Summary

Mesenchymal stromal progenitor cells (MSCs) are multipotent progenitors that can be isolated from numerous tissues. MSCs can undergo osteogenic differentiation under proper stimuli. We have recently demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most osteogenic BMPs. As one of the least studied BMPs, BMP9 has been shown to regulate angiogenesis in endothelial cells. However, it is unclear whether BMP9-regulated angiogenic signaling plays any important role in the BMP9-initiated osteogenic pathway in MSCs. Here, we investigate the functional role of hypoxia-inducible factor 1α (HIF1α)-mediated angiogenic signaling in BMP9-regulated osteogenic differentiation of MSCs. We find that BMP9 induces HIF1α expression in MSCs through Smad1/5/8 signaling. Exogenous expression of HIF1α potentiates BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo. siRNA-mediated silencing of HIF1α or HIF1α inhibitor CAY10585 profoundly blunts BMP9-induced osteogenic signaling in MSCs. HIF1α expression regulated by cobalt-induced hypoxia also recapitulates the synergistic effect between HIF1α and BMP9 in osteogenic differentiation. Mechanistically, HIF1α is shown to exert its synergistic effect with BMP9 by inducing both angiogenic signaling and osteogenic signaling in MSCs. Thus, our findings should not only expand our understanding of the molecular basis behind BMP9-regulated osteoblastic lineage-specific differentiation, but also provide an opportunity to harness the BMP9-induced synergy between osteogenic and angiogenic signaling pathways in regenerative medicine.

Introduction

Mesenchymal stromal progenitor cells (MSCs) are multipotent progenitors which can undergo self-renewal and differentiate into multi-lineages, including osteogenic, chondrogenic, and adipogenic lineages (Deng et al., 2008; Pittenger et al., 1999; Prockop, 1997). Although MSCs have been isolated from numerous tissues, one of the major sources in adults is the bone marrow stromal cells. Osteogenic differentiation is a sequential cascade that recapitulates most, if not all, of the molecular events occurring during embryonic skeletal development (Olsen et al., 2000). Bone morphogenetic proteins (BMPs) play an important role during development (Deng et al., 2008; Luu et al., 2007; Shi and Massagué, 2003) and have been shown to regulate stem cell proliferation and osteogenic differentiation (Varga and Wrana, 2005; Zhang and Li, 2005). BMPs belong to the TGFβ superfamily; and there are at least 14 BMPs in humans and rodents (Deng et al., 2008; Hogan, 1996; Luu et al., 2007; Shi and Massagué, 2003).

We have found that BMP9 is one of the most potent BMPs among the 14 types of BMPs in inducing osteogenic differentiation of MSCs both in vitro and in vivo by regulating several important downstream targets during BMP9-induced osteoblast differentiation (Cheng et al., 2003; Kang et al., 2004; Luo et al., 2004; Luther et al., 2011; Luu et al., 2007; Peng et al., 2003; Peng et al., 2004; Sharff et al., 2009; Tang et al., 2009). BMP9 (also known as growth differentiation factor 2, or GDF-2) was identified in the developing mouse liver (Song et al., 1995). As one of the least studied BMPs, BMP9 has been shown to play some roles in inducing and maintaining the cholinergic phenotype of embryonic basal forebrain cholinergic neurons (López-Coviella et al., 2000), inhibiting hepatic glucose production and inducing the expression of key enzymes of lipid metabolism (Chen et al., 2003), and stimulating hepcidin 1 expression (Truksa et al., 2006).

Osteogenesis usually involves two distinct pathways (Olsen et al., 2000): intramembranous bone formation for the flat bones of the skull and endochondral bone formation for long bones. Endochondral bone formation occurs in close spatial and temporal association and proximity to capillary invasion, so that angiogenesis and osteogenesis must be tightly coupled (Olsen et al., 2000; Wan et al., 2010; Wang et al., 2007). Conflicting results have implicated BMP9 as either an angiogenesis inducer in endothelial cells (Castonguay et al., 2011; Cunha et al., 2010; Mitchell et al., 2010; Park et al., 2012; Scharpfenecker et al., 2007; Suzuki et al., 2010; Yao et al., 2012) or as a potent anti-angiogenic factor (David et al., 2008). Although it is well recognized that osteogenic and angiogenic pathways are well coordinated during bone formation (Wan et al., 2010), it is unclear how these processes are linked in MSCs stimulated by osteogenic factors, such as BMPs.

Here, we investigate whether hypoxia-inducible factor 1α (HIF1α)-mediated angiogenic signaling plays any role in BMP9-regulated osteogenic differentiation of MSCs. HIF1α is a well established regulator of angiogenic cascade, which usually regulates many development processes (Majmundar et al., 2010; Wan et al., 2010). We find that BMP9 directly induces HIF1α expression in MSCs through Smad1/5/8 signaling. Exogenous expression of HIF1α potentiates BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo, while siRNA-mediated silencing of HIF1α or HIF1α inhibitor CAY10585 profoundly blunts BMP9-induced osteogenic signaling in MSCs. HIF1α expression regulated by cobalt-induced hypoxia recapitulates the synergistic effect between HIF1α and BMP9 in osteogenic differentiation. Mechanistically HIF1α is shown to exert its synergistic effect with BMP9 by inducing both angiogenic signaling (e.g. VEGF) and osteogenic signaling in MSCs. Taken together, our findings should not only expand our understanding of the molecular basis behind BMP9-regulated osteoblastic lineage-specific differentiation, but also provide an opportunity to harness BMP9-induced synergy between osteogenic and angiogenic signaling pathways in regenerative medicine.

Results

HIF1α is upregulated in BMP9-stimulated MSCs

Our previous studies have revealed that BMP9 is one of the most osteogenic factors for inducing osteoblastic differentiation in MSCs (Cheng et al., 2003; Deng et al., 2008; Kang et al., 2004; Luther et al., 2011; Luu et al., 2007). However, the molecular mechanisms underlying BMP9 functions in MSCs remain to be fully elucidated. Here, we investigated if HIF1α plays any role in BMP9 osteogenic signaling in MSCs. By using semi-quantitative PCR (sqPCR) analysis, we found that HIF1α was upregulated in MSC line C3H10T1/2 cells at 48 hours after BMP9 transduction (Fig. 1A). Similar results were obtained by western blotting analysis using anti-HIF1α antibody (Fig. 1B). These findings suggest that HIF1α may function as a downstream target of BMP9 signaling.

Fig. 1.

BMP9 regulates HIF1α expression via Smad transcriptional factors in MSCs. (A) Time course expression of HIF-1α upon BMP9 stimulation using sqPCR. Subconfluent C3H10T1/2 cells were cultured in 0.5% FBS DMEM and infected with AdBMP9 or AdGFP. Total RNA was collected at the indicated time points and subjected to sqRT-PCR analysis. All samples were normalized for GAPDH expression. (B) Western blotting analysis of BMP9-induced HIF1α expression. At the indicated time points, AdBMP9- or AdGFP-infected C3H10T1/2 cells were lysed and subjected to western blotting using an anti-HIF1α antibody. The expression level of β-actin was used as a loading control. (C) ChIP analysis of the mouse HIF1α promoter. C3H10T1/2 cells were infected with AdBMP9 or AdGFP for 36 hours followed by formaldehyde crosslinking. The crosslinked cells were lysed and subjected to sonication and immunoprecipitation using anti-Smad1/5/8 or control IgG. The recovered chromatin DNA fragments were used for PCR amplifications with primers specific for the mouse HIF1α promoter.

Fig. 1.

