- Email: [email protected]
Dimethyl sulfoxide as an inducer of differentiation in preosteoblast MC3T3-E1 cells
FEBS Letters 580 (2006) 121–126
Dimethyl sulfoxide as an inducer of differentiation in preosteoblast MC3T3-E1 cells W.M.W. Cheung*,1, W.W. Ng, A.W.C. Kung* Department of Medicine, University of Hong Kong, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China Received 7 September 2005; revised 9 November 2005; accepted 14 November 2005 Available online 6 December 2005 Edited by Veli-Pekka Lehto
Abstract Osteoblastic differentiation is an essential part of bone formation. Dimethyl sulfoxide (DMSO) is a water miscible solvent that is used extensively for receptor ligands in osteoblast studies. However, little is known about its effects on osteoblastogenic precursor cells. In this study, we have used a murine preosteoblast cell line MC3T3-E1 cells to demonstrate that DMSO effectively induces osteoblastic differentiation of MC3T3-E1 cells via the activation of Runx2 and osterix and is dependent upon the protein kinase C (PKC) pathways. We further demonstrated that prolonged activation of PKC pathways is sufficient to induce osteoblastic differentiation, possibly via the activation of PKD/PKCl. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: DMSO; Protein kinase C; PKD; Erk; Proliferation; Osteoblasts
1. Introduction In normal bone remodeling or bone turnover, osteoblastic bone formation and osteoblastic bone resorption is coupled in a precise and orchestrated manner. Osteoblastic differentiation is an essential part of bone formation. Several groups of proteins are necessary for osteoblastic differentiation, such as bone morphogenetic proteins (BMPs) and transcription factors such as Runx2 and osterix (Osx) [1,2]. Dimethyl sulfoxide (DMSO; (CH3)2SO) is a water miscible solvent that serves as powerful solvents that dissolve most water-insoluble drugs [3]. DMSO possesses anti-inflammatory properties [4], as well as the ability to act as a free radical scavenger [5]. Thus, its properties have been exploited in the treatment of dermatological, rheumatic, and renal manifestations of amyloidosis. Depending on the cellular context and proba*
Corresponding authors. Address: Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China. Fax: +852 2816 2187 (A.W.C. Kung). E-mail addresses: [email protected] (W.M.W. Cheung), [email protected] (A.W.C. Kung). 1
Fax: +852 2819 1127.
Abbreviations: DMSO, dimethyl sulfoxide; PKC, protein kinase C; BMP, bone morphogenetic protein; Osx, osterix; GFx, GF 109203X; PMA, phorbol 12-myristate 13-acetate; Tris, Trizma base; TX, TritonX100; ALP, alkaline phosphatase; PNPP, p-nitrophenyl phosphate; MAPKs, MAP kinases
bly via the alteration of splice site selection, DMSO is capable of inducing or inhibiting cell proliferation, apoptosis and/or differentiation [6]. DMSO induces the differentiation of many cell types including leukemia cells and mammary adenocarcinoma LA7 cells [7,8]. DMSO induces differentiation of LA7 cells into mammary cells with milk production, which originally requires the supplementation of lactogenic hormones. Therefore, it is recently developed as an in vitro model system for studying mammary cells differentiation [8]. In osteoblast studies, DMSO is used extensively as a solvent for receptor ligands such as rosiglitazone for peroxisome proliferator-activated receptor gamma [9]. However, limited data is available on the effect of DMSO on the osteoblastic differentiation. In this study, we investigated the effect of DMSO on the proliferation and differentiation of MC3T3-E1 cells, a non-transformed murine cell line derived from neonatal mouse calvariae. Treatment with BMPs, such as BMP2, induces MC3T3-E1 cells to undergo osteoblastic differentiation, which leads to the formation of bone nodules in vitro [10]. We demonstrated that DMSO effectively induces osteoblastic differentiation of MC3T3-E1 cells via the activation of Runx2 and Osx and is dependent upon the protein kinase C (PKC) pathway.
2. Materials and methods 2.1. Chemicals All chemicals were purchased from Sigma–Aldrich Co. (St. Louis, MA, USA) unless otherwise stated. 2.2. Cell culture MC3T3-E1 cells (clone 4; obtained from American Type Culture Collection, Rockville, MD, USA) were maintained in complete modified EagleÕs medium (MEM) alpha medium (aMEM; Invitrogen, San Diego, CA, USA) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS, USA) in a humidified 5% CO2 atmosphere at 37 C. Cells were passaged before reaching confluence. 2.3. Cell differentiation During cell differentiation studies, MC3T3-E1 cells were plated at 5 · 104 cells per cm2, cultivated to complete confluence and then treated with increasing concentrations of DMSO (0.02–1%). For bone nodules formation, MC3T3-E1 cells were cultured in osteogenic medium containing 400 lM ascorbic acid and 5 mM b-glycerophosphate to provide inorganic phosphate. PKC activator, phorbol 12-myristate 13-acetate (PMA) and inhibitor, GF 109203X (GFx), both obtained from Calbiochem-Novabiochem (San Diego, CA, USA) were employed to modulate the activity of PKC in MC3T3-E1 cells. Cells were normally pre-incubated with PMA (1 lM) or GFx (2 lM) for 1 h and then differentiated with DMSO for 6 days in phosphorylation and 15 days in bone nodules formation studies. As DMSO was used as solvent for PMA and GFx, equal volume of DMSO that contributed to less
0014-5793/$32.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.11.062
122 than 0.05% (v/v) was included as solvent control. Fresh culture media containing PKC modulators and DMSO were replenished every 2 days. Cell differentiation experiments were repeated for three times and data from representative experiments are shown. 2.4. Cell proliferation assays Cell proliferation studies were performed as previously described using MTT (GE Healthcare, Piscataway, NJ, USA) assays [11]. After treatment with DMSO (0.02–1%) for 2 days, MTT was added at a final concentration of 0.5 mg/ml for 3 h, cells were solubilized with isopropanol containing 0.1% sodium dodecyl sulfate and 0.04 N hydrochloric acid, and the absorbance measured at 570 nm. All experiments were repeated for three times. 2.5. Total protein extraction, Western blot analysis and antibodies MC3T3-E1 cells were washed with PBS and then lysed with lysis buffer containing 50 mM Trizma base (Tris)–Cl at pH 8, 150 mM sodium chloride, 1% Triton-X100 (TX), 1· aprotinin, 1 mM PMSF, 1 mM sodium orthovanadate, and 1· Complete protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany) at 4 C for 15 min. Western blot analysis was performed as previously described [12] using polyclonal antibodies against phosphorylated p44/ p42 MAP kinase (MAPK) (p-Erk1/2), phosphorylated pan-PKC, phosphorylated PKCh (p-PKCh), phosphorylated PKD/PKCl (p-PKD/PKCl), as well as monoclonal antibodies against Erk2 and different isotypes of PKC that were obtained from Cell Signaling Technology (Beverly, MA, USA) and Transduction Laboratories (Lexington, KY, USA), respectively. Data presented are representative of independent experiments repeated twice with proteins extracted from duplicate treatment. 2.6. Total RNA extraction and gene expression analysis by real time quantitative PCR Transcript expression of Runx2, Osx, and alkaline phosphatase (ALP) was determined using reverse transcription (RT) followed by real-time TaqMan quantitative PCR (qPCR) analysis performed in a ABI 7700 sequence detector (Applied Biosystems Inc., Foster City, CA, USA). Total RNA was isolated using TRI Reagent (Molecular Research Center Inc., Cincinnati, OH, USA). First-strand cDNA was synthesized using random hexamers and M-MLV reverse transcriptase (Promega Co., Madison, MI, USA). TAMRA labeled probes and PCR reagents were obtained from ABI. PCRs were performed in triplicate and data obtained were analyzed using the comparative CT method (ABI) with signals normalized to 18S signal for each sample. All experiments were repeated for 3 times.
