Homeobox B7 promotes the osteogenic differentiation potential of mesenchymal stem cells by activating RUNX2 and transcript of BSP

Abstract

Mesenchymal stem cells (MSCs) are a reliable cell source for tissue regeneration. However, the molecular mechanisms underlying the directed differentiation of MSCs remain unclear; thus, their use is limited. Here, we investigate HOXB7 function in the osteogenic differentiation potentials of MSCs using stem cells from apical papilla (SCAPs) and bone marrow stem cells (BMSCs). The HOXB7 gene is highly expressed in BMSCs compared with dental tissue-derived MSCs. We found that, in vitro, over-expression of HOXB7 in SCAPs enhanced alkaline phosphatase (ALP) activity and mineralization. HOXB7 over-expression affected the mRNA expression of osteonectin (ON), collagen alpha-2(I) chain (COL1A2), bone sialoprotein (BSP), and osteocalcin (OCN), led to the expression of the key transcription factor, runt-related transcription factor 2 (RUNX2), and promoted SCAP osteogenic differentiation in vitro. The knock-down of HOXB7 inhibited ALP activity, mineralization, and the expression of ON, BSP, COL1A2, OCN, and RUNX2 in BMSCs in vitro. In addition, transplant experiments in nude mice confirmed that SCAP osteogenesis was triggered when HOXB7 was activated. Furthermore, Over-expression of HOXB7 significantly increased the levels of HOXB7 associated with the BSP promoter by ChIP assays. Taken together, these results indicate that HOXB7 enhances SCAP osteogenic differentiation by up-regulating RUNX2 and directly activating transcript of BSP. Thus, the activation of HOXB7 signaling might improve tissue regeneration mediated by MSCs. These results provide insight into the mechanism underlying the directed differentiation of MSCs.

Introduction

Mesenchymal stem cells (MSCs) are multipotent cells that were originally isolated from bone marrow. They can differentiate into a variety of cell types, including osteoblasts, chondrocytes, myocytes, and adipocytes. Increasing evidence indicates that MSCs are also present in non-bone marrow tissues [1-3]. Recently, a new population of MSCs was isolated from dental and craniofacial tissues on the basis of their stem cell properties. This population included stem cells from the periodontal ligament (PDLSCs), from dental pulp (DPSCs), from apical papilla (SCAPs), and from human exfoliated deciduous teeth (SHEDs) [4-8]. These isolated cells were multipotent, exhibited osteo/dentinogenic differentiation, and could self-renew. When transplanted into mice or miniature pigs, they generated bone/dentin-like mineralized tissue, and they were capable of repairing tooth defects [8-10]. MSCs are a reliable resource for tissue regeneration, but the molecular mechanisms underlying their directed differentiation remain unclear; thus, their use is limited.

Homeobox genes (HOX) are characterized by a conserved 180 bp DNA sequence coding for a 60aa DNA-binding homeodomain, and the master regulators in determining patterning, specification, and differentiation during embryonic development [11-13]. Homeodomain proteins are transcription factors that can activate or inhibit transcription of downstream genes through the DNA binding. Recent studies show that homeobox genes play crucially regulatory roles in the process of maxillofacial and dental development. In Pbx1-deficient mice, absence of pre-B-cell leukemia homeobox proteins 1 (Pbx1) caused precocious endochondral ossification and abnormal bone formation by perturbing chondrocyte proliferation and differentiation [14]. HOXA10 could mediate chromatin hyperacetylation and trimethyl histone K4 (H3K4) methylation, induce expression of osteogenic genes through activation of RUNX2, or directly regulate other osteoblastic phenotypic genes, and contribute to the onset of osteogenesis and subsequent bone formation [15]. More importantly, recent studies have showed that homeobox gene, such as DLX2, DLX5 and HOXC6 as key regulators in the osteogenic differentiation of MSCs [16-18], indicating that homeobox genes play essential roles in the differentiation of stem cells. Among these homeobox genes, HOXB7 was attracted, since its downregulation in aged MSC was preliminarily observed [19]. HOXB7 regulates several genes playing a significant role in cell proliferation and differentiation [20-23]. Some researchers found that HOXB7 s highly expressed in non-dental tissue-derived MSCs [24], a forced HOXB7 expression was associated with an improved cell growth, a reduction of senescence and an improved osteogenesis [25]. However, at present, functional role of HOXB7 in differentiation of human MSCs, especially, the dental tissue derived MSCs, haven’t been reported.