BMP9 regulates HIF1α expression via Smad transcriptional factors in MSCs. (A) Time course expression of HIF-1α upon BMP9 stimulation using sqPCR. Subconfluent C3H10T1/2 cells were cultured in 0.5% FBS DMEM and infected with AdBMP9 or AdGFP. Total RNA was collected at the indicated time points and subjected to sqRT-PCR analysis. All samples were normalized for GAPDH expression. (B) Western blotting analysis of BMP9-induced HIF1α expression. At the indicated time points, AdBMP9- or AdGFP-infected C3H10T1/2 cells were lysed and subjected to western blotting using an anti-HIF1α antibody. The expression level of β-actin was used as a loading control. (C) ChIP analysis of the mouse HIF1α promoter. C3H10T1/2 cells were infected with AdBMP9 or AdGFP for 36 hours followed by formaldehyde crosslinking. The crosslinked cells were lysed and subjected to sonication and immunoprecipitation using anti-Smad1/5/8 or control IgG. The recovered chromatin DNA fragments were used for PCR amplifications with primers specific for the mouse HIF1α promoter.

HIF1α is a direct target of BMP9/Smad signaling

We next tested if HIF1α is a direct target of BMP9 signaling by analyzing if its genomic promoter can interact with BMP-specific Smad1/5/8. We conducted ChIP analysis using Smad1/5/8 antibody or isotype IgG to pull down genomic DNA from MSCs transduced with BMP9 or GFP. HIF1α promoter-specific primers were shown to yield expected products in BMP9 stimulation- and Smad1/5/8 pulldown-dependent fashion (Fig. 1C). These results suggest that HIF1α expression may be regulated by BMP9 through BMP-specific R-Smad1/5/8.

HIF1α potentiates BMP9-induced osteogenic differentiation of MSCs in vitro

If HIF1α is an important target of BMP9-mediated osteogenic signaling, we hypothesized that exogenous expression of HIF1α would enhance BMP9-induced osteogenic differentiation of MSCs. We found that endogenous HIF1α expression was almost undetectable in MSCs when cultured under low fetal calf serum conditions (Fig. 2Aa). To effectively introduce exogenous HIF1α in MSCs, we constructed a recombinant adenovirus expressing human HIF1α (AdR-HIFα) and demonstrated that AdR-HIF1α mediated a robust transgene expression in MSCs (Fig. 2Ab), which were effectively transduced by adenoviral vectors (Fig. 2Ac).

Fig. 2.

HIF1α potentiates BMP9-induced early and late osteogenic markers in MSCs. (A) Endogenous and exogenous expression of HIF1α in MSCs. For endogenous expression, total RNA was isolated from C3H10T1/2 and iMEF lines (cultured in 1% FBS medium). sqPCR was performed using primers specific for mouse GAPDH and HIF1α (Aa). For adenovirus-mediated exogenous expression of HIF1α, C3H10T1/2 cells were infected with Ad-RFP or AdR-HIF1α. Total RNA was isolated 48 hours and 72 hours after infection and subjected to sqPCR using primer pairs specific for human HIF1α (Ab). The recombinant adenovirus AdR-HIF1α was shown to effectively transduce MSCs, such as C3H10T1/2 and iMEFs (Ac). (B) HIF1α potentiates BMP9-induced ALP activity in MSCs. Subconfluent C3H10T1/2 cells and/or iMEFs were infected with AdBMP9, AdR-HIF1α and/or AdGFP. ALP activity was measured at days 5, 7 and 9 by histochemical staining (C3H10T1/2 shown; Ba) and chemiluminescent assays (Bb). (C) HIF1α augments BMP9-induced late osteogenic markers and mineralization in MSCs. C3H10T1/2 cells were infected with recombinant adenoviruses as indicated. At 12 days post infection, the expression of osteopontin (OPN; Ca) and osteocalcin (OCN; Cb) was assessed by immunohistochemical staining analysis using anti-OPN or anti-OCN antibody. For matrix mineralization assay, C3H10T1/2 cells were infected with the indicated adenoviruses and cultured in mineralization medium. Alizarin Red S staining was performed at 14 days after infection (Cc). Each assay was done in triplicate and/or carried out in at least three independent experiments. Representative results are shown.

Fig. 2.

HIF1α potentiates BMP9-induced early and late osteogenic markers in MSCs. (A) Endogenous and exogenous expression of HIF1α in MSCs. For endogenous expression, total RNA was isolated from C3H10T1/2 and iMEF lines (cultured in 1% FBS medium). sqPCR was performed using primers specific for mouse GAPDH and HIF1α (Aa). For adenovirus-mediated exogenous expression of HIF1α, C3H10T1/2 cells were infected with Ad-RFP or AdR-HIF1α. Total RNA was isolated 48 hours and 72 hours after infection and subjected to sqPCR using primer pairs specific for human HIF1α (Ab). The recombinant adenovirus AdR-HIF1α was shown to effectively transduce MSCs, such as C3H10T1/2 and iMEFs (Ac). (B) HIF1α potentiates BMP9-induced ALP activity in MSCs. Subconfluent C3H10T1/2 cells and/or iMEFs were infected with AdBMP9, AdR-HIF1α and/or AdGFP. ALP activity was measured at days 5, 7 and 9 by histochemical staining (C3H10T1/2 shown; Ba) and chemiluminescent assays (Bb). (C) HIF1α augments BMP9-induced late osteogenic markers and mineralization in MSCs. C3H10T1/2 cells were infected with recombinant adenoviruses as indicated. At 12 days post infection, the expression of osteopontin (OPN; Ca) and osteocalcin (OCN; Cb) was assessed by immunohistochemical staining analysis using anti-OPN or anti-OCN antibody. For matrix mineralization assay, C3H10T1/2 cells were infected with the indicated adenoviruses and cultured in mineralization medium. Alizarin Red S staining was performed at 14 days after infection (Cc). Each assay was done in triplicate and/or carried out in at least three independent experiments. Representative results are shown.

While exogenous HIF1α expression alone did not exert any significant effect on osteogenic early marker alkaline phosphatase (ALP) activity, HIF1α was shown to exhibit a profound synergistic effect on BMP9-induced ALP activity in C3H10T1/2 MSCs (Fig. 2Ba). Quantitatively, HIF1α-mediated synergistic effect on ALP activity in BMP9-transduced C3H10T1/2 cells was increased by 79%, 80% and 175% on days 5, 7, and 9, respectively (Fig. 2Bb). A similar synergistic effect was also found in iMEFs as HIF1α was shown to increase ALP activity in BMP9-transduced iMEFs by 20%, 64%, and 165% on days 3, 5, and 7, respectively (Fig. 2Bc). Moreover, we analyzed the effect of HIF1α on late osteogenic markers osteopotin (OPN) and osteocalcin (OCN), and found that exogenous HIF1α expression augmented BMP9-induced expression of OPN and OCN (Fig. 2Ca,Cb). We also demonstrated that HIF1α enhanced matrix mineralization in BMP9-transduced MSCs (Fig. 2Cc). Taking these results together, HIF1α is shown to potentiate BMP9-induced osteoblastic commitment and terminal differentiation of MSCs in vitro.

HIF1α is an important mediator of BMP9 signaling, and silencing its expression effectively diminishes BMP9-induced osteogenic differentiation in vitro

To further confirm if HIF1α is a crucial mediator of BMP9 osteogenic signaling, we constructed a recombinant adenovirus that expresses a pool of four siRNAs targeting mouse HIF1α coding region using our recently established pSOS system (Luo et al., 2007b), resulting in AdR-simHIF1α, which was shown to effectively silence HIF1α expression in MSCs at as early as 48 hours post infection (Fig. 3A). BMP9-induced ALP activity was significantly decreased in iMEFs by AdR-simHIF1α to 9%, 8%, and 29% of the BMP9 control's on days 3, 5, and 7, respectively (Fig. 3Ba). Qualitatively histochemical staining revealed similar results and the expression of simHIF1α effectively reduced BMP9-induced ALP activity in MSCs (Fig. 3Bb). Furthermore, simHIF1α expression almost completely blunted BMP9-induced matrix mineralization in MSCs as illustrated by Alizarin Red S staining (Fig. 3C). These results strongly suggest that HIF1α may play a crucial role in BMP9-induced osteogenic differentiation of MSCs.

