell types, including osteoblasts, myoblasts, chondrocytes, and adipocytes (Dennis et al., 1999). Adipocytic and osteoblastic cells derived from multipotential stromal cells are reciprocal cell types that are dominant in marrow (Beresford et al., 1992). It is thought that changes in the ratios of these cells are involved in bone volume decreases associated with osteoporosis and age-related osteopenia. One mechanism that may account for the reciprocal relationship between decreased bone density and increased fat formation is an imbalance in the production of osteogenesis and adipogenesis cells in the bone marrow cavity, and an increase in the number of adipocytes occurs at the expense of osteoblasts in osteopenic disorders. Data have suggested that medullary adipocytes are secretory cells that may influence osteogenesis by impairing osteoblast proliferation, differentiation and mineralization, and promoting osteoclasts formation and activation (Gimble, 1990; Mundy, 1996; Sakaguchi et al., 2000). Therefore, inhibition of marrow adipocyte differentiation and a concomitant enhancement
of osteoblastogenesis may provide a strategy for the treatment of osteoporosis. Agents, which inhibit adipogenic and promote osteogenic differentiation of BMSCs, might be helpful in preventing the development of osteoporosis. In the present study, we have examined the effect of tigogenin on adipogenic and osteogenic differentiation of isolated mouse primary BMSCs. To determine whether tigogenin has an ability to decrease adipocyte formation of BMSCs, we detected the adipogenic differentiation using oil red O staining and observed the changes of several differentiation markers. It is found that tigogenin markedly inhibited BMSCs to differentiate into adipocyte and decreased the visfatin secretion and expression levels of PPAR␥ and ap2. Since tigogenin can inhibit the expression of PPAR␥, an early key transcription factor in the signaling cascade during adipogenesis (Gregoire et al., 1998), it possibly prevents adipogenesis of BMSCs in the early phase. Thus, we measured whether tigogenin affected mitotic clonal expansion of adipocyte differentiation which initiates the process of differentiation in response to adipogenic inducers (Tang et al., 2003), and found that tigogenin did not affect this re-entry into the cell cycle. Our previous study also showed that tigogenin treatment
22
H. Zhou et al. / Molecular and Cellular Endocrinology 270 (2007) 17–22
did not alter the expression of C/EBP, the upstream regulator of clonal expansion and PPAR␥ (data not shown). Thus, we deduced from the results that tigogenin could directly act upon the expression of PPAR␥, which might lead to the inhibition of adipocytic differentiation of BMSCs and is at least one major mechanism involved in the inhibition of adipocytic differentiation by tigogenin. To explore whether tigogenin exerted the corresponding influences on osteogenesis of BMSCs, ALP activity, expression levels of Cbfa1, COL I and OCN, and the content of matrix calcium in BMSCs exposed to tigogenin were assayed. We have ever compared the capacity for osteogenesis in BMSCs induced by osteogenic supplements with or without tigogenin in previous studies, and found that tigogenin treatment did not significantly promoted the osteogenesis of BMSCs induced by osteogenic supplements, whereas the osteogenesis could be slightly but significantly elevated when BMSCs were treated with tigogenin only. We speculated that the strong effect of osteogenic inducers on the osteogenesis of BMSCs might make it inapparent the weak effect of tigogenin when both were added to the cells. Thus, in the current study, we employed BMSCs treated without osteogenic inducers. It was found that ALP activity was significantly elevated in the presence of tigogenin. Similarly, the mRNA expression of other osteoblastic phenotypes, such as Cbfa1, COL I and OCN, also increased following the addition of tigogenin. These results in osteoblastic differentiation suggest that tigogenin is able to differentiate BMSCs into an osteoblastic lineage without adding any osteogenic factors, although the effect appears modest. This conclusion is further strengthened by the evidence that tigogenin increased the matrix calcium deposition in BMSCs slightly but significantly. To make it clear the mechanism involved in this stimulatory effect of tigogenin, the role of MAPK in mediating the cell response to tigogenin was assessed. It has been reported that two key MAPK pathways, ERK1/2 and p38, are essential for osteoblastic differentiation of bone cells (Lai et al., 2001; Lai and Cheng, 2002; Chen et al., 2004). In the current study, SB-203580, a specific inhibitor of p38 pathway, remarkably blocked the stimulatory effect of tigogenin on ALP activity of BMSCs, whereas, PD-98059, inhibitor of ERK1/2, barely changed this promotion. Furthermore, we found that the amount of phospo-p38 protein was increased in BMSCs treated with tigogenin. Thus, it is suggested that this promotion of osteoblastic differentiation by tigogenin is at least partially mediated via p38 MAPK pathway. The above results indicate that tigogenin exerts dual influences on BMSCs by inhibiting adipocytic differentiation and promoting osteoblastic differentiation. These results got further substantiated by the fact that tigogenin promoted the proliferation of BMSCs.
