Hypoxia-inducible factor 1α induces osteo/odontoblast differentiation of human dental pulp stem cells via Wnt/β-catenin transcriptional cofactor BCL9

Accelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/β-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers (RUNX2, ALP, OC), BCL9, and Wnt/β-catenin signaling target genes (AXIN2, TCF1) in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and β-catenin expression and BCL9-β-catenin co-localization. In addition, BCL9 formed a complex with β-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/β-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/β-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury.

www.nature.com/scientificreports/ proteasome 12 . However, hypoxic conditions reduce the activity of prolyl hydroxylase 13 , which stabilizes HIF1α in the cytoplasm leading to its translocation to the nucleus. There, HIF1α forms a heterodimer with HIF1β, which is constitutively expressed. The resulting HIF1α and β complex binds to the promoter region of hypoxia-responsive genes, such as vascular endothelial growth factor (VEGF), which further activates the transcription of target genes 14 . Hypoxia-responsive genes possess a cis-acting element, which is called the hypoxia-response element with the core sequence 5′-RCGTG-3′ (R = purine; in most cases 5′-ACGTG-3′) 15 . HIF regulates the expression of multiple genes and is involved in the regulation of energy metabolism 11 , hematopoietic stem cell maintenance 16,17 , angiogenesis 18 , and cancer cell proliferation, apoptosis, invasion, and metastasis 19 . HIF1α signaling is reported to promote osteo/odontoblast differentiation via bone morphogenetic protein (BMP) signaling in stem cells from human exfoliated deciduous teeth extracted from fibrodysplasia ossificans progressiva patients 20 . Wnt/β-catenin signaling plays a pivotal role in cell proliferation, cell differentiation, and tissue homeostasis 21 . In the absence of the ligand Wnt, β-catenin is phosphorylated and degraded by various other factors such as glycogen synthase kinase 3β (GSK3β) and AXIN, but when Wnt binds to the receptor Frizzled, β-catenin in the cytoplasm is stabilized and translocates into the nucleus. β-Catenin then binds to the transcription factors T-cell factor (TCF)/lymphoid enhancer binding factor (LEF) and promotes transcription of target genes such as AXIN2 and TCF1 21 . Studies have disclosed that β-catenin-stabilized mice show excessive dentin and cementum formation 22 , and that Wnt/β-catenin signaling is essential during tooth development 23 . Moreover, direct capping of exposed dental pulp with GSK3β antagonists induces the formation of more reparative dentin than that when using collagen sponges or MTA cement 24 . These findings support the notion that Wnt/β-catenin signaling is crucial to regulate osteo/odontoblast differentiation and the formation of mineralized tissues such as bone and dentin.
Wnt/β-catenin signaling is involved in carcinogenesis not only in colorectal cancer but also in many other cancer entities 25 . HIF1α is also involved in carcinogenesis and tumor growth through the regulation of angiogenesis, glycolytic metabolism, and other biological mechanisms 26 . Recently, hypoxia and HIF1α have been reported to activate B-cell chronic lymphocytic leukemia/lymphoma 9 (BCL9), which is an essential component of canonical Wnt/β-catenin signaling 27,28 . BCL9 acts as a transcriptional cofactor in Wnt/β-catenin signaling, and BCL9 binds to β-catenin and promotes the formation of the β-catenin-TCF complex, which in turn induces the transcription of target genes 29 . BCL9 is also required for activation of the Wnt/β-catenin cascade in adult mammalian myogenic progenitors 30 . BCL9 possesses hypoxia-response elements in its promoter region, and hypoxia and HIF1α induce BCL9 expression in liver, colon, and prostate cancer cells 27,28 . Thus, hypoxia and HIF1α activate Wnt/β-catenin signaling via BCL9, resulting in cancer cell proliferation and metastasis 27 .
We hypothesized that hypoxia and HIF1α are involved in the osteo/odontoblast differentiation of human dental pulp stem cells (hDPSCs) by activating Wnt/β-catenin signaling via BCL9 induced by hypoxia and HIF1α. The aim of this study was to elucidate the role of BCL9 in HIF1α-induced osteo/odontoblast differentiation of hDPSCs.