Fig. 3.

Silencing HIF1α expression effectively diminishes BMP9-induced osteogenic differentiation of MSCs in vitro. (A) Effective silencing of HIF1α expression mediated by adenovirus siRNA expression vector AdR-simHIF1α in MSCs. The iMEFs were infected with AdR-simHIF1α or AdRFP control virus and cultured in 10% FBS completed medium. At 48 hours and 72 hours after infection, total RNA was isolated for sqPCR analysis using primers specific for mouse HIF1α and GAPDH (as a control). (B) Silencing HIF1α inhibits BMP9-induced ALP activity. iMEFs were co-infected with Ad-BMP9, AdR-simHIF1α and/or AdRFP. ALP activity was quantitatively measured at the indicated time points (Ba). ALP staining was also carried out at different time points. The staining results from day 5 are shown (Bb). (C) Alizarin Red S staining. iMEFs cells were infected with adenoviruses as indicated. Alizarin Red S staining was conducted at 10 days after infection. Each assay was done in triplicate and/or carried out in at least three independent experiments. Representative results are shown.

Fig. 3.

Silencing HIF1α expression effectively diminishes BMP9-induced osteogenic differentiation of MSCs in vitro. (A) Effective silencing of HIF1α expression mediated by adenovirus siRNA expression vector AdR-simHIF1α in MSCs. The iMEFs were infected with AdR-simHIF1α or AdRFP control virus and cultured in 10% FBS completed medium. At 48 hours and 72 hours after infection, total RNA was isolated for sqPCR analysis using primers specific for mouse HIF1α and GAPDH (as a control). (B) Silencing HIF1α inhibits BMP9-induced ALP activity. iMEFs were co-infected with Ad-BMP9, AdR-simHIF1α and/or AdRFP. ALP activity was quantitatively measured at the indicated time points (Ba). ALP staining was also carried out at different time points. The staining results from day 5 are shown (Bb). (C) Alizarin Red S staining. iMEFs cells were infected with adenoviruses as indicated. Alizarin Red S staining was conducted at 10 days after infection. Each assay was done in triplicate and/or carried out in at least three independent experiments. Representative results are shown.

HIF1α is crucial to BMP9-induced terminal differentiation of MSCs in vivo

While the above in vitro studies established that HIF1α may play an important role in BMP9-mediated osteogenic signaling, it was imperative to demonstrate if HIF1α played such a role in vivo. Using our previously established stem implantation assay (Chen et al., 2010; Huang et al., 2012a; Huang et al., 2012b; Luo et al., 2010; Zhang et al., 2010), we injected the iMEFs transduced with BMP9 and HIF1α, simHIF1α, or RFP subcutaneously into the flanks of athymic nude mice for 4 weeks. The iMEFs transduced with RFP, HIF1α or simHIF1α alone failed to form any detectable masses (data not shown). We found that HIF1α significantly augmented BMP9-induced bony mass formation whereas simHIF1α inhibited BMP9-induced bone formation (Fig. 4Aa). The gross size differences were further confirmed by iso-surface three-dimensional (3D) analysis of the μCT imaging data (Fig. 4Ab). The hit map analysis of average mineral density revealed that exogenous HIF1α expression increased the average mineral density of the bone masses formed by BMP9-transduced MSCs, and conversely, silencing HIF1α expression significantly decreased the average mineral density (Fig. 4Ac).

Fig. 4.

HIF1α plays an important role in promoting efficient terminal differentiation of MSCs in vivo. (A) BMP9-induced ectopic bone formation is enhanced by exogenous HIF1α and inhibited by silencing of HIF1α. iMEFs were co-transduced with BMP9, RFP, HIF1α and/or simHIF1α adenoviruses for 16 hours and collected for subcutaneous injections into the flanks of athymic nude mice. Bony masses were collected at 4 weeks (Aa) and subjected to microCT analysis to determine the 3D iso-surface (Ab) and the hit map of average mineralization density (Ac). In the hit map analysis, red represents the highest average mineral density and green the lowest. (B) Histologic analysis of the retrieved samples. The retrieved samples were fixed, decalcified, paraffin-embedded and subjected to H&E staining (Ba), Masson's Trichrome staining (Bb), and Alcian Blue staining (Bc). Magnification, 200×. (C) HIF1α expression affects BMP9-induced trabecular structures, including the percentage of trabecular area to the total area (Ca) and the thickness of the trabeculae (Cb), as analyzed using ImageJ software.

Fig. 4.

HIF1α plays an important role in promoting efficient terminal differentiation of MSCs in vivo. (A) BMP9-induced ectopic bone formation is enhanced by exogenous HIF1α and inhibited by silencing of HIF1α. iMEFs were co-transduced with BMP9, RFP, HIF1α and/or simHIF1α adenoviruses for 16 hours and collected for subcutaneous injections into the flanks of athymic nude mice. Bony masses were collected at 4 weeks (Aa) and subjected to microCT analysis to determine the 3D iso-surface (Ab) and the hit map of average mineralization density (Ac). In the hit map analysis, red represents the highest average mineral density and green the lowest. (B) Histologic analysis of the retrieved samples. The retrieved samples were fixed, decalcified, paraffin-embedded and subjected to H&E staining (Ba), Masson's Trichrome staining (Bb), and Alcian Blue staining (Bc). Magnification, 200×. (C) HIF1α expression affects BMP9-induced trabecular structures, including the percentage of trabecular area to the total area (Ca) and the thickness of the trabeculae (Cb), as analyzed using ImageJ software.

The retrieved samples were further subjected to histologic analysis and other special staining. Both H&E staining and trichrome staining revealed that HIF1α significantly enhanced BMP9-induced bone formation and mineralization and that silencing HIF1α inhibited BMP9-induced osteogenesis (Fig. 4Ba,Bb). However, changes in HIF1a expression levels did not affect BMP9's effect on chondrogenesis (Fig. 4Bc). Quantitative analyses indicate that co-expression of BMP9 and HIF1α significantly increased the average thickness of trabeculae and the percentage of trabecular area over total area, whereas knocking down HIF1α expression exhibited an inhibitory effect (Fig. 4Ca,Cb). These in vivo findings are supported by the in vitro studies. Collectively, our results thus far strongly indicate that HIF1α is a crucial mediator of BMP9 osteogenic signaling and that exogenous HIF1α expression augments BMP9-induced osteogenic differentiation of MSCs and produces more mature bone.

BMP9-induced osteogenic differentiation of MSCs can be effectively blunted by HIF1α inhibitor CAY10585

We also studied the functional importance of HIF1α in BMP9 signaling by using CAY10585, a novel small molecule inhibitor of HIF1α accumulation and gene transcriptional activity. We found that BMP9 and/or HIF1α-induced ALP activity in C3H10T1/2 cells was inhibited by CAY10585 in a dose-dependent fashion (Fig. 5Aa). The inhibitory effect of CAY10585 on ALP activity was also confirmed by ALP histochemical staining (Fig. 5Ab). Furthermore, CAY10585 was shown to effectively inhibit BMP9 and HIF1a-induced late stage osteogenic differentiation, as demonstrated by Alizarin Red mineralization staining (Fig. 5B). Thus, these results confirm that HIF1α plays a crucial role in BMP9-induced osteogenic differentiation.

Fig. 5.