W.M.W. Cheung et al. / FEBS Letters 580 (2006) 121–126 2.9. Statistical analysis Statistical analyses were performed using Two-way ANOVA. Bonferroni analyses were used for post hoc examination of the ANOVA results (P < 0.0001). All statistical analyses were performed using the Statview for Macintosh software package (SAS Institute Inc., NC, USA, version 5).
3. Result 3.1. Effect of DMSO on cellular proliferation of MC3T3-E1 cells No significant difference was observed in cell proliferation in cells incubated with DMSO at 0.5% (v/v) or below (Fig. 1). However, 1% DMSO significantly reduced the proliferation of MC3T3-E1 to 93.3 ± 2.0% (Fig. 1; n = 5, *P < 0.001). While osteogenic medium alone increased proliferation to 133.7 ± 4.2%, 1% DMSO significantly suppressed the enhancement to 123.7 ± 3.6% when compared to control. Viable cell count confirmed that osteogenic medium increased the cell number of MC3T3-E1 cells and DMSO significantly reduced the cell number in both normal and osteogenic media (data not shown). 3.2. DMSO-induced formation of bone nodules via increasing number of ALP expressing cells At high concentrations of DMSO (0.5% and 1%), there were noticeable morphological changes and formation of mineralized bone-like nodules (Fig. 2A). The mineralization phenotype was also assessed and confirmed by alizarin red S staining (Fig. 2B). To determine whether DMSO-induced osteoblastic differentiation or only accelerated the rate of mineralization, samples were assayed for ALP activity. MC3T3-E1 cells cultured with DMSO resulted in a significant and dosedependent increase in ALP activity (Fig. 3A). Maximal ALP activity was observed with 0.5% DMSO, which reached a plateau with 1% DMSO (Fig. 3A). ALP staining confirmed DMSO increased ALP-positive cells upon and was comparable to that of control treatment with 50 ng/ml BMP2 (Fig. 3B).
2.7. Assessment of osteoblastic cell differentiation Cells were incubated without or with DMSO (0.02–1%) in normal or osteogenic culture media for selective time periods. ALP activity was measured as p-nitrophenol produced from the hydrolysis of p-nitrophenyl phosphate (PNPP) [13]. In brief, cells were washed with ice-cold PBS, lysed with 50 mM Tris, pH 7.4, and 0.1% TX, and incubated with 2-amino-2-methyl-1-propanol buffer (0.5 M, pH 10.4) containing 10 mM PNPP at 37 C for 15–30 min. The reaction was stopped by the addition of NaOH (final concentration, 0.5 M) and the absorbance measured at 410 nm. Enzyme activity was normalized to the protein content determined using Protein Assay Kit (Bio-Rad). Experiments were performed in triplicate wells and data shown are representative of two independent experiments. 2.8. Histochemical staining Cells were washed twice with ice-cold PBS and then fixed in icecold fixative (10% formalin in PBS) for 15 min. After washed with deionized water, fixed cells were stained with Fast Blue RR Salt and Napthol AS-BI Phosphate for 30 min, and the reaction stopped by washing the cells with surplus deionized water. Mineralized matrix was detected using Von Kossa staining by treating fixed cells with 5% silver nitrate for 30 min, followed by subsequent washes with 5% sodium carbonate in 10% formalin for 1 min and 5% sodium thiosulfate for 5 min. The reaction was stopped by washing the cells thrice with deionized water. Alternatively, fixed cells were stained with 1% alizarin S solution, pH 4.2 for 15 min and then were washed thoroughly with deionized water.
Fig. 1. Effect of DMSO on the proliferation of MC3T3-E1 cells. MC3T3-E1 cells were cultured for 2 days in normal (circle) or osteogenic medium (box) with increasing concentrations of DMSO. Cell proliferation was assessed by MTT reduction. All readings were calculated by comparing with the untreated control cells, which was set as 100%. *P < 0.01 compared with no-DMSO treatment group, unpaired StudentÕs t test. Results are the means ± S.E.M. of 2 independent experiments (n = 5).
W.M.W. Cheung et al. / FEBS Letters 580 (2006) 121–126
123
Fig. 2. DMSO-induced bone nodules formation in MC3T3-E1 cells. (A) Von Kossa staining was performed to visualize mineralization of MC3T3-E1 cells cultured for 15 days in normal (Control) or osteogenic medium (Osteog.) with increasing concentrations of DMSO as indicated. (B) Alizarin Red staining of mineralized nodules in MC3T3-E1 cells treated with 1% (v/v) DMSO. Bar represents 62.5 lm.
3.3. DMSO upregulated the expression of osteoblast-specific transcription factors, Runx2 and Osx Treatment of MC3T3-E1 cells with 1% DMSO increased Runx2 expression significantly by 1.89-fold and Osx expression by 1.39-fold, respectively, (Fig. 4; n = 6, *P < 0.0005). Thus, DMSO-induced osteoblastic differentiation and bone formation through modulation of these 2 bone specific transcription factors that are determinants of bone formation. Low concentration of BMP2 (50 ng/ml) increased ALP expression and upregulated Osx transcription but not mRNA of Runx2. 3.4. Involvement of Raf/MEK/extracellular signal-regulated kinase (Erk) in the DMSO-induced osteoblastic differentiation Erk (also known as p44/p42 MAPK) plays differential roles in proliferation and differentiation and its phosphorylation regulates the transcription of Runx2 [14,15]. Thus, DMSO
Fig. 4. DMSO induced the transcript expression of Runx2 and Osx in MC3T3-E1 cells. MC3T3-E1 cells were cultured for 5 days in normal medium and either left untreated (CTL), or treated with BMP2 (50 ng/ ml; BMP2-50) or 1% DMSO. Results from qPCR analysis of Runx2 (lower panel) and Osx (upper panel) are presented as means ± S.E.M. of 2 independent experiments (each in triplicate). *P < 0.0005 and **P < 0.05, unpaired t test, compared with control (n = 6).
might mediate its biological actions via the activation of Erk. When MC3T3-E1 cells were just confluent, Erk1 and Erk2 were activated and their expression diminished after culturing for 6 days in normal medium (p-Erk1/2; Fig. 5A). However, in the presence of 1% DMSO, higher level of p-Erk1/2 expression was observed, suggesting that DMSO-induced osteoblastic differentiation is associated with the sustained activation of Erk in MC3T3-E1 cells.
Fig. 3. DMSO-induced ALP expression in MC3T3-E1 cells. MC3T3-E1 cells were cultured for 8 days in normal medium (circle) or osteogenic medium (box) with increasing concentrations of DMSO as indicated. (A) ALP activity was determined and normalized to protein content. Data represent the means ± S.E.M. of a representative independent experiment out of 3 (*P < 0.05 compared with no-DMSO control by unpaired StudentÕs t test, n = 4). (B) ALP staining revealed expression of ALP in MC3T3-E1 cells cultured for 8 days either untreated, treated with 1% DMSO or BMP2 (50 ng/ml). Bar represents 62.5 lm.