Based on those previous findings, we hypothesized that HOXB7 might have an effect on human MSCs. In the present study, we studied human SCAPs and bone marrow stem cells (BMSCs) to investigate the effects of HOXB7 and the mechanisms underlying these effects. Our results indicated that HOXB7 could enhance the osteogenic differentiation potential in SCAPs and BMSCs by up-regulating the expression of runt-related transcription factor 2 (RUNX2) and directly activating transcript of bone sialoprotein (BSP). This finding may have practical implications in terms of enhancing the directed differentiation of MSCs for tissue regeneration applications.

Materials and methods

Cell cultures

Tooth tissues were obtained according to approved guidelines set by Beijing Friendship Hospital, Capital Medical University. Informed patient consent was obtained.

Wisdom teeth were disinfected with 75% ethanol and washed with phosphate buffered saline (PBS). SCAPs were gently separated from the apical papilla of the root, PDLSCs were separated from the periodontal ligament in the middle one-third of the root, while DPSCs were separated from crown pulp. Subsequently, the MSCs were digested in a solution of 3 mg/mL collagenase type I (Worthington Biochemical Corp., Lakewood, NJ, USA) and 4 mg/mL dispase (Roche Diagnostics Corp., Indianapolis, IN, USA) for 1 h at 37°C. The digested tissues were passed through a 70-μm strainer (Falcon, BD Labware, Franklin Lakes, NJ, USA) to obtain isolated cell suspensions. Human BMSCs were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA). MSCs were grown in a humidified incubator under 5% CO₂ at 37°C in DMEM alpha modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA), supplemented with 15% fetal bovine serum (FBS; Invitrogen), 2 mmol/l glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). The culture medium was changed every 3 days. Cells from passages 2-4 were used in further experiments.

Human embryonic kidney 293T (HEK 293T) (American Type Culture Collection, Manassas, VA, USA) cells were maintained in complete DMEM medium with 10% FBS (Invitrogen), 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen). These cells were utilized for packaging viral constructs.

Plasmid construction and viral infection

The plasmids were constructed according to standard methods, and all constructs were verified with the appropriate restriction digestion analysis and/or sequencing. Human full-length HOXB7 cDNA was obtained by isolating total RNA from cultured BMSCs, then performing reverse-transcription and PCR amplification (RT-PCR). Next, the HOXB7 cDNA sequence was fused to a Flag-tag sequence (Flag-HOXB7), with AgeI and EcoR1 restriction sites, and subcloned into the T-easy vector (Promega, Madison, WI, USA). The final sequence was confirmed with DNA sequencing. Next, the Flag- HOXB7 was subcloned into the pQCXIN retroviral vector (BD Clontech, Mountain View, CA, USA) with the AgeI and EcoR1 restriction sites, and that sequence was also confirmed by DNA sequencing.