HIF1α inhibitor CAY10585 effectively blunts BMP9-induced osteogenic differentiation of MSCs. (A) CAY10585 inhibits BMP9-induced osteogenic signaling in a dose-dependent fashion. C3H10T1/2 cells were infected with BMP9, GFP and/or HIF1α and treated with or without CAY10585 (25 nM and/or 50 nM). At the indicated time points, ALP activity was quantitatively measured (Aa) and histochemically stained (Ab). (B) CAY10585 effectively reduces BMP9-induced matrix mineralization of MSCs. C3H10T1/2 cells were infected with BMP9, GFP and/or HIF1α; treated with CAY10585 (50 nM) or DMSO; and maintained in mineralization medium. The CAY10585- or DMSO-containing mineralization medium was changed every 2 days for the first 6 days. At day 12, Alizarin Red S staining was conducted on the treated cells. Each assay was done in triplicate and/or carried out in at least three independent experiments. Representative results are shown.

Fig. 5.

HIF1α inhibitor CAY10585 effectively blunts BMP9-induced osteogenic differentiation of MSCs. (A) CAY10585 inhibits BMP9-induced osteogenic signaling in a dose-dependent fashion. C3H10T1/2 cells were infected with BMP9, GFP and/or HIF1α and treated with or without CAY10585 (25 nM and/or 50 nM). At the indicated time points, ALP activity was quantitatively measured (Aa) and histochemically stained (Ab). (B) CAY10585 effectively reduces BMP9-induced matrix mineralization of MSCs. C3H10T1/2 cells were infected with BMP9, GFP and/or HIF1α; treated with CAY10585 (50 nM) or DMSO; and maintained in mineralization medium. The CAY10585- or DMSO-containing mineralization medium was changed every 2 days for the first 6 days. At day 12, Alizarin Red S staining was conducted on the treated cells. Each assay was done in triplicate and/or carried out in at least three independent experiments. Representative results are shown.

Cobalt chloride-induced hypoxia potentiates the BMP9-regulated osteogenic differentiation of MSCs

HIF1α is frequently induced in hypoxia condition. Cobalt chloride has been widely used a hypoxia inducer. We tested if BMP9-induced osteogenic differentiation would be affected under hypoxia condition. HIF1α was induced in the MSCs treated with cobalt chloride, which could be effectively silenced by AdR-simHIF1α (Fig. 6A). In fact, cobalt chloride treatment in MSCs synergized with BMP9 in ALP induction in a dosage and time-dependent manner (Fig. 6B). The synergistic effect between cobalt and BMP9 on ALP activity (Fig. 6Ca) and matrix mineralization (Fig. 6Bb) could be effectively reduced by siRNAs targeting HIF1α. These results suggest that HIF1α may play a crucial role in mediating the synergistic effect of cobalt-induced hypoxia with BMP9 in MSCs.

Fig. 6.

Cobalt chloride-induced hypoxia potentiates BMP9-regulated osteogenic differentiation of MSCs. (A) Cobalt upregulates HIF1α, which can be silenced by simHIF1α in MSCs. C3H10T1/2 cells were infected with AdR-simHIF1a or AdRFP in the presence or absence of cobalt chloride (50 µM). Total cell lysate was collected at 48 hours and subjected to SDS-PAGE and western blotting with HIF1α antibody or β-actin control. (B) Cobalt potentiates BMP9-induced ALP activity in a time- and dose-dependent fashion. The iMEFs were infected with AdBMP9 or control AdGFP in the presence of different concentrations of CoCl2 (0, 50 and 100 µM). ALP activity was assayed on days 5 and 7. P-values are indicated between groups. (C) The synergy between BMP9 and cobalt chloride on osteogenic differentiation can be blunted by siRNAs targeting HIF1α. MSC lines C3H10T1/2 and iMEFs were infected with AdBMP9, AdGFP control and/or AdR-simHIF1α in the presence of 100 µM CoCl2. ALP activity (Ca) was assessed on day 5 (iMEFs) or day 7 (C3H10T1/2). Matrix mineralization was determined by Alizarin Red S staining (Cb) on day 10. Representative results are shown.

Fig. 6.

Cobalt chloride-induced hypoxia potentiates BMP9-regulated osteogenic differentiation of MSCs. (A) Cobalt upregulates HIF1α, which can be silenced by simHIF1α in MSCs. C3H10T1/2 cells were infected with AdR-simHIF1a or AdRFP in the presence or absence of cobalt chloride (50 µM). Total cell lysate was collected at 48 hours and subjected to SDS-PAGE and western blotting with HIF1α antibody or β-actin control. (B) Cobalt potentiates BMP9-induced ALP activity in a time- and dose-dependent fashion. The iMEFs were infected with AdBMP9 or control AdGFP in the presence of different concentrations of CoCl2 (0, 50 and 100 µM). ALP activity was assayed on days 5 and 7. P-values are indicated between groups. (C) The synergy between BMP9 and cobalt chloride on osteogenic differentiation can be blunted by siRNAs targeting HIF1α. MSC lines C3H10T1/2 and iMEFs were infected with AdBMP9, AdGFP control and/or AdR-simHIF1α in the presence of 100 µM CoCl2. ALP activity (Ca) was assessed on day 5 (iMEFs) or day 7 (C3H10T1/2). Matrix mineralization was determined by Alizarin Red S staining (Cb) on day 10. Representative results are shown.

HIF1α-regulated angiogenic signaling plays an important role in BMP9-induced osteogenic differentiation

HIF1α is an established angiogenic regulator; but its role in MSCs is not well defined. We found that HIF1α in MSCs was able to induce known target genes, such as VEGF and β-catenin (Fig. 7Aa), and late osteogenic marker OPN (to a lesser extent, OCN) (Fig. 7Ab). Expression of these genes was further enhanced by BMP9 in MSCs. We further examined the expression of angiogenic marker von Willebrand factor (vWF) in the ectopic bone masses retrieved from the studies described in Fig. 4, and found that co-expression of BMP9 and HIF1α increased von Willebrand factor expression, which was inhibited by simHIF1α (Fig. 7B). Taking our in vitro and in vivo findings together, our results strongly indicate that BMP9-regulated HIF1α expression not only plays an important role in transducing angiogenic signaling but also functions as a crucial mediator of osteogenic signaling in BMP9-stimulated MSCs (Fig. 7C).

Fig. 7.

HIFα-regulated angiogenic signaling plays an important role in BMP9-induced osteogenic differentiation. (A) HIF1α synergizes with BMP9 in regulating early target genes and late osteogenic markers. MSCs iMEFs were infected with AdBMP9, AdGFP and/or AdR-HIF1α. Total RNA was isolated on day 3 or 7 and subjected to sqPCR with primers specific for early known targets of HIF1α VEGF and β-catenin (Aa) and late osteogenic markers OCN and OPN (Ab). All samples were normalized with GAPDH expression level. (B) Immunohistochemical staining of Factor VIII levels in ectopic bone masses with HIF1α overexpression or silencing. Ectopic bone masses were established and retrieved in a similar fashion as shown in Fig. 4. The retrieved masses were subjected to H&E staining and von Willebrand factor (vWF) antibody immunohistochemical staining. Isotype IgG was used as a negative control (not shown). Positive staining for Factor VIII is indicated by arrows. Representative results are shown. Magnification, 400×. (C) A proposed model of action. BMP9 upregulates HIF1α expression in MSCs, which in turn induces osteogenic factors and angiogenic factor VEGF. A converge of osteogenic and angiogenic signaling leads to efficient bone formation.

Fig. 7.