124
W.M.W. Cheung et al. / FEBS Letters 580 (2006) 121–126
Fig. 5. DMSO activates the phosphorylation of Erks and PKCs in MC3T3-E1 cells. MC3T3-E1 cells were cultured for 6 days in normal medium and either left untreated or with 1% DMSO. Total proteins collected on day 0 is denoted as ‘‘-’’. Activation of (A) Erk1/2; (B) PKCa and -d; (C) PKCh was detected by phospho-specific and respective non-phosphorylation state-specific antibodies as indicated; (D) PKD/PKCl activation was examined in MC3T3-E1 cells cultured for 5 days in normal medium ( ) or with 1% DMSO (+) and with either control solvent (Sol), GFx (2 lM) or PMA (1 lM) as indicated. Expression of b-actin was used as internal control as indicated.
3.5. Requirement of PKC for DMSO-induced osteoblastic differentiation of MC3T3-E1 cells PKC comprises a family of serine–threonine kinases that play pivotal roles in the regulation of cell proliferation and differentiation [16]. Using DMSO-induced osteoblastic differentiation as model system, we evaluated the role of PKC pathways in mediating osteoblastic differentiation. Both PKCa and -d were activated in confluent MC3T3-E1 cells, but their expression was not altered by DMSO treatment (Fig. 5B). In contrast, PKCh was activated by DMSO but not by cell confluence (p-PKCh; Fig. 5C). There was also a slight increase in p-PKCh when MC3T3-E1 cells were cultured in normal medium for 6 days. The activation paralleled the expression of ALP and bone nodules formation. Total PKCa, -d, and -h expression was not affected. Prolonged DMSO treatment also upregulated the expression of p-PKD (also known as phosphoPKCl) (Fig. 5D). However, unlike PKCh, the expression of total PKD/PKCl was also upregulated (Fig. 5D, middle panel). PKD is activated via PKC-dependent pathways [17]. Thus, we further investigated the involvement of PKD in DMSO-induced osteoblastic differentiation by modulating the PKC activities. As expected, the expression of p-PKD and total PKD was markedly attenuated by PKC inhibitor GFx but augmented by PKC activator PMA (Fig. 5D). Next, we examined the prolonged effect of PKD inhibition on the bone nodules formation. DMSO-induced formation of bone nodules was completely blocked by either exposing MC3T3-E1 cells to GFx for 5 days or throughout the differentiation period (15 days) (Fig. 6A). In contrast, PMA enhanced the bone forming effect of DMSO (Fig. 6A). PMA alone could also induce bone nodules formation though less effective than combining with DMSO. It is noteworthy that formation of bone nodules could partially be reversed upon GFx washout at day 5 when there was a continuous supplementation of DMSO. This supports the hypothesis that DMSO-induced osteoblastic differentiation via the activation of PKD. Our findings were confirmed by measuring the ALP activity. DMSO alone increased ALP activity by 1.5-fold (Fig. 6B). GFx suppressed the induced ALP activity to basal expression level, whereas PMA augmented ALP activity to 2.9-fold that of the control cells. PMA alone upregulated the ALP activity by 1.8-fold. Taken together, our results suggest the involvement of PKD in mediating the DMSO-induced osteoblastic differentiation.
Fig. 6. Effect of PKC modulation on osteoblastic differentiation of MC3T3-E1 cells. MC3T3-E1 cells were cultured for 15 days in osteogenic medium with 1% DMSO 5 or 15 days. GFx (2 lM; GFx-2) or PMA (1 lM; PMA-1) were supplemented as indicated. (A) Alizarin Red staining of bone nodules and (B) ALP activity was determined. Mean ALP activity obtained using control solvent (0.05% DMSO) was designated as 1. *P < 0.01 compared to control, by Bonferroni post hoc test when ANOVA (P < 0.0001); **P < 0.01 compared to DMSO treatment; #P < 0.01 compared to PMA alone.
4. Discussion This report shows that DMSO significantly inhibited cell proliferation of MC3T3-E1 cells at a concentration of 1% (v/ v) and DMSO-induced osteoblastic differentiation of MC3T3-E1 cells. The osteoblastogenic effect of DMSO was
W.M.W. Cheung et al. / FEBS Letters 580 (2006) 121–126
mediated via Runx2 and Osx and was dependent upon the activation of PKD via the PKC pathways. The preosteoblast MC3T3-E1 cell line is an excellent cell differentiation model that simulates the events of early osteoblastogenesis. Differentiation of MC3T3-E1 cells to osteoblasts by exposure to BMP2 was shown to be associated with induction of the bone-specific transcription factors such as Runx2 and Osx [18,19]. Runx2 and Osx are hypothesized to act on different stages of osteoblastic differentiation, i.e., commitment and terminal differentiation, respectively [19]. In this work, treatment with DMSO resulted in a significant induction of both Runx2 and Osx in MC3T3-E1 cells, suggesting that DMSO is capable of inducing the differentiation of preosteoblast cells into mature osteoblasts. We hypothesize the use of DMSO-induced osteoblastic differentiation of MC3T3-E1 cells as an in vitro model system for the study of mechanisms controlling osteoblastic differentiation. Activity of Erks regulates the transcription of Runx2 during the extracellular matrices induced osteoblastic differentiation of MC3T3-E1 cells [20]. In line with previous findings, we detected a noticeable increase in the expression of activated Erks. The functional roles of Erk in cellular differentiation have been paradoxical. Dependent upon the cellular context, the Erk pathway suppresses cellular differentiation such as adipogenesis [21] while induces differentiation of pheochromocytoma PC12 cells [22]. Likewise, in osteoblastic differentiation, activation of MAPKs enhances the expression of ALP [23]. Using DMSO-induced osteoblastic differentiation of preosteoblast cells as model system, we have also examined the role of PKD in osteoblastic differentiation. Functional roles of PKC pathways in regulating the transcriptional activity and posttranslational modification of Runx2 have previously been demonstrated [24]. However, little is known on the phenotypic changes associated with prolonged PKC activation. This report provides the first evidence that prolonged activation of PKC pathways alone is sufficient to induce osteoblastic differentiation, possibly via the activation of PKD/PKCl. PKD/PKCl is a serine protein kinase that responds to PMA treatment [25]. Unlike other PKC isotypes, PKD lacks C2 domain that plays essential roles in the Ca2+ sensitivity [26]. Activation of PKD in turn requires the upstream PKCh signaling. Treatment of MC3T3-E1 cells with GFx, which is capable of inhibiting PKC but not PKD activities [9], abolished the DMSO-mediated expression and activation of PKD (Fig. 5D; lanes 3 and 4). Concomitantly, GFx inhibited DMSO-induced ALP expression and bone nodules formation in MC3T3-E1 cells. As it has previously been suggested that PKCh is the upstream kinase of PKD/ PKCl [27], GFx might inhibit PKCh, thereby indirectly inhibited the phosphorylation of PKD. Consistent with previous findings that inhibition of PKD markedly reduced the BMP2induced expression of ALP and osteocalcin [28], GFx inhibits DMSO-induced bone nodules formation. Our studies further demonstrate that the activation of PKD enhances both ALP expression and bone nodules formation via PKC activation. In conclusion, we have demonstrated that DMSO is a powerful inducer of osteoblastic differentiation in MC3T3-E1 cells. Our results also show that DMSO-induced osteoblastic differentiation of MC3T3-E1 cells represents a model system to study the functional roles of biochemical pathways in inducing or regulating osteoblastic differentiation and provides clues to alternative therapeutic approaches by providing additional pathways to bone formation.