Short hairpin RNAs (shRNA) with sequences complementary to those of the target genes were subcloned into the pLKO.1 lentiviral vector (Addgene, Cambridge, MA, USA). Viral packaging was performed, according to the manufacturer’s protocol, with HEK 293T cells (BD Clontech). For viral infections, SCAPs were plated overnight, and then, infected with retroviruses or lentiviruses in the presence of polybrene (6 µg/mL, Sigma-Aldrich, St. Louis, MO, USA) for 6 h. After 48 h, infected cells were selected with the appropriate antibiotics. BMSCs were infected with lentiviruses expressing SMAD4 shRNA (SMAD4sh), expressed HOXB7 shRNA (HOXB7sh) or Scramsh, and selected with 2 μg/mL puromycin for 7 days. SCAPs were infected with a retrovirus that carried the wild type HOXB7 fused to a Flag-tag or an empty vector, and selected with 600 µg/mL G418 for 10 days. The target sequences for the shRNAs were as follows: HOXB7shRNA (HOXB7sh), 5’-gaggaagagacatgagaaa-3’. A scrambled shRNA (Scramsh) was used as a control (Addgene). The PCR primers used for cloning the HOXB7 gene were as follows: HOXB7 Forward, 5’-tcgaaccggtatggattacaaggacgacgatgacaagatgagttcattgtattatgc-3’; and HOXB7 Reverse, 5’-acgtgaattctcactcttcctcttcctcct-3’.

Western blot analysis

Cells were lysed in RIPA buffer (10 mM Tris-HCL, 1 mM EDTA, 1% sodium dodecyl sulfate [SDS], 1% NP-40, 1:100 proteinase inhibitor cocktail, 50 mM β-glycerophosphate, 50 mM sodium fluoride). The samples were separated on a 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes with a semi-dry transfer apparatus (BioRad, Hercules, CA, USA). The membranes were blotted with 5% dehydrated milk for 2 h and then incubated with primary antibodies overnight. Next, membranes were incubated with anti-mouse IgG secondary antibodies conjugated to horseradish peroxidase (Promega), and the immune complexes were visualized with SuperSignal reagents (Pierce, Rockford, IL, USA). The primary antibodies were: monoclonal anti-Flag M2 (Clone No.9A3, Cat No.8146, Cell Signaling Technology, Beverly, MA, USA), and a monoclonal antibody specific for the housekeeping protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Clone No.GAPDH-71.1, Cat No.G8795, Sigma-Aldrich).

Alkaline phosphatase and Alizarin Red staining

MSCs were grown in osteogenic-inducing medium that contained 100 µM/mL ascorbic acid, 2 mM β-glycerophosphate, 1.8 mM KH₂PO₄, and 10 nM dexamethasone. After induction, for ALP staining, the cells were fixed with 4% paraformaldehyde. Cells were stained with a solution of 0.25% naphthol AS-BI phosphate and 0.75% Fast Red (in an ALP kit), according to the manufacturer’s protocol (Sigma-Aldrich). ALP activity was assayed with an ALP activity kit, according to the manufacturer’s protocol (Sigma-Aldrich). Signals were normalized based on protein concentration. To detect mineralization, cells were induced for 3 weeks, fixed with 70% ethanol, and stained with 2% Alizarin Red (Sigma-Aldrich). To quantitatively determine calcium content, cells stained with Alizarin Red were destained with 10% cetylpyridinium chloride in 10 mM sodium phosphate for 30 min at room temperature. The calcium concentration was determined by measuring absorbance at 562 nm on a multiplate reader and comparing the reading to a standard calcium curve, constructed with calcium diluted in the same solution. The final calcium level in each group was normalized to the total protein concentration detected in a duplicate plate [26].

Real time, reverse transcriptase-PCR

Total RNA was isolated from MSCs with Trizol reagent (Invitrogen). We synthesized cDNA from 2-µg aliquots of RNA with random hexamers or oligo (dT) and reverse transcriptase, according to the manufacturer’s protocol (Invitrogen). Real-time PCR reactions were performed with the QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany) and an Icycler iQ Multi-color Real-time PCR Detection System. The expression of genes changes was calculated by the method of 2^-ΔΔCT. The primers used for specific genes are shown in Table S1.