HIFα-regulated angiogenic signaling plays an important role in BMP9-induced osteogenic differentiation. (A) HIF1α synergizes with BMP9 in regulating early target genes and late osteogenic markers. MSCs iMEFs were infected with AdBMP9, AdGFP and/or AdR-HIF1α. Total RNA was isolated on day 3 or 7 and subjected to sqPCR with primers specific for early known targets of HIF1α VEGF and β-catenin (Aa) and late osteogenic markers OCN and OPN (Ab). All samples were normalized with GAPDH expression level. (B) Immunohistochemical staining of Factor VIII levels in ectopic bone masses with HIF1α overexpression or silencing. Ectopic bone masses were established and retrieved in a similar fashion as shown in Fig. 4. The retrieved masses were subjected to H&E staining and von Willebrand factor (vWF) antibody immunohistochemical staining. Isotype IgG was used as a negative control (not shown). Positive staining for Factor VIII is indicated by arrows. Representative results are shown. Magnification, 400×. (C) A proposed model of action. BMP9 upregulates HIF1α expression in MSCs, which in turn induces osteogenic factors and angiogenic factor VEGF. A converge of osteogenic and angiogenic signaling leads to efficient bone formation.

Discussion

HIF1α is a well established regulator of angiogenic cascade, which usually coordinates with many developmental processes, including skeletal development (Majmundar et al., 2010; Wan et al., 2010). However, it is not clear if HIF1 exerts any effect on BMP9-regulated osteogenic differentiation. In this report, we investigate if HIF1α-mediated angiogenic signaling plays any role in BMP9-regulated osteogenic differentiation of MSCs. We have found that BMP9 upregulates HIF1α expression in MSCs through Smad1/5/8 signaling. Exogenous expression of HIF1α potentiates BMP9-induced osteogenic differentiation of MSCs while silencing HIF1α or HIF1α inhibitor CAY10585 profoundly blocks BMP9-induced osteogenic signaling in MSCs. Furthermore, HIF1α expression regulated by cobalt-induced hypoxia recapitulates the synergistic effect between HIF1α and BMP9 in osteogenic differentiation. Mechanistically HIF1α is shown to exert its synergistic effect with BMP9 by inducing both angiogenic signaling (e.g. VEGF) and osteogenic signaling in MSCs. Thus, our results strongly indicate that HIF1α-induced angiogenic signaling plays a crucial role in BMP9-initiated osteogenic differentiation of MSCs.

Hypoxia-inducible factors (HIFs) regulate target genes that mediate adaptive responses, such as angiogenesis, to reduced oxygen availability (Wan et al., 2010). HIF complex has 1 of 3 α subunits (HIF1α, HIF2α, or HIF3α) bound to the aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIFβ). The level of HIF1α and HIF2α proteins is regulated by ongoing ubiquitination and proteasomal degradation following enzymatic prolyl hydroxylation on an oxygen-dependent degradation domain (ODD) (Wan et al., 2010). Under normoxic conditions HIF1α undergoes prolyl hydroxylation and is ligated by the E3 ubiquitin ligase von Hippel-Lindau protein (pVHL), and then processed for degradation by the proteosome. Prolyl hydroxylases require oxygen, iron, and 2-oxoglutarate as cofactors (Wan et al., 2010). Under hypoxia, HIF1α prolyl hydroxylation is inhibited, and dimerizes with the ARNT HIF-1, which in turn complexes with coactivator p300 and regulates downstream targets (Wan et al., 2010).

It has been reported that angiogenesis and osteogenesis are well coordinated processes during bone development (Wan et al., 2010; Wang et al., 2007). Osteogenesis usually involves two distinct pathways (Olsen et al., 2000). For intramembranous bone formation, which gives rise to the flat bones of the skull, MSCs differentiate directly into osteoblasts. However, during endochondral bone formation bones are formed through the formation of a chondrocyte anlage, onto which osteoblasts differentiate and deposit bone. Endochondral bone formation occurs in close spatial and temporal association and proximity to capillary invasion, so that angiogenesis and osteogenesis must be tightly coupled (Olsen et al., 2000, Wan et al., 2010).

During osteogenesis MSCs elicit angiogenic signals to recruit new blood vessels into bone. Overexpression of HIF1 in osteoblasts through disruption of the pVHL increases angiogenesis and osteogenesis, which involve the action of VEGF on the endothelial cells (Wan et al., 2010). In fact, mice overexpressing HIF1α in osteoblasts through selective deletion of the Vhl expressed high levels of VEGF and developed extremely dense, heavily vascularized long bones (Wang et al., 2007). Conversely, mice lacking HIF1α in osteoblasts had significantly thinner and less vascularized long bones were than those of controls (Wang et al., 2007). Thus, the activation of HIF1α pathway in developing bone may increase bone modeling events through cell-nonautonomous mechanisms to coordinate the timing, direction, and degree of new blood vessel formation in bone (Wan et al., 2010). Furthermore, HIF1α pathway has been shown as a crucial mediator of neoangiogenesis for skeletal regeneration and bone healing (Wan et al., 2008).

Although it is well recognized that osteogenic and angiogenic pathways are well coordinated during bone formation, it is unclear how these processes are linked in MSCs stimulated osteogenic factors, such as BMPs. We have demonstrated that BMP9 directly upregulates HIF1α expression in MSCs, which in turn induces both osteogenic factors and angiogenic factor VEGF. Thus, potent osteogenic factors, such as BMP9, may induce a tightly-regulated convergence of osteogenic and angiogenic signaling in MSCs, and subsequently lead to efficient bone formation.

Materials and Methods

Cell culture and chemicals

HEK-293 and C3H10T1/2 cells were from ATCC (Manassas, VA). The iMEFs were immortalized mouse embryonic fibroblasts as previously described (Huang et al., 2012a). The cell lines were maintained in the conditions as described (Cheng et al., 2003; Luo et al., 2008; Peng et al., 2003; Tang et al., 2009). HIF1α inhibitor CAY10585 was purchased from Cayman Chemical (Ann Arbor, MI), and was prepared in DMSO (as 50 µM stock solution, kept at −80°C). Unless indicated otherwise, all chemicals were purchased from Sigma-Aldrich (St Louis, MO) or Fisher Scientific (Pittsburgh, PA).

Recombinant adenoviruses expressing BMP9, HIF1α, simHIF1α, GFP and RFP

Recombinant adenoviruses were generated using AdEasy technology as described (Cheng et al., 2003; He et al., 1998; Kang et al., 2009; Kang et al., 2004; Luo et al., 2007a). The coding regions of human BMP9 and HIF1α were PCR amplified and cloned into an adenoviral shuttle vector and subsequently used to generate recombinant adenoviruses in HEK-293 cells. The siRNA target sites against mouse HIF1α coding region were cloned into the pSES adenoviral shuttle vector (Luo et al., 2007b) to generate recombinant adenoviruses. The resulting adenoviruses were designated as AdBMP9, AdR-HIF1α, or AdR-simHIF1α. AdBMP9 also expresses GFP, whereas AdR-HIF1α and AdR-simHIF1α express RFP as a marker for monitoring infection efficiency. Analogous adenovirus expressing only monomeric RFP (AdRFP) or GFP (AdGFP) were used as controls (He et al., 1998; Luo et al., 2007a; Luo et al., 2004; Peng et al., 2004; Sharff et al., 2009; Si et al., 2006; Tang et al., 2009).

RNA isolation and semi-quantitative RT-PCR

Total RNA was isolated using TRIZOL Reagents (Invitrogen) and used to generate cDNA templates by RT reaction with hexamer and M-MuLV Reverse Transcriptase (New England Biolabs, Ipswich, MA). The first strand cDNA products were further diluted five- to tenfold and used as PCR templates. Semi-quantitative RT-PCR (sqPCR) was carried out as described (Huang et al., 2009; Rastegar et al., 2010; Su et al., 2011; Zhang et al., 2010; Zhu et al., 2009). PCR primers (supplementary material Table S1) were designed by using the Primer3 program to amplify the genes of interest (∼150–180 bp). A touchdown cycling program was as follows: 94°C for 2 minutes for 1 cycle; 92°C for 20 seconds, 68°C for 30 seconds, and 72°C for 12 cycles decreasing 1°C per cycle; and then at 92°C for 20 seconds, 57°C for 30 seconds, and 72°C for 20 seconds for 20–25 cycles, depending on the abundance of a given gene. PCR products were resolved on 1.5% agarose gels. All samples were normalized by the expression level of GAPDH.