125 Acknowledgements: We are thankful to Lilian Jin, Patrick Chu and Hardy Chan for their technical assistance. We thank Dr. Susan Yung for critical review of the manuscript. This study was supported by the Seed Funding Programme for Basic Research, HKU.
References [1] Banerjee, C., Javed, A., Choi, J.Y., Green, J., Rosen, V., van Wijnen, A.J., Stein, J.L., Lian, J.B. and Stein, G.S. (2001) Differential regulation of the two principal Runx2/Cbfa1 n-terminal isoforms in response to bone morphogenetic protein-2 during development of the osteoblast phenotype. Endocrinology 142, 4026–4039. [2] Nakashima, K., Zhou, X., Kunkel, G., Zhang, Z., Deng, J.M., Behringer, R.R. and de Crombrugghe, B. (2002) The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17–29. [3] Santos, N.C., Figueira-Coelho, J., Martins-Silva, J. and Saldanha, C. (2003) Multidisciplinary utilization of dimethyl sulfoxide: pharmacological, cellular, and molecular aspects. Biochem. Pharmacol. 65, 1035–1041. [4] Melchior, D., Packer, C.S., Johnson, T.C. and Kaefer, M. (2003) Dimethyl sulfoxide: does it change the functional properties of the bladder wall? J. Urol. 170, 253–258. [5] Xing, L. and Remick, D.G. (2005) Mechanisms of dimethyl sulfoxide augmentation of IL-1 beta production. J. Immunol. 174, 6195–6202. [6] Bolduc, L., Labrecque, B., Cordeau, M., Blanchette, M. and Chabot, B. (2001) Dimethyl sulfoxide affects the selection of splice sites. J. Biol. Chem. 276, 17597–17602. [7] Lee, Y.R., Shim, H.J., Yu, H.N., Song, E.K., Park, J., Kwon, K.B., Park, J.W., Rho, H.W., Park, B.H., Han, M.K. and Kim, J.S. (2005) Dimethylsulfoxide induces upregulation of tumor suppressor protein PTEN through nuclear factor-kappaB activation in HL-60 cells. Leuk. Res. 29, 401–405. [8] Zucchi, I., Bini, L., Albani, D., Valaperta, R., Liberatori, S., Raggiaschi, R., Montagna, C., Susani, L., Barbieri, O., Pallini, V., Vezzoni, P. and Dulbecco, R. (2002) Dome formation in cell cultures as expression of an early stage of lactogenic differentiation of the mammary gland. Proc. Natl. Acad. Sci. USA 99, 8660–8665. [9] Lecka-Czernik, B., Moerman, E.J., Grant, D.F., Lehmann, J.M., Manolagas, S.C. and Jilka, R.L. (2002) Divergent effects of selective peroxisome proliferator-activated receptor-gamma 2 ligands on adipocyte versus osteoblast differentiation. Endocrinology 143, 2376–2384. [10] Zhu, W., Rawlins, B.A., Boachie-Adjei, O., Myers, E.R., Arimizu, J., Choi, E., Lieberman, J.R., Crystal, R.G. and Hidaka, C. (2004) Combined bone morphogenetic protein-2 and -7 gene transfer enhances osteoblastic differentiation and spine fusion in a rodent model. J. Bone Miner. Res. 19, 2021–2032. [11] Cheung, W.M.W., Hui, W.S., Chu, P.W.K., Chiu, S.W. and Ip, N.Y. (2000) Ganoderma extract activates MAP kinases and induces the neuronal differentiation of rat pheochromocytoma PC12 cells. FEBS Lett. 486, 291–296. [12] Cheung, W.M.W., Chu, A.H., Chu, P.W.K. and Ip, N.Y. (2001) Cloning and expression of a novel nuclear matrix-associated protein that is regulated during the retinoic acid-induced neuronal differentiation. J. Biol. Chem. 276, 17083–17091. [13] Cheng, S.L., Zhang, S.F., Nelson, T.L., Warlow, P.M. and Civitelli, R. (1994) Stimulation of human osteoblast differentiation and function by ipriflavone and its metabolites. Calcif. Tissue Int. 55, 356–362. [14] Gatti, A. (2003) Divergence in the upstream signaling of nerve growth factor (NGF) and epidermal growth factor (EGF). Neuroreport 14, 1031–1035. [15] Xiao, G., Jiang, D., Gopalakrishnan, R. and Franceschi, R.T. (2002) Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, Cbfa1/Runx2. J. Biol. Chem. 277, 36181–36187. [16] Newton, A.C. (1995) Protein kinase C: structure, function, and regulation. J. Biol. Chem. 270, 28495–28498.
126 [17] Zugaza, J.L., Sinnett-Smith, J., Van Lint, J. and Rozengurt, E. (1996) Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway. EMBO J. 15, 6220–6230. [18] Spinella-Jaegle, S., Roman-Roman, S., Faucheu, C., Dunn, F.W., Kawai, S., Gallea, S., Stiot, V., Blanchet, A.M., Courtois, B., Baron, R. and Rawadi, G. (2001) Opposite effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on osteoblast differentiation. Bone 29, 323–330. [19] Lee, M.H., Kwon, T.G., Park, H.S., Wozney, J.M. and Ryoo, H.M. (2003) BMP-2-induced osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem. Biophys. Res. Commun. 309, 689–694. [20] Xiao, G., Gopalakrishnan, R., Jiang, D., Reith, E., Benson, M.D. and Franceschi, R.T. (2002) Bone morphogenetic proteins, extracellular matrix, and mitogen-activated protein kinase signaling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells. J. Bone Miner. Res. 17, 101–110. [21] Hu, E., Kim, J.B., Sarraf, P. and Spiegelman, B.M. (1996) Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPARc. Science 274, 2100–2103. [22] Cowley, S., Paterson, H., Kemp, P. and Marshall, C.J. (1994) Activation of MAP kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77, 841–852.
W.M.W. Cheung et al. / FEBS Letters 580 (2006) 121–126 [23] Takeuchi, Y., Suzawa, M., Kikuchi, T., Nishida, E., Fujita, T. and Matsumoto, T. (1997) Differentiation and transforming growth factor-beta receptor down-regulation by collagen-alpha2beta1 integrin interaction is mediated by focal adhesion kinase and its downstream signals in murine osteoblastic cells. J. Biol. Chem. 272, 29309–29316. [24] Kim, H.J., Kim, J.H., Bae, S.C., Choi, J.Y., Kim, H.J. and Ryoo, H.M. (2003) The protein kinase C pathway plays a central role in the fibroblast growth factor-stimulated expression and transactivation activity of Runx2. J. Biol. Chem. 278, 319–326. [25] Van Lint, J.V., Sinnett-Smith, J. and Rozengurt, E. (1995) Expression and characterization of PKD, a phorbol ester and diacylglycerol-stimulated serine protein kinase. J. Biol. Chem. 270, 1455–1461. [26] Johannes, F.J., Prestle, J., Eis, S., Oberhagemann, P. and Pfizenmaier, K. (1994) PKCl is a novel, atypical member of the protein kinase C family. J. Biol. Chem. 269, 6140–6148. [27] Yuan, J., Bae, D., Cantrell, D., Nel, A.E. and Rozengurt, E. (2002) Protein kinase D is a downstream target of protein kinase Ctheta. Biochem. Biophys. Res. Commun. 291, 444– 452. [28] Lemonnier, J., Ghayor, C., Guicheux, J. and Caverzasio, J. (2004) Protein kinase C-independent activation of protein kinase D is involved in BMP-2-induced activation of stress mitogen-activated protein kinases JNK and p38 and osteoblastic cell differentiation. J. Biol. Chem. 279, 259–264.
