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Introduction

The Distal-less (Dlx) protein is a homeobox domain (HD) protein. In vertebrates, there are six Dlx genes arranged in three clusters (Dlx1/Dlx2, Dlx3/Dlx4, Dlx5/Dlx6) [1]. Dlx is expressed in limb bud, brachial arch and epi-mesoderm during development and is required for the development and maintenance of these structures. Among the Dlx genes, Dlx3 and Dlx5 are known to be a positive regulator of osteoblast differentiation [2,3]. Dlx3 is expressed in pre-osteoblast, osteoblast and osteocyte in bone [4]. Bone morphogenetic protein 2 (BMP2), an inducer of osteoblast differentiation, increases Dlx3 expression and Dlx3 expression is induced earlier than that of Runx2 [5]. Dlx3 over-expression promotes the expression of osteoblast marker genes and directly re­gulates the transcription of Runx2 and osteocalcin by binding to their promoters [2,6]. However, in vivo function of the Dlx3 in osteoblast differentiation and bone formation is not clear because the dlx3^-/- mice are embryonic lethal due to placental deficiency [7]. Dlx5 expression is also induced by BMP2 [8]. Over-expression of Dlx5 induces the expression of Runx2, alkaline phosphatase (ALP) and osteocalcin and increases mineralized nodule formation in vitro [2,3]. Dlx5­deficient mice showed reduced calcification and abnormal craniofacial development [9].

Mesenchymal stem cells are multipotent that can give rise not only to osteoblasts, but also to a range of other cell types, including adipocytes, chondrocytes and myoblasts [10]. Among these fates, differentiation to the osteoblast and adipocyte lineages has particular relevance to the maintenance of normal bone homeostasis. Because osteoblasts and adipo­cytes differentiate from a common progenitor, inducers of differentiation toward one lineage may inhibit cell differ­entiation into another lineage. Osteoporosis and aging-related osteopenia were shown to be associated with an increased adipocyte accumulation in bone marrow, suggesting that there are the inverse relationships between osteoblast differentiation and adipocyte differentiation in bone marrow [11-13].

Although there were a significant number of reports about the role of Dlx3 and Dlx5 in relation to osteoblast differ­entiation, we could not find the report about the regulatory role of Dlx3 and Dlx5 in adipocyte differentiation. Therefore, in this study, we examined the effect of Dlx3 and Dlx5 onadipocyte differentiation in mesenchymal stem cells derived from human dental pulp tissues.  

Materials and methods

Reagents

 Alpha modified Eagle’s medium (αMEM) and fetal bovine serum (FBS) was purchased from BioWhittaker (Walkersville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Hyclone (Logan, UT, USA). BMP2 was purchased from R&D Systems (Minneapolis, MN, USA). The easy-BLUE^TM for total RNA extraction and StarTaq^TM polymerase for polymerase chain reaction (PCR) amplifica­tion were obtained from iNtRON Biotechnology (Sungnam, Korea). AccuPower RT-PreMix for the first-strand cDNA synthesis was from Bioneer (Daejeon, Korea). PCR primers were synthesized by TaKaRa Korea (Seoul, Korea). Ascorbic acid, β-glycerophosphate, dexamethasone, indomethacin, isobutylmethylxanthin and insulin were purchased from Sigma (St. Louis, MO, USA).

Cell culture and transient transfection

 Human dental pulp stem cells were prepared from exfoliated deciduous pulp tissues as previously described [14]. Single-cell suspensions were cultured in α-MEM supplemented with 10% FBS and 1% antibiotics-antimycotics. Medium was changed every 3 days and cells were sub-cultured at 70% confluency. Murine preadipocytic 3T3-L1 cells (Korean cell line bank; Seoul, Korea) were cultured in DMEM sup­plemented with 10% FBS and 1% antibiotics-antimycotics.

For the transient transfection of Dlx3 and Dlx5 expression plasmids, electroporation was performed using a Gene Pulser X Cell (Bio-Rad laboratories, Richmond, CA, USA) according to the manufacturer's instructions. The construc­tion of Dlx3 and Dlx5 expression vectors has been described previously [15,16].