Chromatin immunoprecipitation (ChIP) analysis

Subconfluent C3H10T1/2 cells were infected with AdGFP or AdBMP9. At 30 hours after infection, cells were cross-linked and subjected to ChIP analysis as previously described (Sharff et al., 2009; Si et al., 2006; Tang et al., 2009). Smad1/5/8 antibody (Santa Cruz Biotechnology) or control IgG was used to pull down the protein-DNA complexes. The presence of HIF1α promoter sequence was detected by using two pairs of primers corresponding to mouse HIF1α promoter region.

Alkaline phosphatase (ALP) activity assays

ALP activity was assessed by a modified Great Escape SEAP Chemiluminescence assay (BD Clontech, Mountain View, CA) and/or histochemical staining assay (using a mixture of 0.1 mg/ml napthol AS-MX phosphate and 0.6 mg/ml Fast Blue BB salt) as described (Chen et al., 2010; Cheng et al., 2003; Kang et al., 2009; Kang et al., 2004; Luo et al., 2004; Luo et al., 2008; Peng et al., 2004; Sharff et al., 2009; Tang et al., 2009; Zhang et al., 2010). For the chemiluminescence assays, each assay condition was performed in triplicate. The results were repeated in at least three independent experiments.

Alizarin Red S staining

C3H10T1/2 cells and iMEFs were seeded in 24-well cell culture plates and infected with adenoviruses and/or treated with inhibitors. The cells were cultured in the presence of ascorbic acid (50 µg/mL) and β-glycerophosphate (10 mM) for 10–14 days. Mineralized matrix nodules were stained for calcium precipitation by means of Alizarin Red S staining as described previously (Cheng et al., 2003; Kang et al., 2009; Kang et al., 2004; Luo et al., 2004; Luo et al., 2008; Peng et al., 2004; Sharff et al., 2009; Tang et al., 2009).

Western blotting analysis

Western blotting was carried out as previously described (Huang et al., 2009; Rastegar et al., 2010; Su et al., 2011; Zhang et al., 2010; Zhu et al., 2009). Briefly, cells were collected in Lysis Buffer. Cleared total cell lysate was denatured by boiling and resolved by 10% SDS-PAGE. After electrophoretic separation, proteins were transferred to an Immobilon-P membrane. Membrane was blocked with SuperBlock Blocking Buffer, and probed with anti-HIF1α or anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with a secondary antibody conjugated with horseradish peroxidase. The proteins of interest were detected by using SuperSignal West Pico Chemiluminescent Substrate kit.

Immunohistochemical staining

The cultured cells were fixed with 10% formalin, washed with PBS, and permeabilized with 1% NP-40. The fixed cells were blocked and incubated with an anti-osteocalcin, or osteopontin antibody (Santa Cruz Biotechnology). After being washed, cells were incubated with biotin-labeled secondary antibody for 30 minutes, followed by incubating cells with streptavidin-HRP conjugate for 20 minutes at room temperature. For von Willebrand factor expression, immunohistochemical staining on paraffin-embedded tissues was also carried out with a von Willebrand factor (vWF) antibody (DaKO, Carpinteria, CA). The presence of the expected protein was visualized by DAB staining and examined under a microscope. Stains with control IgG, were used as negative controls.

Stem cell implantation and μCT analysis

The iMEFs were infected with AdBMP9/AdRFP, AdBMP9/AdR-HIF1α, or AdBMP9/AdR-simHIF1α. At 16 hours post infection, cells were harvested, and resuspended in PBS for subcutaneous injection (5×106 cells/injection) into the flanks of athymic nude (nu/nu) mice (5 animals/group, 4-6 weeks old, female, Harlan Sprague-Dawley). At 4 weeks post implantation, animals were sacrificed. Implantation sites were retrieved for μCT analysis, histologic evaluation, and other stains. All specimens were imaged using the μCT component of the GE Triumph (GE Healthcare, Piscataway, NJ, USA) trimodality preclinical imaging system. All image data analysis was performed using Amira 5.3 (Visage Imaging, San Diego, CA); and 3D volumetric data and bone mean density hit maps were obtained as previously described (Chen et al., 2010; Luo et al., 2010; Zhang et al., 2010).

Hematoxylin and eosin, Trichrome and Alcian Blue staining

Retrieved tissues were fixed, decalcified in 10% formalin and embedded in paraffin. Serial sections of the embedded specimens were stained with hematoxylin and eosin (H&E). Masson Trichrome and Alcian Blue stains were carried out as previously described (Chen et al., 2010; Kang et al., 2009; Kang et al., 2004; Luo et al., 2010; Luo et al., 2004; Luo et al., 2008; Sharff et al., 2009; Tang et al., 2009; Zhang et al., 2010).

Statistical analysis

All quantitative experiments were performed in triplicate and/or repeated three times. Data were expressed as mean ± s.d. Statistical significances between vehicle treatment versus drug-treatment were determined by one-way analysis of variance and the Student's t-test. A value of P<0.05 was considered statistically significant.

Acknowledgements

The authors wish to thank Chad Hanley of the Department of Radiology at The University of Chicago for his assistance and advice on μCT scanning and imaging analysis. The authors declare no conflict of interest.

Funding

This work was supported in part by research grants from the National Institutes of Health [grant numbers AR50142, AR054381, and AT004418-01A1 to R.C.H., T.-C.H. and H.H.L.]; the 973 Program of the Ministry of Science and Technology of China [grant number 2011CB707906 to T.-C.H.]; and the Natural Science Foundation of China [grant numbers 31070875 to W.H., 30901530 to X. Luo and 81171685 to D.J.]. Deposited in PMC for release after 12 months.