Dimethyl sulfoxide as an inducer of differentiation in preosteoblast MC3T3-E1 cells W.M.W. Cheung*,1, W.W. Ng, A.W.C. Kung* Department of Medicine, University of Hong Kong, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China Received 7 September 2005; revised 9 November 2005; accepted 14 November 2005 Available online 6 December 2005 Edited by Veli-Pekka Lehto
Abstract Osteoblastic differentiation is an essential part of bone formation. Dimethyl sulfoxide (DMSO) is a water miscible solvent that is used extensively for receptor ligands in osteoblast studies. However, little is known about its effects on osteoblastogenic precursor cells. In this study, we have used a murine preosteoblast cell line MC3T3-E1 cells to demonstrate that DMSO effectively induces osteoblastic differentiation of MC3T3-E1 cells via the activation of Runx2 and osterix and is dependent upon the protein kinase C (PKC) pathways. We further demonstrated that prolonged activation of PKC pathways is sufficient to induce osteoblastic differentiation, possibly via the activation of PKD/PKCl. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: DMSO; Protein kinase C; PKD; Erk; Proliferation; Osteoblasts
1. Introduction In normal bone remodeling or bone turnover, osteoblastic bone formation and osteoblastic bone resorption is coupled in a precise and orchestrated manner. Osteoblastic differentiation is an essential part of bone formation. Several groups of proteins are necessary for osteoblastic differentiation, such as bone morphogenetic proteins (BMPs) and transcription factors such as Runx2 and osterix (Osx) [1,2]. Dimethyl sulfoxide (DMSO; (CH3)2SO) is a water miscible solvent that serves as powerful solvents that dissolve most water-insoluble drugs [3]. DMSO possesses anti-inflammatory properties [4], as well as the ability to act as a free radical scavenger [5]. Thus, its properties have been exploited in the treatment of dermatological, rheumatic, and renal manifestations of amyloidosis. Depending on the cellular context and proba*
Corresponding authors. Address: Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China. Fax: +852 2816 2187 (A.W.C. Kung). E-mail addresses: [email protected] (W.M.W. Cheung), [email protected] (A.W.C. Kung). 1
Fax: +852 2819 1127.
Abbreviations: DMSO, dimethyl sulfoxide; PKC, protein kinase C; BMP, bone morphogenetic protein; Osx, osterix; GFx, GF 109203X; PMA, phorbol 12-myristate 13-acetate; Tris, Trizma base; TX, TritonX100; ALP, alkaline phosphatase; PNPP, p-nitrophenyl phosphate; MAPKs, MAP kinases
bly via the alteration of splice site selection, DMSO is capable of inducing or inhibiting cell proliferation, apoptosis and/or differentiation [6]. DMSO induces the differentiation of many cell types including leukemia cells and mammary adenocarcinoma LA7 cells [7,8]. DMSO induces differentiation of LA7 cells into mammary cells with milk production, which originally requires the supplementation of lactogenic hormones. Therefore, it is recently developed as an in vitro model system for studying mammary cells differentiation [8]. In osteoblast studies, DMSO is used extensively as a solvent for receptor ligands such as rosiglitazone for peroxisome proliferator-activated receptor gamma [9]. However, limited data is available on the effect of DMSO on the osteoblastic differentiation. In this study, we investigated the effect of DMSO on the proliferation and differentiation of MC3T3-E1 cells, a non-transformed murine cell line derived from neonatal mouse calvariae. Treatment with BMPs, such as BMP2, induces MC3T3-E1 cells to undergo osteoblastic differentiation, which leads to the formation of bone nodules in vitro [10]. We demonstrated that DMSO effectively induces osteoblastic differentiation of MC3T3-E1 cells via the activation of Runx2 and Osx and is dependent upon the protein kinase C (PKC) pathway.
2. Materials and methods 2.1. Chemicals All chemicals were purchased from Sigma–Aldrich Co. (St. Louis, MA, USA) unless otherwise stated. 2.2. Cell culture MC3T3-E1 cells (clone 4; obtained from American Type Culture Collection, Rockville, MD, USA) were maintained in complete modified EagleÕs medium (MEM) alpha medium (aMEM; Invitrogen, San Diego, CA, USA) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS, USA) in a humidified 5% CO2 atmosphere at 37 C. Cells were passaged before reaching confluence. 2.3. Cell differentiation During cell differentiation studies, MC3T3-E1 cells were plated at 5 · 104 cells per cm2, cultivated to complete confluence and then treated with increasing concentrations of DMSO (0.02–1%). For bone nodules formation, MC3T3-E1 cells were cultured in osteogenic medium containing 400 lM ascorbic acid and 5 mM b-glycerophosphate to provide inorganic phosphate. PKC activator, phorbol 12-myristate 13-acetate (PMA) and inhibitor, GF 109203X (GFx), both obtained from Calbiochem-Novabiochem (San Diego, CA, USA) were employed to modulate the activity of PKC in MC3T3-E1 cells. Cells were normally pre-incubated with PMA (1 lM) or GFx (2 lM) for 1 h and then differentiated with DMSO for 6 days in phosphorylation and 15 days in bone nodules formation studies. As DMSO was used as solvent for PMA and GFx, equal volume of DMSO that contributed to less
0014-5793/$32.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.11.062
122 than 0.05% (v/v) was included as solvent control. Fresh culture media containing PKC modulators and DMSO were replenished every 2 days. Cell differentiation experiments were repeated for three times and data from representative experiments are shown. 2.4. Cell proliferation assays Cell proliferation studies were performed as previously described using MTT (GE Healthcare, Piscataway, NJ, USA) assays [11]. After treatment with DMSO (0.02–1%) for 2 days, MTT was added at a final concentration of 0.5 mg/ml for 3 h, cells were solubilized with isopropanol containing 0.1% sodium dodecyl sulfate and 0.04 N hydrochloric acid, and the absorbance measured at 570 nm. All experiments were repeated for three times. 2.5. Total protein extraction, Western blot analysis and antibodies MC3T3-E1 cells were washed with PBS and then lysed with lysis buffer containing 50 mM Trizma base (Tris)–Cl at pH 8, 150 mM sodium chloride, 1% Triton-X100 (TX), 1· aprotinin, 1 mM PMSF, 1 mM sodium orthovanadate, and 1· Complete protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany) at 4 C for 15 min. Western blot analysis was performed as previously described [12] using polyclonal antibodies against phosphorylated p44/ p42 MAP kinase (MAPK) (p-Erk1/2), phosphorylated pan-PKC, phosphorylated PKCh (p-PKCh), phosphorylated PKD/PKCl (p-PKD/PKCl), as well as monoclonal antibodies against Erk2 and different isotypes of PKC that were obtained from Cell Signaling Technology (Beverly, MA, USA) and Transduction Laboratories (Lexington, KY, USA), respectively. Data presented are representative of independent experiments repeated twice with proteins extracted from duplicate treatment. 2.6. Total RNA extraction and gene expression analysis by real time quantitative PCR Transcript expression of Runx2, Osx, and alkaline phosphatase (ALP) was determined using reverse transcription (RT) followed by real-time TaqMan quantitative PCR (qPCR) analysis performed in a ABI 7700 sequence detector (Applied Biosystems Inc., Foster City, CA, USA). Total RNA was isolated using TRI Reagent (Molecular Research Center Inc., Cincinnati, OH, USA). First-strand cDNA was synthesized using random hexamers and M-MLV reverse transcriptase (Promega Co., Madison, MI, USA). TAMRA labeled probes and PCR reagents were obtained from ABI. PCRs were performed in triplicate and data obtained were analyzed using the comparative CT method (ABI) with signals normalized to 18S signal for each sample. All experiments were repeated for 3 times.