To induce osteogenic differentiation, human dental pulp stem cells and 3T3-L1 cells were cultured in osteogenic me­dium (growth medium supplemented with 0.1 µM dexa­methasone, 10 mM β-glycerophosphate and 50 µg/ml ascorbic acid) for the indicated period. When indicated, BMP2 (300 ng/ml) was added to osteogenic medium. To induce adipogenic differentiation, cells were incubated in adipogenic medium (growth medium supplemented with 0.1 µM dexa­methasone, 50 µM indomethacin, 0.5 mM isobutylmethy­lxanthine and 10 µg/ml insulin) for the indicated period. At the end of the culture period, osteogenic and adipogenic differentiation was confirmed by Alizarin red S and Oil red O staining, respectively. 

Fluorescense-assisted cell sorter (FACS) analysis

 Expression of stem cell surface markers in human dental pulp stem cells was determined by FACS analysis as pre­viously described [14]. The cells were detached and washed with phosphate-buffered saline supplemented with 2% FBS. Quantities of 100,000-500,000 cells were incubated with the primary antibody for 30 min on ice. The following antibodies were used; FITC-conjugated mouse anti-human CD14, CD31, CD44 and CD45 antibodies; PE-conjugated mouse anti-human CD29, CD73 and CD117 antibodies; PE.Cy5-conjugated mouse anti-human CD90 antibody; APC-conjugated mouse anti-human CD34 and HLA-DR antibodies (all from BD PharMingen, San Jose, CA, USA); APC-conjugated mouse anti-human CD105 antibody (eBioscience, San Diego, CA, USA) and biotin-conjugated mouse anti­human HLA class I antibody (BD PharMingen). After washing, cells were fixed with 4% paraformaldehyde at 4^oC before analysis. Fluorescence intensity was measured on a FACS Calibur (BD PharMingen), and data were analyzed using FLOWJO software (Tree Star, Inc., Ashland, OR, USA).

Reverse Transcription-PCR (RT-PCR)

 To evaluate mRNA expression, semi-quantitative RT-PCR was performed in the range of linear amplification. Total RNA was isolated using easy-BLUE^TM RNA Extraction Re­agents. cDNA was synthesized from total RNA using the AccuPower^TM RT-PreMix and was subsequently subjected to PCR amplification using StarTaq^TM polymerase. The PCR products were electrophoresed in a 1.2% agarose gel and visualized under UV light by ethidium bromide staining. Human genes and their primer sequences for PCR were as follows (in parentheses, the annealing temperature used for the PCR reaction and the product size are described): cAMP response element binding protein (CREB) forward (f) 5’­ACCAGCAGAGTGGAGATGCT-3’, CREB reverse (r) 5’­CTGCCCACTGCTAGTTTGGT-3’ (58^oC, 476 bp); peroxi­some proliferator-activated receptor γ (PPARγ)-f 5’-GAC CACTCCCACTCCTTTGA-3’, PPARγ-r 5’-CGACATTC AATTGCCATGAG-3’ (58^oC, 257 bp); adipocyte protein 2 (aP2)-f 5’-CATCAGTGTGAATGGGGATG-3’, aP2-r 5’­GTGGAAGTGACGCCTTTCAT-3’ (58^oC, 541 bp); lipopro­tein lipase (LPL)-f 5’-ACAGGGCGGCCACAAGTTTT-3’, LPL-r 5’-ATGGAGAGCAAAGCCCTGCTC-3’ (60^oC, 717 bp); CCAAT/enhancer-binding proteins α (C/EBPα)-f 5’­CACGAAGCACGATCAGTCCAT-3’, C/EBPα-r 5’-GGC ACAGAGGCCAGATACAAG-3’ (58^oC, 110 bp); Dlx3-f 5’­AAGGTCCGAAAGCCGCGTA-3’, Dlx3-r 5’-CTGCTGCTGTAAGTGGGGT-3’ (55^oC, 414 bp) GAPDH-f 5’-CCATCTTCCAGGAGCGAGATC-3’ and GAPDH-r 5’-GCCTTCTCCATGGTGGTGAA-3’ (56^oC, 321 bp). Mouse genes and their primer sequences for PCR are as follows Runx2-f 5’-CCGCACGACAACCGCACCAT-3’, Runx2-r 5’-CGCTCCGGCCCACAAATCTC-3’ (56^oC, 289 bps); bone sialoprotein(BSP)-f 5’-GTCAACGGCACCAGCACCAA-3’, BSP-r 5’-GTAGCTGTATTCGTCCTCAT-3’ (58^oC, 375 bps); ALP-f 5’-AGGCAGGATTGACCACGG-3’, ALP-r 5’-TGTAGTTCTGCTCATGGA-3’ (56^oC, 439 bps); Dlx3-f 5’-ATGAGTGGCTCCTTCGATCGCAAGCT-3’, Dlx3-r 5’-TCAGTACACAGCCCCAGGGTTGGGC-3’ (60^oC, 421 bp) Dlx5-f 5’-TCTCTAGGACTGACGCAAACA-3’, Dlx5-r 5’-GTTACACGCCATAGGGTCGC-3’ (56^oC, 120 bp) GAPDHf 5’-TCACCATCTTCCAGGAGCG-3’ and GAPDH-r 5’-CTGCTTACCACCTTCTTGA-3’ (56^oC, 571 bps).