HIF1α protein expression was higher in hDPSCs cultured under hypoxic conditions for 48 h than in those cultured under normoxic ones (Fig. 2a). The mRNA expression of VEGF, a target gene of HIF1α, and runtrelated transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteocalcin (OC), osteo/odontoblast differentiation markers, was significantly upregulated in hDPSCs cultured under hypoxic conditions compared with that in hDPSCs cultured under normoxic ones (p < 0.05, Fig. 2b,c). Overexpression of HIF1α in hDPSCs transfected with a HIF1α-expression vector induced increased expression of HIF1α protein (Fig. 2d) and significant upregulation of the mRNA expression of VEGF, RUNX2, ALP, and OC (p < 0.05, Fig. 2e,f).

Discussion
Hypoxia and HIF1α are reported to promote osteo/odontoblast differentiation 20 and mineralization 8 , but the precise mechanisms behind these effects remain unclear. Here, we revealed that hypoxia/HIF1α-dependent osteo/ odontoblast differentiation was partially dependent on the activation of Wnt/β-catenin signaling, and that BCL9 acted as a key mediator between HIF1α and Wnt/β-catenin signaling. In this study, hypoxia (1% O 2 ) promoted the mRNA expression of RUNX2, ALP, and OC in hDPSCs, which are representative osteo/odontoblast differentiation markers 31 (Fig. 2c). This result is consistent with a previous report showing that hypoxic culture promotes osteo/odontoblastic gene expression and mineralization of human dental pulp cells 8 . Moreover, the overexpression of HIF1α, an essential transcription factor activated in hypoxic conditions, also upregulated the mRNA expression of RUNX2, ALP, and OC in hDPSCs (Fig. 2f). HIF1α is known to induce the upregulation of BMP signaling, which has been reported to be responsible for osteo/odontoblast differentiation in stem cells from human exfoliated deciduous teeth 20 . However, we failed to detect the upregulation of mRNAs for BMP2 and BMP4 and the phosphorylation of SMAD4 in hDPSCs cultured under hypoxic conditions and HIF1α-overexpressing hDPSCs (data not shown). This led us to the notion that mechanisms other than BMP signaling are involved in the hypoxia/HIF1α-dependent osteo/odontoblast differentiation in hDPSCs. Then, we focused on Wnt/β-catenin signaling, which is one of the essential signaling pathways involved in the differentiation of mineralized tissue-forming cells including hDPSCs 24 . Our study revealed that the mRNA expression of TCF1, a target gene of Wnt/β-catenin signaling, was promoted in hDPSCs cultured under hypoxic conditions and HIF1α-overexpressing hDPSCs (Fig. 3b,d). AXIN2 is also regarded as a major target gene of Wnt/β-catenin signaling, although the degradation of β-catenin is induced by AXIN2 32 . We failed to detect the upregulation of AXIN2 in hDPSCs cultured under hypoxic conditions, although its expression was promoted in HIF1α-overexpressing hDPSCs. Various factors other than HIF1α are activated in hypoxic conditions; for example, hypoxia activates notch signaling 33 (Figs. 3b and 4b), which might be explained by the influence of negative signals to Wnt pathways on the hypoxic-cultured cells. The intracellular domain of Notch1 limits β-catenin-induced transcription of genes including TCF1 through the formation of a complex that requires its interaction with RBPjκ 35 . Moreover, a high expression of BCL9 in SW480, a primary human adenocarcinoma of the colon, is accompanied with a high expression of TCF1 36 . Furthermore, blocking of BCL9 expression induces downregulation of AXIN2 and, in a greater extent,TCF1 in SW480 37 . These findings suggest synergic actions of BCL9 and TCF1. However, we detected the upregulation of TCF1, a major target gene of Wnt/β-catenin signaling, in hDPSCs cultured under hypoxic conditions and the upregulation of AXIN2 and TCF1 in HIF1α-overexpressing hDPSCs. These results suggest that crosstalk between HIF1α and Wnt/β-catenin signaling may be induced in hDPSCs cultured under hypoxic conditions and HIF1α-overexpressing hDPSCs. Hypoxia is a typical and common feature of tumor microenvironment primarily caused by an inadequate and heterogeneous vascular network 38 , and hypoxic condition characterizes the properties of tumors 39 . HIF1α and Wnt/β-catenin signaling are involved in carcinogenesis 