W.M.W. Cheung et al. / FEBS Letters 580 (2006) 121–126 2.9. Statistical analysis Statistical analyses were performed using Two-way ANOVA. Bonferroni analyses were used for post hoc examination of the ANOVA results (P < 0.0001). All statistical analyses were performed using the Statview for Macintosh software package (SAS Institute Inc., NC, USA, version 5).
3. Result 3.1. Effect of DMSO on cellular proliferation of MC3T3-E1 cells No significant difference was observed in cell proliferation in cells incubated with DMSO at 0.5% (v/v) or below (Fig. 1). However, 1% DMSO significantly reduced the proliferation of MC3T3-E1 to 93.3 ± 2.0% (Fig. 1; n = 5, *P < 0.001). While osteogenic medium alone increased proliferation to 133.7 ± 4.2%, 1% DMSO significantly suppressed the enhancement to 123.7 ± 3.6% when compared to control. Viable cell count confirmed that osteogenic medium increased the cell number of MC3T3-E1 cells and DMSO significantly reduced the cell number in both normal and osteogenic media (data not shown). 3.2. DMSO-induced formation of bone nodules via increasing number of ALP expressing cells At high concentrations of DMSO (0.5% and 1%), there were noticeable morphological changes and formation of mineralized bone-like nodules (Fig. 2A). The mineralization phenotype was also assessed and confirmed by alizarin red S staining (Fig. 2B). To determine whether DMSO-induced osteoblastic differentiation or only accelerated the rate of mineralization, samples were assayed for ALP activity. MC3T3-E1 cells cultured with DMSO resulted in a significant and dosedependent increase in ALP activity (Fig. 3A). Maximal ALP activity was observed with 0.5% DMSO, which reached a plateau with 1% DMSO (Fig. 3A). ALP staining confirmed DMSO increased ALP-positive cells upon and was comparable to that of control treatment with 50 ng/ml BMP2 (Fig. 3B).
2.7. Assessment of osteoblastic cell differentiation Cells were incubated without or with DMSO (0.02–1%) in normal or osteogenic culture media for selective time periods. ALP activity was measured as p-nitrophenol produced from the hydrolysis of p-nitrophenyl phosphate (PNPP) [13]. In brief, cells were washed with ice-cold PBS, lysed with 50 mM Tris, pH 7.4, and 0.1% TX, and incubated with 2-amino-2-methyl-1-propanol buffer (0.5 M, pH 10.4) containing 10 mM PNPP at 37 C for 15–30 min. The reaction was stopped by the addition of NaOH (final concentration, 0.5 M) and the absorbance measured at 410 nm. Enzyme activity was normalized to the protein content determined using Protein Assay Kit (Bio-Rad). Experiments were performed in triplicate wells and data shown are representative of two independent experiments. 2.8. Histochemical staining Cells were washed twice with ice-cold PBS and then fixed in icecold fixative (10% formalin in PBS) for 15 min. After washed with deionized water, fixed cells were stained with Fast Blue RR Salt and Napthol AS-BI Phosphate for 30 min, and the reaction stopped by washing the cells with surplus deionized water. Mineralized matrix was detected using Von Kossa staining by treating fixed cells with 5% silver nitrate for 30 min, followed by subsequent washes with 5% sodium carbonate in 10% formalin for 1 min and 5% sodium thiosulfate for 5 min. The reaction was stopped by washing the cells thrice with deionized water. Alternatively, fixed cells were stained with 1% alizarin S solution, pH 4.2 for 15 min and then were washed thoroughly with deionized water.
Fig. 1. Effect of DMSO on the proliferation of MC3T3-E1 cells. MC3T3-E1 cells were cultured for 2 days in normal (circle) or osteogenic medium (box) with increasing concentrations of DMSO. Cell proliferation was assessed by MTT reduction. All readings were calculated by comparing with the untreated control cells, which was set as 100%. *P < 0.01 compared with no-DMSO treatment group, unpaired StudentÕs t test. Results are the means ± S.E.M. of 2 independent experiments (n = 5).
W.M.W. Cheung et al. / FEBS Letters 580 (2006) 121–126
123
Fig. 2. DMSO-induced bone nodules formation in MC3T3-E1 cells. (A) Von Kossa staining was performed to visualize mineralization of MC3T3-E1 cells cultured for 15 days in normal (Control) or osteogenic medium (Osteog.) with increasing concentrations of DMSO as indicated. (B) Alizarin Red staining of mineralized nodules in MC3T3-E1 cells treated with 1% (v/v) DMSO. Bar represents 62.5 lm.
3.3. DMSO upregulated the expression of osteoblast-specific transcription factors, Runx2 and Osx Treatment of MC3T3-E1 cells with 1% DMSO increased Runx2 expression significantly by 1.89-fold and Osx expression by 1.39-fold, respectively, (Fig. 4; n = 6, *P < 0.0005). Thus, DMSO-induced osteoblastic differentiation and bone formation through modulation of these 2 bone specific transcription factors that are determinants of bone formation. Low concentration of BMP2 (50 ng/ml) increased ALP expression and upregulated Osx transcription but not mRNA of Runx2. 3.4. Involvement of Raf/MEK/extracellular signal-regulated kinase (Erk) in the DMSO-induced osteoblastic differentiation Erk (also known as p44/p42 MAPK) plays differential roles in proliferation and differentiation and its phosphorylation regulates the transcription of Runx2 [14,15]. Thus, DMSO
Fig. 4. DMSO induced the transcript expression of Runx2 and Osx in MC3T3-E1 cells. MC3T3-E1 cells were cultured for 5 days in normal medium and either left untreated (CTL), or treated with BMP2 (50 ng/ ml; BMP2-50) or 1% DMSO. Results from qPCR analysis of Runx2 (lower panel) and Osx (upper panel) are presented as means ± S.E.M. of 2 independent experiments (each in triplicate). *P < 0.0005 and **P < 0.05, unpaired t test, compared with control (n = 6).
might mediate its biological actions via the activation of Erk. When MC3T3-E1 cells were just confluent, Erk1 and Erk2 were activated and their expression diminished after culturing for 6 days in normal medium (p-Erk1/2; Fig. 5A). However, in the presence of 1% DMSO, higher level of p-Erk1/2 expression was observed, suggesting that DMSO-induced osteoblastic differentiation is associated with the sustained activation of Erk in MC3T3-E1 cells.
Fig. 3. DMSO-induced ALP expression in MC3T3-E1 cells. MC3T3-E1 cells were cultured for 8 days in normal medium (circle) or osteogenic medium (box) with increasing concentrations of DMSO as indicated. (A) ALP activity was determined and normalized to protein content. Data represent the means ± S.E.M. of a representative independent experiment out of 3 (*P < 0.05 compared with no-DMSO control by unpaired StudentÕs t test, n = 4). (B) ALP staining revealed expression of ALP in MC3T3-E1 cells cultured for 8 days either untreated, treated with 1% DMSO or BMP2 (50 ng/ml). Bar represents 62.5 lm.