CHIP assays

We used a ChIP assay kit (Merck Millipore, Darmstadt, Germany) following the manufacturer’s protocol. Briefly, cells were incubated with 1% formaldehyde for 10 min at 37°C. Each ChIP reaction was performed using 2.0 × 10⁶ cells. For DNA precipitation, we added 2 µg monoclonal anti-Flag M2 (Clone No.9A3, Cat No.8146, Cell Signaling Technology, Beverly, MA, USA). The precipitated DNA samples were quantified by real-time PCR with primers targeting the HOXB7-binding region of the BSP promoter (HOXB7 binding site): forward, 5’-gcaagctttcctttctttcg-3’ and reverse, 5’-gaaaacccccaaacttcaac-3’; and primers targeting the open reading frame (ORF) region of the BSP (Negative control): forward, 5’-gaaagtgtcactggagccaa-3’ and reverse, 5’-tggagaggttgttgtcttcg-3’. Quantification data are expressed as the percentage of input DNA.

Transplantation into nude mice

The present study was approved by an animal care and use committee at the Beijing Friendship Hospital, Capital Medical University. Our care and use of animals followed the guidelines of the Experimental Animal Management Ordinance. We used 10-week old, immunocompromised beige mice (nu/nu) female nude mice. All animals were purchased from the Institute of Animal Science of the Vital River Co., Ltd. The animals had not received any drugs or previous procedures.

Approximately 4.0 × 10⁶ SCAPs were mixed with 40 mg of ceramic particles made of hydroxyapatite/tricalcium phosphate (HA/TCP; Engineering Research Center for Biomaterials, Sichuan University, Chengdu, China). The mixture was transplanted subcutaneously into the dorsal surface of immunocompromised beige mice. All procedures were performed in accordance with the specifications of an approved animal protocol. Eight weeks after transplantation, the transplanted cells were harvested, fixed in 10% formalin, decalcified with buffered 10% EDTA (pH 8.0), embedded in paraffin, and sliced in 5-µm sections. For analysis, the sections were deparaffinized, hydrated, and stained with hematoxylin and eosin (H&E) [26-28]. Qualitative measurements of tissue mineralization were performed with the Image-Pro Plus 6.0 program (Media Cybernetics, Rockville, MD, USA).

Statistics

All statistical calculations were performed with SPSS10 statistical software. The student’s t-test or one-way ANOVA were utilized to determine statistically significant differences between groups. A P-value ≤ .05 was considered significant.

Results

HOXB7 over-expression enhanced osteogenic differentiation potential in SCAPs in vitro

We used real-time RT-PCR to test the HOXB7 mRNA levels in BMSCs and dental tissue-derived MSCs. We found higher HOXB7 expression in BMSCs than in SCAPs, DPSCs, and PDLSCs (Figure 1A). Then we investigated the function of HOXB7 in SCAPs with HOXB7 overexpression. First, we inserted the Flag-HOXB7 sequence into a retroviral vector. This construct induced overexpression of ectopic HOXB7 when transduced into SCAPs via retroviral infection. Ectopic HOXB7 expression was confirmed by Western blot analysis (Figure 1B). Next, SCAPs transduced with the Flag-HOXB7 or with the empty vector alone (controls) were cultured in osteogenic-inducing medium to determine the osteogenic differentiation potential. The results indicated that over-expression of HOXB7 strongly increased ALP activity, one of the earliest markers of osteogenic differentiation in SCAPs (Figure 1C, 1D). Accordingly, Alizarin Red staining and quantitative calcium measurements showed that mineralization was markedly enhanced in SCAPs infected with Flag-HOXB7 compared to control-infected cells (Figure 1E, 1F). Consistent with this finding, real-time RT-PCR results showed that the osteogenic marker genes, which encodes extracellular matrix proteins in bone and dentin tissues were changed. The results showed that the overexpression of Flag-HOXB7 in SCAPs was associated with elevated expression of osteonectin (ON) and collagen alpha-2(I) chain (COL1A2) at 7 days (Figure 2A, 2D), and bone sialoprotein (BSP) and osteocalcin (OCN) at 3, 7, and 14 days after induction (Figure 2B, 2C).