25,26 , and BCL9 is reported to participate in the HIF1α-derived activation of Wnt/β-catenin signaling during tumorigenesis in the liver 27 and intestine 28 . BCL9 is required for activation of the Wnt/β-catenin cascade in adult mammalian myogenic progenitors 30 . BCL9 possesses hypoxia-responsive elements in its promoter region, and hypoxia and HIF1α induce BCL9 expression in liver, colon, and prostate cancer cells 28 . BCL9, in turn, binds to β-catenin and promotes the formation of the β-catenin-TCF complex, triggering the transcription of target genes 29 . Hypoxia and HIF1α activate Wnt/βcatenin signaling via the expression of BCL9, resulting in cancer cell proliferation and metastasis 27 . Hypoxic condition is involved in ischemia-induced ectopic dental pulp mineralization, where osteo/odontoblastic differentiation of DPSCs is promoted 8,9 , and Wnt signaling is one of essential signaling pathways in odontoblast differentiation 40 . Thus, it seems reasonable to assume that DPSCs and cancer cells share a common intracellular signaling mechanism, i.e., hypoxic-induced activation of Wnt signaling, which is now revealed as a new differentiation mechanism in DPSCs.
In this study, mRNA expression of BCL9 was upregulated in hDPSCs cultured under hypoxic conditions and HIF1α-overexpressing hDPSCs (Fig. 3a,c). We then performed the overexpression and suppression of BCL9 in hDPSCs to examine the function of BCL9 in hDPSCs. The overexpression of BCL9 promoted the mRNA expression of AXIN2 and TCF1 (Fig. 4b) and the transcriptional activity of TCF/LEF (Fig. 4d), whereas the suppression of BCL9 induced downregulation of these genes (Fig. 5b). The formation of BCL9/β-catenin is considered to be essential for the nuclear translocation of β-catenin 29 . We revealed that BCL9 bound to β-catenin by using an immunoprecipitation assay (Fig. 4e), and confirmed that BCL9 and β-catenin proteins translocate to the nucleus in hDPSCs (Fig. 4f). Moreover, ex vivo hypoxic culture of mouse dental pulp tissue revealed the upregulation and nuclear translocation of Hif1α (Fig. 6a), Bcl9, and β-catenin (Fig. 6b), and Bcl9 and β-catenin were co-localized in the nucleus (Fig. 6b). Collectively, these findings indicated that BCL9 is involved in the activation of Wnt/βcatenin signaling as a molecule downstream of HIF1α in hDPSCs. However, BCL9 is reported to interact with www.nature.com/scientificreports/ not only β-catenin but other proteins such as clathrin and the components in Wnt destruction complex 41 , and it cannot be ruled out that BCL9 activates intracellular signaling pathways other than Wnt/β-catenin signaling [41][42][43] .
Overexpression and suppression of BCL9 induced up-and downregulation, respectively, of the mRNA expression of RUNX2, ALP, and OC in hDPSCs (Figs. 4c, 5c), which indicates that BCL9 is involved in the osteo/ odontoblast differentiation of hDPSCs via Wnt/β-catenin signaling. This notion was also supported by the finding that a Wnt pathway inhibitor suppressed osteo/odontoblast differentiation and Wnt/β-catenin signaling of hDPSCs overexpressing BCL9 or HIF1α (Supplemental Fig. 1). The involvement of Bcl9 in enamel formation was previously reported, and conditional deletion of both Bcl9 and Bcl9l was shown to induce abnormal enamel formation 42 . Our study is the first to reveal the involvement of BCL9 in the osteo/odontoblast differentiation of www.nature.com/scientificreports/ hDPSCs. However, further study is necessary to reveal the involvement of HIF1α/BCL9/Wnt signaling in each stage of the osteo/odontoblastic differentiation of hDPSCs. In summary, our results indicate that, in hDPSCs, hypoxia induces the stabilization of HIF1α and HIF1α stimulates the expression of BCL9, which in turn activates Wnt/β-catenin signaling and induces osteo/odontoblast differentiation (Fig. 7), although involvement of HIF1α/BCL9 signaling in all stages of their differentiation has not been fully revealed. These findings provide new insight into the mechanism of pulp tissue mineralization, particularly following traumatic dental injury. The inhibition of BCL9 could be a new therapeutic approach to prevent the excessive dental pulp mineralization and root canal obliteration that can develop after traumatic dental injury.