124
W.M.W. Cheung et al. / FEBS Letters 580 (2006) 121–126
Fig. 5. DMSO activates the phosphorylation of Erks and PKCs in MC3T3-E1 cells. MC3T3-E1 cells were cultured for 6 days in normal medium and either left untreated or with 1% DMSO. Total proteins collected on day 0 is denoted as ‘‘-’’. Activation of (A) Erk1/2; (B) PKCa and -d; (C) PKCh was detected by phospho-specific and respective non-phosphorylation state-specific antibodies as indicated; (D) PKD/PKCl activation was examined in MC3T3-E1 cells cultured for 5 days in normal medium ( ) or with 1% DMSO (+) and with either control solvent (Sol), GFx (2 lM) or PMA (1 lM) as indicated. Expression of b-actin was used as internal control as indicated.
3.5. Requirement of PKC for DMSO-induced osteoblastic differentiation of MC3T3-E1 cells PKC comprises a family of serine–threonine kinases that play pivotal roles in the regulation of cell proliferation and differentiation [16]. Using DMSO-induced osteoblastic differentiation as model system, we evaluated the role of PKC pathways in mediating osteoblastic differentiation. Both PKCa and -d were activated in confluent MC3T3-E1 cells, but their expression was not altered by DMSO treatment (Fig. 5B). In contrast, PKCh was activated by DMSO but not by cell confluence (p-PKCh; Fig. 5C). There was also a slight increase in p-PKCh when MC3T3-E1 cells were cultured in normal medium for 6 days. The activation paralleled the expression of ALP and bone nodules formation. Total PKCa, -d, and -h expression was not affected. Prolonged DMSO treatment also upregulated the expression of p-PKD (also known as phosphoPKCl) (Fig. 5D). However, unlike PKCh, the expression of total PKD/PKCl was also upregulated (Fig. 5D, middle panel). PKD is activated via PKC-dependent pathways [17]. Thus, we further investigated the involvement of PKD in DMSO-induced osteoblastic differentiation by modulating the PKC activities. As expected, the expression of p-PKD and total PKD was markedly attenuated by PKC inhibitor GFx but augmented by PKC activator PMA (Fig. 5D). Next, we examined the prolonged effect of PKD inhibition on the bone nodules formation. DMSO-induced formation of bone nodules was completely blocked by either exposing MC3T3-E1 cells to GFx for 5 days or throughout the differentiation period (15 days) (Fig. 6A). In contrast, PMA enhanced the bone forming effect of DMSO (Fig. 6A). PMA alone could also induce bone nodules formation though less effective than combining with DMSO. It is noteworthy that formation of bone nodules could partially be reversed upon GFx washout at day 5 when there was a continuous supplementation of DMSO. This supports the hypothesis that DMSO-induced osteoblastic differentiation via the activation of PKD. Our findings were confirmed by measuring the ALP activity. DMSO alone increased ALP activity by 1.5-fold (Fig. 6B). GFx suppressed the induced ALP activity to basal expression level, whereas PMA augmented ALP activity to 2.9-fold that of the control cells. PMA alone upregulated the ALP activity by 1.8-fold. Taken together, our results suggest the involvement of PKD in mediating the DMSO-induced osteoblastic differentiation.
Fig. 6. Effect of PKC modulation on osteoblastic differentiation of MC3T3-E1 cells. MC3T3-E1 cells were cultured for 15 days in osteogenic medium with 1% DMSO 5 or 15 days. GFx (2 lM; GFx-2) or PMA (1 lM; PMA-1) were supplemented as indicated. (A) Alizarin Red staining of bone nodules and (B) ALP activity was determined. Mean ALP activity obtained using control solvent (0.05% DMSO) was designated as 1. *P < 0.01 compared to control, by Bonferroni post hoc test when ANOVA (P < 0.0001); **P < 0.01 compared to DMSO treatment; #P < 0.01 compared to PMA alone.
4. Discussion This report shows that DMSO significantly inhibited cell proliferation of MC3T3-E1 cells at a concentration of 1% (v/ v) and DMSO-induced osteoblastic differentiation of MC3T3-E1 cells. The osteoblastogenic effect of DMSO was
W.M.W. Cheung et al. / FEBS Letters 580 (2006) 121–126
mediated via Runx2 and Osx and was dependent upon the activation of PKD via the PKC pathways. The preosteoblast MC3T3-E1 cell line is an excellent cell differentiation model that simulates the events of early osteoblastogenesis. Differentiation of MC3T3-E1 cells to osteoblasts by exposure to BMP2 was shown to be associated with induction of the bone-specific transcription factors such as Runx2 and Osx [18,19]. Runx2 and Osx are hypothesized to act on different stages of osteoblastic differentiation, i.e., commitment and terminal differentiation, respectively [19]. In this work, treatment with DMSO resulted in a significant induction of both Runx2 and Osx in MC3T3-E1 cells, suggesting that DMSO is capable of inducing the differentiation of preosteoblast cells into mature osteoblasts. We hypothesize the use of DMSO-induced osteoblastic differentiation of MC3T3-E1 cells as an in vitro model system for the study of mechanisms controlling osteoblastic differentiation. Activity of Erks regulates the transcription of Runx2 during the extracellular matrices induced osteoblastic differentiation of MC3T3-E1 cells [20]. In line with previous findings, we detected a noticeable increase in the expression of activated Erks. The functional roles of Erk in cellular differentiation have been paradoxical. Dependent upon the cellular context, the Erk pathway suppresses cellular differentiation such as adipogenesis [21] while induces differentiation of pheochromocytoma PC12 cells [22]. Likewise, in osteoblastic differentiation, activation of MAPKs enhances the expression of ALP [23]. Using DMSO-induced osteoblastic differentiation of preosteoblast cells as model system, we have also examined the role of PKD in osteoblastic differentiation. Functional roles of PKC pathways in regulating the transcriptional activity and posttranslational modification of Runx2 have previously been demonstrated [24]. However, little is known on the phenotypic changes associated with prolonged PKC activation. This report provides the first evidence that prolonged activation of PKC pathways alone is sufficient to induce osteoblastic differentiation, possibly via the activation of PKD/PKCl. PKD/PKCl is a serine protein kinase that responds to PMA treatment [25]. Unlike other PKC isotypes, PKD lacks C2 domain that plays essential roles in the Ca2+ sensitivity [26]. Activation of PKD in turn requires the upstream PKCh signaling. Treatment of MC3T3-E1 cells with GFx, which is capable of inhibiting PKC but not PKD activities [9], abolished the DMSO-mediated expression and activation of PKD (Fig. 5D; lanes 3 and 4). Concomitantly, GFx inhibited DMSO-induced ALP expression and bone nodules formation in MC3T3-E1 cells. As it has previously been suggested that PKCh is the upstream kinase of PKD/ PKCl [27], GFx might inhibit PKCh, thereby indirectly inhibited the phosphorylation of PKD. Consistent with previous findings that inhibition of PKD markedly reduced the BMP2induced expression of ALP and osteocalcin [28], GFx inhibits DMSO-induced bone nodules formation. Our studies further demonstrate that the activation of PKD enhances both ALP expression and bone nodules formation via PKC activation. In conclusion, we have demonstrated that DMSO is a powerful inducer of osteoblastic differentiation in MC3T3-E1 cells. Our results also show that DMSO-induced osteoblastic differentiation of MC3T3-E1 cells represents a model system to study the functional roles of biochemical pathways in inducing or regulating osteoblastic differentiation and provides clues to alternative therapeutic approaches by providing additional pathways to bone formation.
125 Acknowledgements: We are thankful to Lilian Jin, Patrick Chu and Hardy Chan for their technical assistance. We thank Dr. Susan Yung for critical review of the manuscript. This study was supported by the Seed Funding Programme for Basic Research, HKU.