Figure 1.

Wild type HOXB7 over-expression enhances osteogenic differentiation of SCAPs. (A) HOXB7 expression in BMSCs, SCAPs, DPSCs, and PDLSCs according to the results of real-time RT-PCR. GAPDH was used as an internal control. The one-way ANOVA was performed to determine statistical significance. (B) Western blot analysis showed that HOXB7 over-expression in SCAPs. GAPDH was used as an internal control. (C, D) ALP activity assay (C) and ALP (D) staining results show that HOXB7 over-expression increased ALP activity in SCAPs. Bar: 100 µm. (E, F) Alizarin Red staining (E) and quantitative calcium analysis results (F) show that HOXB7 over-expression enhanced mineralization in SCAPs. Bar: 100 µm. The student’s t test was performed to determine statistical significance. All error bars represent s.d. (n = 3). **P ≤ 0.01.

Figure 2.

Wild type HOXB7 over-expression enhances the mRNA levels of osteogenic differentiation markers in SCAPs. Real-time RT-PCR results showed that HOXB7 over-expression up-regulated the expression of ON (A), BSP (B), OCN (C), COL1 (D) and RUNX2 (E) in SCAPs. GAPDH was used as an internal control. The student’s t test was performed to determine statistical significance. All error bars represent s.d. (n = 3). *P ≤ 0.05. **P ≤ 0.01.

Next, we examined the expression of key transcription factors that regulate osteogenic differentiation, including runt-related transcription factor 2 (RUNX2) and osterix (OSX). We found that the mRNA level of RUNX2 was significantly increased at 3, 7, and 14 days after induction in Flag-HOXB7 overexpressed SCAPs compared to SCAP-Vector cells (Figure 2E). However, the expression levels of OSX were not significantly different between these cell groups (data not shown).

HOXB7 over-expression enhanced osteogenic differentiation potential in SCAPs in vivo

Next, we investigated whether the overexpression of HOXB7 would affect SCAP osteogenesis in vivo. We transplanted control or Flag-HOXB7 SCAPs subcutaneously into nude mice. Eight weeks after transplantation, the transplanted tissues were harvested. The H&E staining revealed that more bone-like mineralized tissue was present in tissues transplanted with Flag-HOXB7 SCAPs compared to tissues transplanted with control SCAPs (Figure 3A). Qualitative measurements of the mineralization showed much more mineral in tissues transplanted with Flag-HOXB7 than those transplanted with control SCAPs (Figure 3B). These transplantation experiments demonstrated that Flag-HOXB7 SCAPs generated more bone-like mineralized tissue than control SCAPs in vivo. Taken together, these results showed that HOXB7 expression substantially triggered osteogenic differentiation in SCAPs.

Figure 3.

HOXB7 over-expression enhances mineralized tissue formation in vivo. SCAPs infected with either the empty vector (control) or with Flag-HOXB7 (HOXB7 overexpression) were transplanted subcutaneously into the dorsal surfaces of 10-week-old immunocompromised beige mice. (A) Tissue sections were prepared 8 weeks after transplantation. Sections were stained with (A) H&E. (B) Qualitative measurement of mineralization in tissue samples after transplantation with SCAPs that overexpressed HOXB7 or control SCAPs. The mineralization area in the tissue sections was calculated based on the results of H&E staining. The student’s t test was performed to determine statistical significance. All error bars represent s.d. (n = 5). **P ≤ 0.01. Bar: 100 µm. B, Bone-like tissues; HA, hydroxyapatite tricalcium carrier; CT, connective tissue.