References

Castonguay
R.
,
Werner
E. D.
,
Matthews
R. G.
,
Presman
E.
,
Mulivor
A. W.
,
Solban
N.
,
Sako
D.
,
Pearsall
R. S.
,
Underwood
K. W.
,
Seehra
J.
et al.  (
2011
).
Soluble endoglin specifically binds bone morphogenetic proteins 9 and 10 via its orphan domain, inhibits blood vessel formation, and suppresses tumor growth.
J. Biol. Chem.
286
,
30034
30046
.
Chen
C.
,
Grzegorzewski
K. J.
,
Barash
S.
,
Zhao
Q.
,
Schneider
H.
,
Wang
Q.
,
Singh
M.
,
Pukac
L.
,
Bell
A. C.
,
Duan
R.
et al.  (
2003
).
An integrated functional genomics screening program reveals a role for BMP-9 in glucose homeostasis.
Nat. Biotechnol.
21
,
294
301
.
Chen
L.
,
Jiang
W.
,
Huang
J.
,
He
B. C.
,
Zuo
G. W.
,
Zhang
W.
,
Luo
Q.
,
Shi
Q.
,
Zhang
B. Q.
,
Wagner
E. R.
et al.  (
2010
).
Insulin-like growth factor 2 (IGF-2) potentiates BMP-9-induced osteogenic differentiation and bone formation.
J. Bone Miner. Res.
25
,
2447
2459
.
Cheng
H.
,
Jiang
W.
,
Phillips
F. M.
,
Haydon
R. C.
,
Peng
Y.
,
Zhou
L.
,
Luu
H. H.
,
An
N.
,
Breyer
B.
,
Vanichakarn
P.
et al.  (
2003
).
Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs).
J. Bone Joint Surg. Am.
85–A
,
1544
1552
.
Cunha
S. I.
,
Pardali
E.
,
Thorikay
M.
,
Anderberg
C.
,
Hawinkels
L.
,
Goumans
M. J.
,
Seehra
J.
,
Heldin
C. H.
,
ten Dijke
P.
,
Pietras
K.
(
2010
).
Genetic and pharmacological targeting of activin receptor-like kinase 1 impairs tumor growth and angiogenesis.
J. Exp. Med.
207
,
85
100
.
David
L.
,
Mallet
C.
,
Keramidas
M.
,
Lamandé
N.
,
Gasc
J. M.
,
Dupuis–Girod
S.
,
Plauchu
H.
,
Feige
J. J.
,
Bailly
S.
(
2008
).
Bone morphogenetic protein-9 is a circulating vascular quiescence factor.
Circ. Res.
102
,
914
922
.
Deng
Z. L.
,
Sharff
K. A.
,
Tang
N.
,
Song
W. X.
,
Luo
J.
,
Luo
X.
,
Chen
J.
,
Bennett
E.
,
Reid
R.
,
Manning
D.
et al.  (
2008
).
Regulation of osteogenic differentiation during skeletal development.
Front. Biosci.
13
,
2001
2021
.
He
T. C.
,
Zhou
S.
,
da Costa
L. T.
,
Yu
J.
,
Kinzler
K. W.
,
Vogelstein
B.
(
1998
).
A simplified system for generating recombinant adenoviruses.
Proc. Natl. Acad. Sci. USA
95
,
2509
2514
.
Hogan
B. L.
(
1996
).
Bone morphogenetic proteins: multifunctional regulators of vertebrate development.
Genes Dev.
10
,
1580
1594
.
Huang
J.
,
Bi
Y.
,
Zhu
G. H.
,
He
Y.
,
Su
Y.
,
He
B. C.
,
Wang
Y.
,
Kang
Q.
,
Chen
L.
,
Zuo
G. W.
et al.  (
2009
).
Retinoic acid signalling induces the differentiation of mouse fetal liver-derived hepatic progenitor cells.
Liver Int.
29
,
1569
1581
.
Huang
E.
,
Bi
Y.
,
Jiang
W.
,
Luo
X.
,
Yang
K.
,
Gao
J. L.
,
Gao
Y.
,
Luo
Q.
,
Shi
Q.
,
Kim
S. H.
et al.  (
2012a
).
Conditionally immortalized mouse embryonic fibroblasts retain proliferative activity without compromising multipotent differentiation potential.
PLoS ONE
7
,
e32428
.
Huang
E.
,
Zhu
G.
,
Jiang
W.
,
Yang
K.
,
Gao
Y.
,
Luo
Q.
,
Gao
J. L.
,
Kim
S. H.
,
Liu
X.
,
Li
M.
et al.  (
2012b
).
Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway in murine multilineage cells.
J. Bone Miner. Res.
27
,
1566
1575
.
Kang
Q.
,
Sun
M. H.
,
Cheng
H.
,
Peng
Y.
,
Montag
A. G.
,
Deyrup
A. T.
,
Jiang
W.
,
Luu
H. H.
,
Luo
J.
,
Szatkowski
J. P.
et al.  (
2004
).
Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery.
Gene Ther.
11
,
1312
1320
.
Kang
Q.
,
Song
W. X.
,
Luo
Q.
,
Tang
N.
,
Luo
J.
,
Luo
X.
,
Chen
J.
,
Bi
Y.
,
He
B. C.
,
Park
J. K.
et al.  (
2009
).
A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells.
Stem Cells Dev.
18
,
545
558
.
López–Coviella
I.
,
Berse
B.
,
Krauss
R.
,
Thies
R. S.
,
Blusztajn
J. K.
(
2000
).
Induction and maintenance of the neuronal cholinergic phenotype in the central nervous system by BMP-9.
Science
289
,
313
316
.
Luo
Q.
,
Kang
Q.
,
Si
W.
,
Jiang
W.
,
Park
J. K.
,
Peng
Y.
,
Li
X.
,
Luu
H. H.
,
Luo
J.
,
Montag
A. G.
et al.  (
2004
).
Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells.
J. Biol. Chem.
279
,
55958
55968
.
Luo
J.
,
Deng
Z. L.
,
Luo
X.
,
Tang
N.
,
Song
W. X.
,
Chen
J.
,
Sharff
K. A.
,
Luu
H. H.
,
Haydon
R. C.
,
Kinzler
K. W.
et al.  (
2007a
).
A protocol for rapid generation of recombinant adenoviruses using the AdEasy system.
Nat. Protoc.
2
,
1236
1247
.
Luo
Q.
,
Kang
Q.
,
Song
W. X.
,
Luu
H. H.
,
Luo
X.
,
An
N.
,
Luo
J.
,
Deng
Z. L.
,
Jiang
W.
,
Yin
H.
et al.  (
2007b
).
Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing.
Gene
395
,
160
169
.
Luo
X.
,
Chen
J.
,
Song
W. X.
,
Tang
N.
,
Luo
J.
,
Deng
Z. L.
,
Sharff
K. A.
,
He
G.
,
Bi
Y.
,
He
B. C.
et al.  (
2008
).
Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects.
Lab. Invest.
88
,
1264
1277
.
Luo
J.
,
Tang
M.
,
Huang
J.
,
He
B. C.
,
Gao
J. L.
,
Chen
L.
,
Zuo
G. W.
,
Zhang
W.
,
Luo
Q.
,
Shi
Q.
et al.  (
2010
).
TGFbeta/BMP type I receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in mesenchymal stem cells.
J. Biol. Chem.
285
,
29588
29598
.
Luther
G.
,
Wagner
E. R.
,
Zhu
G.
,
Kang
Q.
,
Luo
Q.
,
Lamplot
J.
,
Bi
Y.
,
Luo
X.
,
Luo
J.
,
Teven
C.
et al.  (
2011
).
BMP-9 induced osteogenic differentiation of mesenchymal stem cells: molecular mechanism and therapeutic potential.
Curr. Gene Ther.
11
,
229
240
.
Luu
H. H.
,
Song
W. X.
,
Luo
X.
,
Manning
D.
,
Luo
J.
,
Deng
Z. L.
,
Sharff
K. A.
,
Montag
A. G.
,
Haydon
R. C.
,
He
T. C.
(
2007
).
Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells.
J. Orthop. Res.
25
,
665
677
.
Majmundar
A. J.
,
Wong
W. J.
,
Simon
M. C.
(
2010
).
Hypoxia-inducible factors and the response to hypoxic stress.
Mol. Cell
40
,
294
309
.
Mitchell
D.
,
Pobre
E. G.
,
Mulivor