References [1] Banerjee, C., Javed, A., Choi, J.Y., Green, J., Rosen, V., van Wijnen, A.J., Stein, J.L., Lian, J.B. and Stein, G.S. (2001) Differential regulation of the two principal Runx2/Cbfa1 n-terminal isoforms in response to bone morphogenetic protein-2 during development of the osteoblast phenotype. Endocrinology 142, 4026–4039. [2] Nakashima, K., Zhou, X., Kunkel, G., Zhang, Z., Deng, J.M., Behringer, R.R. and de Crombrugghe, B. (2002) The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17–29. [3] Santos, N.C., Figueira-Coelho, J., Martins-Silva, J. and Saldanha, C. (2003) Multidisciplinary utilization of dimethyl sulfoxide: pharmacological, cellular, and molecular aspects. Biochem. Pharmacol. 65, 1035–1041. [4] Melchior, D., Packer, C.S., Johnson, T.C. and Kaefer, M. (2003) Dimethyl sulfoxide: does it change the functional properties of the bladder wall? J. Urol. 170, 253–258. [5] Xing, L. and Remick, D.G. (2005) Mechanisms of dimethyl sulfoxide augmentation of IL-1 beta production. J. Immunol. 174, 6195–6202. [6] Bolduc, L., Labrecque, B., Cordeau, M., Blanchette, M. and Chabot, B. (2001) Dimethyl sulfoxide affects the selection of splice sites. J. Biol. Chem. 276, 17597–17602. [7] Lee, Y.R., Shim, H.J., Yu, H.N., Song, E.K., Park, J., Kwon, K.B., Park, J.W., Rho, H.W., Park, B.H., Han, M.K. and Kim, J.S. (2005) Dimethylsulfoxide induces upregulation of tumor suppressor protein PTEN through nuclear factor-kappaB activation in HL-60 cells. Leuk. Res. 29, 401–405. [8] Zucchi, I., Bini, L., Albani, D., Valaperta, R., Liberatori, S., Raggiaschi, R., Montagna, C., Susani, L., Barbieri, O., Pallini, V., Vezzoni, P. and Dulbecco, R. (2002) Dome formation in cell cultures as expression of an early stage of lactogenic differentiation of the mammary gland. Proc. Natl. Acad. Sci. USA 99, 8660–8665. [9] Lecka-Czernik, B., Moerman, E.J., Grant, D.F., Lehmann, J.M., Manolagas, S.C. and Jilka, R.L. (2002) Divergent effects of selective peroxisome proliferator-activated receptor-gamma 2 ligands on adipocyte versus osteoblast differentiation. Endocrinology 143, 2376–2384. [10] Zhu, W., Rawlins, B.A., Boachie-Adjei, O., Myers, E.R., Arimizu, J., Choi, E., Lieberman, J.R., Crystal, R.G. and Hidaka, C. (2004) Combined bone morphogenetic protein-2 and -7 gene transfer enhances osteoblastic differentiation and spine fusion in a rodent model. J. Bone Miner. Res. 19, 2021–2032. [11] Cheung, W.M.W., Hui, W.S., Chu, P.W.K., Chiu, S.W. and Ip, N.Y. (2000) Ganoderma extract activates MAP kinases and induces the neuronal differentiation of rat pheochromocytoma PC12 cells. FEBS Lett. 486, 291–296. [12] Cheung, W.M.W., Chu, A.H., Chu, P.W.K. and Ip, N.Y. (2001) Cloning and expression of a novel nuclear matrix-associated protein that is regulated during the retinoic acid-induced neuronal differentiation. J. Biol. Chem. 276, 17083–17091. [13] Cheng, S.L., Zhang, S.F., Nelson, T.L., Warlow, P.M. and Civitelli, R. (1994) Stimulation of human osteoblast differentiation and function by ipriflavone and its metabolites. Calcif. Tissue Int. 55, 356–362. [14] Gatti, A. (2003) Divergence in the upstream signaling of nerve growth factor (NGF) and epidermal growth factor (EGF). Neuroreport 14, 1031–1035. [15] Xiao, G., Jiang, D., Gopalakrishnan, R. and Franceschi, R.T. (2002) Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, Cbfa1/Runx2. J. Biol. Chem. 277, 36181–36187. [16] Newton, A.C. (1995) Protein kinase C: structure, function, and regulation. J. Biol. Chem. 270, 28495–28498.
126 [17] Zugaza, J.L., Sinnett-Smith, J., Van Lint, J. and Rozengurt, E. (1996) Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway. EMBO J. 15, 6220–6230. [18] Spinella-Jaegle, S., Roman-Roman, S., Faucheu, C., Dunn, F.W., Kawai, S., Gallea, S., Stiot, V., Blanchet, A.M., Courtois, B., Baron, R. and Rawadi, G. (2001) Opposite effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on osteoblast differentiation. Bone 29, 323–330. [19] Lee, M.H., Kwon, T.G., Park, H.S., Wozney, J.M. and Ryoo, H.M. (2003) BMP-2-induced osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem. Biophys. Res. Commun. 309, 689–694. [20] Xiao, G., Gopalakrishnan, R., Jiang, D., Reith, E., Benson, M.D. and Franceschi, R.T. (2002) Bone morphogenetic proteins, extracellular matrix, and mitogen-activated protein kinase signaling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells. J. Bone Miner. Res. 17, 101–110. [21] Hu, E., Kim, J.B., Sarraf, P. and Spiegelman, B.M. (1996) Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPARc. Science 274, 2100–2103. [22] Cowley, S., Paterson, H., Kemp, P. and Marshall, C.J. (1994) Activation of MAP kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77, 841–852.
W.M.W. Cheung et al. / FEBS Letters 580 (2006) 121–126 [23] Takeuchi, Y., Suzawa, M., Kikuchi, T., Nishida, E., Fujita, T. and Matsumoto, T. (1997) Differentiation and transforming growth factor-beta receptor down-regulation by collagen-alpha2beta1 integrin interaction is mediated by focal adhesion kinase and its downstream signals in murine osteoblastic cells. J. Biol. Chem. 272, 29309–29316. [24] Kim, H.J., Kim, J.H., Bae, S.C., Choi, J.Y., Kim, H.J. and Ryoo, H.M. (2003) The protein kinase C pathway plays a central role in the fibroblast growth factor-stimulated expression and transactivation activity of Runx2. J. Biol. Chem. 278, 319–326. [25] Van Lint, J.V., Sinnett-Smith, J. and Rozengurt, E. (1995) Expression and characterization of PKD, a phorbol ester and diacylglycerol-stimulated serine protein kinase. J. Biol. Chem. 270, 1455–1461. [26] Johannes, F.J., Prestle, J., Eis, S., Oberhagemann, P. and Pfizenmaier, K. (1994) PKCl is a novel, atypical member of the protein kinase C family. J. Biol. Chem. 269, 6140–6148. [27] Yuan, J., Bae, D., Cantrell, D., Nel, A.E. and Rozengurt, E. (2002) Protein kinase D is a downstream target of protein kinase Ctheta. Biochem. Biophys. Res. Commun. 291, 444– 452. [28] Lemonnier, J., Ghayor, C., Guicheux, J. and Caverzasio, J. (2004) Protein kinase C-independent activation of protein kinase D is involved in BMP-2-induced activation of stress mitogen-activated protein kinases JNK and p38 and osteoblastic cell differentiation. J. Biol. Chem. 279, 259–264.