HOXB7 depletion inhibited the osteogenic differentiation potential of BMSCs in vitro

To further elucidate the function of HOXB7 in MSCs, we designed a shRNA to target and inhibit HOXB7 expression. We introduced this shRNA (BMSC-HOXB7sh) or a Scramble control shRNA (BMSC-Scramsh) into BMSCs by lentiviral infection. After antibiotic selection of infected BMSCs, we determined that the knockdown efficiency in BMSC-HOXB7sh cells was 70% with real-time RT-PCR (Figure 4A). To determine whether HOXB7 intrinsically affected osteogenesis in BMSCs, we cultured these transgenic BMSCs in osteogenic-inducing medium. We found that ALP activity was reduced in BMSC-HOXB7sh cells compared to BMSC-Scramsh cells (Figure 4B). Two weeks after induction, Alizarin Red staining and quantitative calcium measurements revealed that mineralization was also significantly lower in BMSC-HOXB7sh cells compared to BMSC-Scramsh cells (Figure 4C, 4D). Consistent with this finding, real-time RT-PCR results showed that the osteogenic marker genes, ON was down-regulated in HOXB7-depleted cells at 3, 7, and 14 21 days after induction (Figure 5A); BSP was down-regulated in HOXB7-depleted cells at 7, and 14 days after induction (Figure 5B); OCN was strongly inhibited in HOXB7-depleted cells at 0, 3, 7, and 14 days after induction (Figure 5C); COL1A2 was inhibited in HOXB7-depleted cells at 3, and 14 days after induction (Figure 5D). Next, we examined the expression of key transcription factor that regulate osteogenic differentiation, RUNX2. We found that the mRNA level of RUNX2 was significantly lower at 3, 7, and 14 days after induction in BMSC-HOXB7sh cells compared to BMSC-Scramsh cells (Figure 5E).

Figure 4.

HOXB7 knock-down inhibits the osteogenic potential of BMSCs. (A) The real-time RT-PCR result showed that HOXB7 knock-down in BMSCs. GAPDH was used as an internal control. (B) ALP activity assay result showed that, when HOXB7 was knocked-down in BMSCs, the ALP activity was reduced. (C, D) Alizarin Red staining (C) and quantitative calcium analysis (D) showed that, when HOXB7 was knocked-down in BMSCs, mineralization was reduced. Bar: 100 µm. The student’s t test was performed to determine statistical significance. All error bars represent s.d. (n = 3). *P ≤ 0.05. **P ≤ 0.01.

Figure 5.

HOXB7 knock-down decreases the mRNA levels of osteogenic differentiation markers in BMSCs. Real-time RT-PCR results show that HOXB7 knock-down in BMSCs caused reduced expression of ON (A), BSP (B), OCN (C), COL1 (D) and RUNX2 (E). GAPDH was used as an internal control. The student’s t test was performed to determine statistical significance. All error bars represent s.d. (n = 3). *P ≤ 0.05. **P ≤ 0.01.

HOXB7 over-expression enhanced BSP transcription by increasing the binding of HOXB7 in BSP promoter

Next, we investigated how HOXB7 regulated the expression of BSP. We analysis the promoter of BSP, and identified the specific sequence for HOXB7 binding (tcaattaa) in the BSP promoter at +3010 bp up~+3003 bp up upstream of the transcription start site (Figure 6A). Then we investigated whether the HOXB7 could bind on the BSP promoter by a ChIP assay. However, despite repeated efforts, the available commercial anti- HOXB7 antibodies were ineffective in our ChIP assays. To overcome this problem, we constructed retroviruses to express Flag-HOXB7 or the empty Vector in SCAPs (Figure 1A), and we performed ChIP assays with anti-Flag antibodies. The results indicated that, compared to the Vector group, significantly higher levels of Flag-HOXB7 were on associated with the BSP promoter at 3 days after induction (Figure 6B, 6C).

Figure 6.