A. W.
,
Grinberg
A. V.
,
Castonguay
R.
,
Monnell
T. E.
,
Solban
N.
,
Ucran
J. A.
,
Pearsall
R. S.
,
Underwood
K. W.
et al.  (
2010
).
ALK1-Fc inhibits multiple mediators of angiogenesis and suppresses tumor growth.
Mol. Cancer Ther.
9
,
379
388
.
Olsen
B. R.
,
Reginato
A. M.
,
Wang
W.
(
2000
).
Bone development.
Annu. Rev. Cell Dev. Biol.
16
,
191
220
.
Park
J. E.
,
Shao
D.
,
Upton
P. D.
,
Desouza
P.
,
Adcock
I. M.
,
Davies
R. J.
,
Morrell
N. W.
,
Griffiths
M. J.
,
Wort
S. J.
(
2012
).
BMP-9 induced endothelial cell tubule formation and inhibition of migration involves Smad1 driven endothelin-1 production.
PLoS ONE
7
,
e30075
.
Peng
Y.
,
Kang
Q.
,
Cheng
H.
,
Li
X.
,
Sun
M. H.
,
Jiang
W.
,
Luu
H. H.
,
Park
J. Y.
,
Haydon
R. C.
,
He
T. C.
(
2003
).
Transcriptional characterization of bone morphogenetic proteins (BMPs)-mediated osteogenic signaling.
J. Cell. Biochem.
90
,
1149
1165
.
Peng
Y.
,
Kang
Q.
,
Luo
Q.
,
Jiang
W.
,
Si
W.
,
Liu
B. A.
,
Luu
H. H.
,
Park
J. K.
,
Li
X.
,
Luo
J.
et al.  (
2004
).
Inhibitor of DNA binding/differentiation helix-loop-helix proteins mediate bone morphogenetic protein-induced osteoblast differentiation of mesenchymal stem cells.
J. Biol. Chem.
279
,
32941
32949
.
Pittenger
M. F.
,
Mackay
A. M.
,
Beck
S. C.
,
Jaiswal
R. K.
,
Douglas
R.
,
Mosca
J. D.
,
Moorman
M. A.
,
Simonetti
D. W.
,
Craig
S.
,
Marshak
D. R.
(
1999
).
Multilineage potential of adult human mesenchymal stem cells.
Science
284
,
143
147
.
Prockop
D. J.
(
1997
).
Marrow stromal cells as stem cells for nonhematopoietic tissues.
Science
276
,
71
74
.
Rastegar
F.
,
Gao
J. L.
,
Shenaq
D.
,
Luo
Q.
,
Shi
Q.
,
Kim
S. H.
,
Jiang
W.
,
Wagner
E. R.
,
Huang
E.
,
Gao
Y.
et al.  (
2010
).
Lysophosphatidic acid acyltransferase β (LPAATβ) promotes the tumor growth of human osteosarcoma.
PLoS ONE
5
,
e14182
.
Scharpfenecker
M.
,
van Dinther
M.
,
Liu
Z.
,
van Bezooijen
R. L.
,
Zhao
Q.
,
Pukac
L.
,
Löwik
C. W.
,
ten Dijke
P.
(
2007
).
BMP-9 signals via ALK1 and inhibits bFGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis.
J. Cell Sci.
120
,
964
972
.
Sharff
K. A.
,
Song
W. X.
,
Luo
X.
,
Tang
N.
,
Luo
J.
,
Chen
J.
,
Bi
Y.
,
He
B. C.
,
Huang
J.
,
Li
X.
et al.  (
2009
).
Hey1 basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells.
J. Biol. Chem.
284
,
649
659
.
Shi
Y.
,
Massagué
J.
(
2003
).
Mechanisms of TGF-beta signaling from cell membrane to the nucleus.
Cell
113
,
685
700
.
Si
W.
,
Kang
Q.
,
Luu
H. H.
,
Park
J. K.
,
Luo
Q.
,
Song
W. X.
,
Jiang
W.
,
Luo
X.
,
Li
X.
,
Yin
H.
et al.  (
2006
).
CCN1/Cyr61 is regulated by the canonical Wnt signal and plays an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells.
Mol. Cell. Biol.
26
,
2955
2964
.
Song
J. J.
,
Celeste
A. J.
,
Kong
F. M.
,
Jirtle
R. L.
,
Rosen
V.
,
Thies
R. S.
(
1995
).
Bone morphogenetic protein-9 binds to liver cells and stimulates proliferation.
Endocrinology
136
,
4293
4297
.
Su
Y.
,
Wagner
E. R.
,
Luo
Q.
,
Huang
J.
,
Chen
L.
,
He
B. C.
,
Zuo
G. W.
,
Shi
Q.
,
Zhang
B. Q.
,
Zhu
G.
et al.  (
2011
).
Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma.
Oncogene
30
,
3907
3917
.
Suzuki
Y.
,
Ohga
N.
,
Morishita
Y.
,
Hida
K.
,
Miyazono
K.
,
Watabe
T.
(
2010
).
BMP-9 induces proliferation of multiple types of endothelial cells in vitro and in vivo.
J. Cell Sci.
123
,
1684
1692
.
Tang
N.
,
Song
W. X.
,
Luo
J.
,
Luo
X.
,
Chen
J.
,
Sharff
K. A.
,
Bi
Y.
,
He
B. C.
,
Huang
J. Y.
,
Zhu
G. H.
et al.  (
2009
).
BMP-9-induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/beta-catenin signalling.
J. Cell. Mol. Med.
13
,
2448
2464
.
Truksa
J.
,
Peng
H.
,
Lee
P.
,
Beutler
E.
(
2006
).
Bone morphogenetic proteins 2, 4, and 9 stimulate murine hepcidin 1 expression independently of Hfe, transferrin receptor 2 (Tfr2), and IL-6.
Proc. Natl. Acad. Sci. USA
103
,
10289
10293
.
Varga
A. C.
,
Wrana
J. L.
(
2005
).
The disparate role of BMP in stem cell biology.
Oncogene
24
,
5713
5721
.
Wan
C.
,
Gilbert
S. R.
,
Wang
Y.
,
Cao
X.
,
Shen
X.
,
Ramaswamy
G.
,
Jacobsen
K. A.
,
Alaql
Z. S.
,
Eberhardt
A. W.
,
Gerstenfeld
L. C.
et al.  (
2008
).
Activation of the hypoxia-inducible factor-1alpha pathway accelerates bone regeneration.
Proc. Natl. Acad. Sci. USA
105
,
686
691
.
Wan
C.
,
Shao
J.
,
Gilbert
S. R.
,
Riddle
R. C.
,
Long
F.
,
Johnson
R. S.
,
Schipani
E.
,
Clemens
T. L.
(
2010
).
Role of HIF-1alpha in skeletal development.
Ann. N. Y. Acad. Sci.
1192
,
322
326
.
Wang
Y.
,
Wan
C.
,
Deng
L.
,
Liu
X.
,
Cao
X.
,
Gilbert
S. R.
,
Bouxsein
M. L.
,
Faugere
M. C.
,
Guldberg
R. E.
,
Gerstenfeld
L. C.
et al.  (
2007
).
The hypoxia-inducible factor alpha pathway couples angiogenesis to osteogenesis during skeletal development.
J. Clin. Invest.
117
,
1616
1626
.
Yao
Y.
,
Jumabay
M.
,
Ly
A.
,
Radparvar
M.
,
Wang
A. H.
,
Abdmaulen
R.
,
Boström
K. I.
(
2012
).
Crossveinless 2 regulates bone morphogenetic protein 9 in human and mouse vascular endothelium.
Blood
119
,
5037
5047
.
Zhang
J.
,
Li
L.
(
2005
).
BMP signaling and stem cell regulation.
Dev. Biol.
284
,
1
11
.
Zhang
W.
,
Deng
Z. L.
,
Chen
L.
,
Zuo
G. W.
,
Luo
Q.
,
Shi
Q.
,
Zhang
B. Q.
,
Wagner
E. R.
,
Rastegar
F.
,
Kim
S. H.
et al.  (
2010
).
Retinoic acids potentiate BMP9-induced osteogenic differentiation of mesenchymal progenitor cells.
PLoS ONE
5
,
e11917
.
Zhu
G. H.
,
Huang
J.
,
Bi
Y.
,
Su
Y.
,
Tang
Y.
,
He
B. C.
,
He
Y.
,
Luo
J.
,
Wang
Y.
,
Chen
L.
et al.  (
2009
).
Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells.
Differentiation
78
,
195
204
.

Supplementary information