Methods
Cell culture. All procedures were approved by the Ethical Committee of Tokyo Medical and Dental University (#D2014-039-03) and performed in accordance with the Ethical Guidelines for Clinical Studies. Informed consent was obtained from all participants in accordance with the Ethical Guidelines for Clinical Studies. Human dental pulp stem cells (hDPSCs) were obtained from freshly extracted wisdom teeth and were cultured in alphamodified minimum essential medium (α-MEM; Fujifilm Wako Pure Chemical, Osaka, Japan) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and an antibiotic and antifungal solution (penicillin-streptomycin-amphotericin B suspension; Fujifilm Wako Pure Chemical) at 37 °C and 5% CO 2 in air (referred to as 21% O 2 ). Hypoxic conditions were maintained with a gas mixture of 1% O 2 , 5% CO 2 , and 94% N 2 (referred to as 1% O 2 ) for 48 h. All methods used in this study were in accordance with the Declaration of Helsinki. Quantitative RT-PCR. hDPSCs were plated at a concentration of 2.0 × 10 5 cells/well in a six-well culture plate. Total RNA was isolated with QuickGene-Mini80 (Kurabo, Tokyo, Japan), and cDNA was synthesized from 150 ng of total RNA using PrimeScript™ RT Master Mix (Takara Bio, Kusatsu, Japan). Quantitative RT-PCR was performed with GoTaq qPCR Master Mix (Promega). β-Actin was used as an internal control. The specific primers used in this study are shown in Table 1.
Luciferase assay. hDPSCs were plated at a concentration of 5.0 × 10 4 cells/well in a 24-well plate and were lysed 24 h after transfection using 100 μL of lysis buffer (Cell Culture Lysis Reagent; Promega). Luciferase activity was measured using a luciferase assay substrate (Luciferase Assay System; Promega) and a luminometer (AB-2200; ATTO, Tokyo, Japan).
Co-immunoprecipitation. hDPSCs (5 × 10 5 cells/dish) in 60 mm dishes were lysed in 1 ml of RIPA buffer after the overexpression of BCL9 (FLAG Tag) and β-catenin (V5 Tag) for 24 h. V5 mouse IgG2bκ isotype antibody (M215-7; MBL) was added as a primary antibody and reacted at 4 °C for 1 h, and then reacted with Protein G PLUS-Agarose (Santa Cruz Biotechnology) at 4 °C overnight under rotation. The samples were then centrifuged at 2500 rpm for 5 min, and the supernatant was aspirated and washed four times. The immunoprecipitated samples were checked for protein expression by western blotting using FLAG mouse IgG2aκ isotype antibody Immunocytochemistry. Pulp tissues were fixed with 4% paraformaldehyde at 4 °C overnight, after which they were embedded in Tissue-Tek ® O.C.T. Compound (Sakura Finetek, Tokyo, Japan). Six-micrometer-thick frozen sections were prepared. Anti-HIF1α rabbit polyclonal antibody (1:500, GTX127309; GeneTex), anti-BCL9 rabbit polyclonal antibody (1:100, PA5-93229; Thermo Fisher Scientific), and anti-β-catenin mouse IgG1κ monoclonal antibody (1:3200, 37447; Cell Signaling Technology) were used for primary antibodies, and samples were incubated with one of the primary antibodies overnight at 4 °C. Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500; Molecular Probes, Eugene, OR, USA) or Alexa Fluor 594-conjugated goat anti-mouse IgG (1:500; Molecular Probes) was used as a secondary antibody and applied for 60 min at room temperature. Mounting Medium with DAPI (Abcam, Cambridge, UK) was used for nuclear staining.
Statistical analysis. Statistical analysis was conducted using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). F test, Mann-Whitney's U test, and Student's t test were performed with significance set at p < 0.05.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.