Taken together, we deduced from our results that tigogenin might have protective effect on bone and be helpful in preventing the development of osteoporosis by inhibiting adipocyte formation and stimulating osteoblast formation from BMSCs. References Ahdjoudj, S., Fromigue, O., Marie, P.J., 2004. Plasticity and regulation of human bone marrow stromal osteoprogenitor cells: potential implication in the treatment of age-related bone loss. Histopathology 19, 151–157. Beresford, J.N., Bennett, J.H., Devlin, C., Leboy, P., Owen, M.E., 1992. Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J. Cell. Sci. 102, 341–351. Chen, Y.J., Kuo, Y.R., Yang, K.D., Wang, C.J., Chen, S.M., Huang, H.C., Yang, Y.J., 2004. Activation of extracellular signal-regulated kinase (ERK) and p38 kinase in shock wave-promoted bone formation of segmental defect in rats. Bone 34 (3), 466–477. Dennis, J.E., Merriam, A., Awadallah, A., Yoo, J.U., Johnstone, B., Caplan, A.I., 1999. A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J. Bone Miner. Res. 14, 700–709. Dominici, M., Hofmann, T.J., Horwitz, E.M., 2001. Bone marrow mesenchymal cells: biological properties and clinical applications. J. Biol. Regul. Homeost. Agents 15, 28–37. Gimble, J.M., 1990. The function of adipocytes in the bone marrow stroma. New Biol. 2, 304–312. Gregoire, F.M., Smas, C.M., Sul, H.S., 1998. Understanding adipocyte differentiation. Physiol. Rev. 78, 783–809. Lai, C.F., Cheng, S.L., 2002. Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor- in normal human osteoblastic cells. J. Biol. Chem. 277, 15514–15522. Lai, C.F., Chaudhary, L., Fausto, A., Halstead, L.R., Ory, D.S., Avioli, L.V., Cheng, S.L, 2001. Erk is essential for growth, differentiation, integrin expression, and cell function in human osteoblastic cells. J. Biol. Chem. 276, 14443–144509. Li, X., Cui, Q., Kao, C., Wang, G.J., Balian, G., 2003. Lovastatin inhibits adipogenic and stimulates osteogenic differentiation by suppressing PPAR␣2 and increasing Cbfa1/Runx2 expression in bone marrow mesenchymal cell cultures. Bone. 33, 652–659. Mundy, G.R., 1996. Cytokines and bone remodeling. In: Marcus, R., Feldman, D., Kelsey, J. (Eds.), Osteoporosis. Academic Press, San Diego, p. 301. Nuttall, M.E., Gimble, J.M., 2000. Is there a therapeutic opportunity to either prevent or treat osteopenic disorders by inhibiting marrow adipogenesis? Bone 27, 177–184. Rosen, C.J., Bouxsein, M.L., 2006. Mechanism of disease: is osteoporosis the obesity of bone. Nat. Clin. Pract. Rheumatol. 2 (1), 35–43. Sakaguchi, K., Morita, I., Murota, S., 2000. Relationship between the ability to support differentiation of osteoclast-like cells and adipogenesis in murine stromal cells derived from bone marrow. Prostag. Leukotr. Ess. 62, 319–327. Tang, Q.Q., Otto, T.C., Lane, M.D., 2003. Mitotic clonal expansion: a synchronous process required for adipogenesis. Proc. Natl. Acad. Sci. U.S.A. 100, 44–49. Verma, S., Rajaratnam, J.H., Denton, J., Hoyland, J.A., Byer, R.J., 2002. Adipocytic proportion of bone marrow is inversely related to bone formation in osteoporosis. J. Clin. Pathol. 55, 693–698. Wada, T., McKee, M.D., Steitz, S., Giachelli, C.M., 1999. Calcification of vascular smooth muscle cell cultures inhibited by osteopontin. Circ. Res. 84, 166–178.