Results and discussion

 Dental pulp tissues have been shown to be an excellent source for stem cells because it can be obtained from the deciduous dentition requiring extraction as part of a planned serial extraction for management of occlusion [17]. Previous studies have shown that in vitro culture conditions can induce dental pulp stem cells to differentiate into osteogenic, chondrogenic, adipogenic and myogenic cells [17,18]. Therefore, in this study, we used dental pulp stem cells derived from human exfoliated deciduous pulp tissues. The cells showed fibroblast-like morphology (Fig. 1A). Immunephenotyping of the cells of passage number 3 showed that they were negative for hematopoietic markers (CD14, CD34, CD45, CD117 and HLA-DR) and endothelial marker (CD31), whereas more than 90% of cells were positive for mesenchymal stem cell markers (CD29, CD44, CD73, CD90, CD105, CD73 and HLA-I) (Fig. 1B). These results are consistent with the previous report about stem cells from human exfoliated deciduous teeth, and indicate that these cells are mesenchymal stem cells [19].

We then examined the effect of Dlx3 and Dlx5 overexpression on adipogenic differentiation. Dental pulp stem cells were transiently transfected with the expression vector for Dlx3 or Dlx5 and incubated under the adipogenic conditions for 21 days. Oil red O staining showed that the accumulation of lipid droplets was diminished in Dlxtransfected cells compared with pcDNA-transfected cells (Fig. 2). As a control, these cells were also incubated in the osteogenic medium for 21 days, and osteogenic induction was verified by Alizarin red staining (Fig. 2). Consistent with the previous reports, over-expression of Dlx3 or Dlx5 enhanced osteogenic differentiation and matrix mineralization [2,5]. 

Fig. 1. Human dental pulp stem cells showed fibroblast-like morphology and mesenchymal stem cell surface marker expression. (A) Microscopic images of human dental pulp stem cells of passage number three. (B) Results of immune-phenotyping of human dental pulp stem cells (passage 3) for detection of surface marker expression.

Fig. 2. Over-expression of Dlx3 or Dlx5 inhibited adipogenic differentiation of human dental pulp stem cells while stimulating osteogenic differentiation. The cells were transiently transfected with pcDNA or with expression vector for Dlx3 or Dlx5 and incubated in adipogenic medium (AM) or osteogenic medium (OM) for 21 days with medium change every other day. At the end of the culture period, Oil red O and Alizarin red staining was performed to examine adipogenic and osteogenic differentiation, respectively. Microscopic images of Oil red O- or Alizarin red-stained cells were presented (× 100). (Upper left panel) Higher magnification image (× 400) of Oil red O-stained pcDNA-transfected cells.

Fig. 3. Over-expression of Dlx3 and Dlx5 suppressed adipogenic marker gene expression of human dental pulp stem cells. The cells were transiently transfected with pcDNA or with expression vector for Dlx3 (A) or Dlx5 (B) and incubated in adipogenic medium for 21 days. At the end of the culture period, total RNA was isolated and semi-quantitative RT-PCR was performed.