HOXB7 over-expression increased the binding of HOXB7 in the BSP promoter. A. HOXB7 binding site in the BSP promoter. B. Location of ChIP assay primers targeting the BSP promoter at +3157 to +2908 bp upstream, and ORF at -708 to -791 bp downstream of the transcription start site (TSS). C. ChIP assay showed that HOXB7 over-expression led to changes in the binding of HOXB7 in the BSP promoter at 3days after cultured in osteogenic-inducing medium. Student’s t test was performed to determine statistical significance. Error bars represent SD (n = 3). **P ≤ 0.01.

Discussion

MSCs can differentiate into several cell types, including osteoblasts. The molecular mechanisms underlying directed differentiation remain unclear. HOXB7 is a critical member of homeobox genes and plays a key role in regulating cell fate. Evidence for the potential function of HOXB7 in controlling differentiation of stem cells involved in its critical roles in osteogenesis and age of stem cells [19,25]. Here, we present evidence to extend the functions of HOXB7 to be a critical regulator in maintaining osteogenic differentiation of human post-natal MSCs.

In this study, we investigated the function of HOXB7 in the differentiation of MSCs derived from human dental tissues and bone marrow. First, we found that HOXB7 enhanced ALP activity and mineralization in vitro. Moreover, HOXB7 overexpression enhanced osteogenesis in vivo. These findings indicated that HOXB7 might be a key transcription factor that controls the osteogenic differentiation potential of MSCs.

The commitment of MSCs to an osteogenic lineage requires coordinated inhibition of differentiation toward other lineages. Thus, the activation of multiple transcription factors is associated with MSC differentiation [29,30]. Two of the key transcription factors, RUNX2 and OSX, are necessary for osteogenic differentiation [31-33]. RUNX2 directs the differentiation of MSCs into an osteoblastic lineage, and inhibits them from differentiating into adipocytic and chondrocytic lineages. After differentiating into preosteoblasts, OSX and RUNX2 can drive the cells to become immature osteoblasts and produce bone matrix. As a downstream gene of RUNX2, OSX is a second transcription factor that is essential for osteogenic differentiation; it is specifically expressed in all developing bones [31-34]. Previous study showed that HOXA10 was markedly stimulated in osteoblasts after BMP2 treatment, coincident with the robust expression of RUNX2 [35]. Also, we showed that HOXB7 evoked the significant up-regulation of RUNX2, but didn’t affect OSX expression, indicating that regulation of HOXB7 on osteogenesis mediated by RUNX2. Furthermore, HOXB7 induced expression of the ON, COL1A2, BSP, and OCN genes, which encode extracellular matrix proteins. These results suggested that HOXB7 is a key enhancer of osteogenic differentiation.

In addition, compared with other osteogenic marker genes, BSP was significantly induced by HOXB7 after osteogenic induction, suggested HOXB7 might regulated BSP transcript directly. We analysis the promoter of BSP, and identified the specific sequence for HOXB7 binding (tcaattaa) in the BSP promoter. Furthermore, Over-expression of HOXB7 significantly increased the levels of HOXB7 associated with the BSP promoter by ChIP assays at 3 days after osteogenic induction. These results indicated that HOXB7 could directly bind with the promoter of BSP and promote its transcript.

In conclusion, this study found that HOXB7 was a positive effector for the osteogenic differentiation of MSCs via up-regulation of RUNX2 and directly activating transcript of BSP. These results suggested that activation of HOXB7 signaling might improve tissue regeneration mediated by MSCs. These results have provided insights into the mechanism underlying directed differentiation of MSCs.

Disclosure of conflict of interest

None.

Abbreviations

MSCs

mesenchymal stem cells

SCAPs

stem cells from apical papilla

PDLSCs

periodontal ligament stem cells

DPSCs

dental pulp stem cells

BMSCs

bone marrow stromal stem cells

ALP

alkaline phosphatase

ARS

Alizarin red staining

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

OCN

osteocalcin

OSX

osterix

RUNX2

runt-related transcription factor 2

BSP

bone sialoprotein

OPN

osteopontin