Fig. 4. Adipogenic stimuli suppressed the expression levels of Dlx3 and Dlx5 whereas osteogenic stimuli increased their expression levels in 3T3-L1 cells. (A) The cells were incubated in osteogenic medium in the presence or absence of BMP2 (300 ng/ml) for 6 days. (B) The cells were incubated in growth medium, adipogenic medium (AM) or osteogenic medium (OM) supplemented with BMP2 (300 ng/ml) for 48 h. At the end of the culture period, total RNA was isolated and semi-quantitative RT-PCR was performed. BSP, bone sialoprotein; ALP, alkaline phosphatase Anti-adipogenic effect of Dlx3 and Dlx5 35

 To further verify the inhibitory effect of Dlx3 and Dlx5 on adipogenic differentiation, we examined the expression levels of adipogenic marker genes. After the incubation for 21days in adipogenic medium, the cells were harvested for RTPCR. As expected, over-expression of Dlx3 and Dlx5 suppressed the expression of adipogenic marker genes such as C/EBPα, PPARγ, aP2 and LPL but not that of CREB (Fig. 3). These results indicate that Dlx3 and Dlx5 inhibit adipogenic differentiation through the inhibition of adipogenic gene expression.

 Compared with pcDNA-transfected cells, the level of Dlx3 or Dlx5 was higher in Dlx-transfected cells (Fig. 3). Because we used transient transfection method to over-express Dlx, it is unlikely that Dlx3 or Dlx5 mRNA in 21 day-cultured cells is mainly derived from exogenously introduced expression vectors. Therefore, we examined whether adipogenic differentiation itself regulates the expression levels of Dlx3 and Dlx5. For this experiment, we used 3T3-L1 preadipocyte cell line. 3T3-L1 cells were incubated in the growth medium, adipogenic medium or osteogenic medium for 48 h and we examined the expression levels of Dlx3 and Dlx5 by RT-PCR. Adipogenic stimuli suppressed the expression of both Dlx3 and Dlx5 whereas osteogenic stimuli increased their expression levels (Fig. 4B). To confirm that osteogenic condition can induce osteogenic differentiation of 3T3-L1 cells, we incubated the cells under osteogenic condition for 6 days and examined the expression levels of osteogenic marker genes. RT-PCR results showed that the levels of Runx2, BSP and ALP mRNA increased by osteogenic stimuli (Fig. 4A). These results suggest that increased expression of Dlx3 and Dlx5 mRNA observed in Fig. 3 is mainly indirect effect of inhibition of adipogenic differentiation of dental pulp stem cells.

 Differentiation of mesenchymal stem cells to the specific lineage depends on the phenotype-specific transcription factors. The representative transcription factors involved in osteoblast differentiation include Runx2, Osterix, ATF4, Dlx3 and Dlx5 [20]. As for adipocyte differentiation, transcription factors including C/EBPα, C/EBPβ, PPARγ and CREB are the main determinants of adipogenic differentiation [21]. Among them, C/EBPα and PPARγ are regarded as the essential transcription factors because the deletion of any of these transcription factors blocked adipogenic differentiation [21,22]. As described above, there are inverse relationships between adipogenesis and osteogenesis in the bone marrow. Several transcription factors have been suggested to be involved in this reciprocal regulation, including Runx2 and PPARγ [23]. Osteogenic stimuli, such as mechanical stress, increase the expression and transcriptional activity of Runx2 while suppressing PPARγ expression, resulting in the shift of mesenchymal stem cell fate toward the osteogenic lineage [24,25]. In contrast, adipogenic stimuli, such as immobilization and an antidiabetic medicine rosiglitazone, show opposite effects; that is, enhancement of PPARγ expression and suppression of Runx2 expression are observed, resulting in enhanced adipogenesis in place of osteogenic differentiation [26,27]. Therefore, the transcription factors that regulate the expression of Runx2 and/or PPARγ will exert an effect on stem cell fate determination toward osteoblast and/or adipocyte lineage. Previous studies have shown that Dlx3 and Dlx5 mediate BMP2-induced Runx2 expression in preosteoblastic cells [2,5,28]. In this study, we showed that Dlx3 and Dlx5 suppressed the expression of PPARγ. Furthermore, we also demonstrated that expression levels of Dlx3 and Dlx5 were up-regulated by osteogenic stimuli while being down-regulated by adipogenic stimuli. Taken together with previous reports, these results suggest that Dlx3 and Dlx5 play an important regulatory role in fate determination of mesenchymal stem cells toward osteoblast lineage by inhibition of adipogenesis as well as by stimulation of osteoblastogenesis. Furthermore, it is suggested that downregulation of Dlx3 and Dlx5 is necessary for proper adipogenic differentiation.

Acknowledgement

 This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0068779).