INTRODUCTION
Bone differentiation is controlled by signaling networking including the bone morphogenetic proteins (BMPs)/transforming growth factor (TGF-β) and Wnt pathways. Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, activate its receptors and initiate phosphorylation of the downstream effector proteins, known as receptor-regulated Smads, leading to signal transduction. BMPs play pivotal roles and are involved in nearly all processes associated with skeletal morphogenesis (
ten Dijke, 2006).
Wnt/β-catenin signaling is another key signaling pathway required for bone formation and bone homeostasis. In the presence of Wnt ligand, frizzled (Fz) and low-density lipoprotein receptor-related protein-5 or 6 (LRP-5/6) co-receptors recruit Disheveled (Dvl) to the plasma membrane along with Axin-GSK3β, inhibiting the formation of β-catenin destruction complex, allowing β-catenin levels to increase and the activation of Wnt/β-catenin pathway (
Behrens et al., 1996;
Brunner et al., 1997;
Zhang and Ma., 2010). Wnt/β-catenin pathway directly targets Runx2 expression and promotes mineralization during osteoblastogenesis (
Gaur et al., 2005). The canonical Wnt blocks apoptosis and osteoclastogenesis by increasing the OPG/RANKL ratio (
Glass et al., 2005;
Holmen et al., 2005;
Spencer et al., 2006). Mutations in LRP-5 profoundly affect skeletal development and result in low bone mass (
Gong et al., 2001;
Little et al., 2002). The Dickkopf-1 (Dkk-1) resistant LRP5V171 mutation leads to high bone density (
Boyden et al., 2002). Conditional deletion of the β-catenin gene in osteoblasts leads to reduced bone mass, and osteocyte-specific β-catenin-deficient mice develop low bone mass phenotype (
Glass et al., 2005;
Kramer et al., 2010). Increasing evidences also indicate that autocrine Wnt signaling is required for BMP in inducing bone formation (
Rawadi et al., 2003;
Fujita and Janz, 2007;
Qiang et al., 2008;
Tang et al., 2009;
Honda, et al., 2010).
As a critical kinase involved in controlling both Wnt/β-catenin and BMP signaling, glycogen synthase kinase 3β (GSK3β) serves as an effective therapeutic target for bone disease treatment. In this study, we show that through suppressing GSK3β kinase activity, bisindoylmaleimide I (BIM) promotes Wnt/β-catenin signaling and prolongs duration of BMP induced Smad pathway in preosteoblast cells. Functional read out measured it as a bone-specific alkaline phosphatase (ALP) activator and mineralization assay uncovered it as a potent osteogenic inducer, which is further proved to regulate osteoblast differentiation from mesenchymal stem cells.
RESULTS
BIM stimulates cytosolic β-catenin accumulation in preosteoblast cells
Forming of osteoblasts contributes to bone resorption and treatment of bone diseases. We started this project with a screen to identify kinase inhibitors that could elevate cytosolic β-catenin level in preosteoblast cells. KS483 cells were treated with suboptimal doses of kinase inhibitors and then assayed for cytosolic β-catenin expression. Among all the 40 compounds tested, BIM appeared to be the most potent in triggering cytosolic β-catenin accumulation (Fig. 1). We then focused our attention on its applications in osteogenic differentiation.
BIM enhances osteogenic differentiation
It is well established that cytosolic β-catenin accumulation is a key factor that determines Wnt induced osteoblast differentiation and bone formation. We then investigated C2C12 myoblast and KS483 preosteoblast cells that were treated with BIM. As shown in Fig. 2, BIM treatment dose-dependently increased ALP activity in C2C12 cells (Fig. 2A). Quantitative real-time RT-PCR analysis indicated that osteogenic marker genes, such as OPG and Runx2, were elevated upon BIM treatment (Fig. 2B). And in KS483 cells, BIM significantly induced mineralization (Fig. 2C). Taken these data together, BIM may act as a potent osteogenic inducer through Wnt/β-catenin signaling pathway.
BIM activates Wnt/β-catenin signaling by suppressing GSK3β in preosteoblast cells
We next examined the underlying mechanism of BIM on canonical Wnt signaling. Reporter assay showed that the activity of Wnt-responsive TopFlash-Luc (
Korinek et al., 1997) and LEF-Luc (
Hsu et al., 1998) were dose-dependently increased by BIM in C2C12 and KS483 cells whereas BMP-responsive BRE-Luc (
Korchynskyi and ten Dijke, 2002) was not affected (Fig. 3A–3D). The activation of Topflash-luciferase by BIM was Wnt-specific, since the control reporter Fopflash-luciferase was not stimulated (data not shown). Furthermore, Wnt3a-induced TopFlash luciferase activity could be inhibited by DKK1, active GSK3β and dominant negative LEF-1 (dnLEF-1), whereas BIM-induced reporter activity could only be inhibited by active GSK3β and dnLEF-1, but not by DKK1. This indicates that BIM may not play a role by stimulating Wnt secretion but act on the downstream signaling cascades (Fig. 3E and 3F). To prove this hypothesis, we treated KS483 cells with BIM and performed immunoblot analysis. Consistent with previous findings, β-catenin was accumulated in BIM treated cells. Notably, western blot analysis with phospho-specific anti-β-catenin antibodies showed that Wnt3a-CM and SB216763 inhibited the phosphorylation of β-catenin at the Ser33/37/Thr41 residues (Fig. 3G). Treatment with BIM also inhibited Ser33/37/Thr41 phosphorylation without affecting the phosphorylation of β-catenin at Ser552 and Ser675 (Fig. 3G), two previously identified phosphorylation sites by Akt and PKA respectively (
Hino et al., 2005;
Taurin et al., 2006;
Fang et al., 2007;
He et al., 2007). Additionally, phosphorylation of GSK3β at Ser9 was observed to be downregulated by BIM. These data indicate that BIM inhibits GSK3β kinase activity, and thus allows canonical Wnt activation.
BIM induces osteogenesis via Wnt/β-catenin pathway
We showed that BIM activates Wnt/β-catenin signaling and promotes osteogenic differentiation. To further test whether β-catenin mediated signaling is required for BIM-induced osteogenesis, siRNAs that specifically silence β-catenin were used to serve this purpose. Firstly, BIM as well as Wnt3a stimulated Wnt reporter activity was abolished by si-β-catenin (Fig. 4A). Secondly in KS483 cells, measurement of ALP activity and Alizarin red staining analysis showed that efficient depletion of endogenous β-catenin in KS483 cells partially blocked BIM-induced ALP activity and mineralization (Fig. 4B and 4C). These data indicate that BIM induces bone formation by activating Wnt/β-catenin pathway.
BIM enhances duration of BMP-induced signaling
Although basal BMP reporter was not stimulated by BIM (Fig. 3D), we observed elevated BMP responses when BIM was co-treated with BMP ligand in cells (Fig. 5A). This suggests that BIM facilitates BMP ligand-induced signaling. As Smad1 phosphorylation in linker region by GSK3β induces its instability (
Fuentealba et al., 2007), we investigated whether BIM affects BMP/Smad1 signaling as well. Immunoblot analysis showed that after BMP6 stimulation (30 min treatment and then washed away as shown in Fig. 5B top panel), the duration of BMP receptor induced C-terminal phosphorylated Smad1 was stabilized by BIM, which is consistent with the fact that BIM could suppress GSK3β kinase activity.
BIM enhances BMP-induced mesenchymal stem cell differentiation into osteoblast
Requirement for bone therapy is to increase bone formation by stimulating stem cells to commit to the osteoblast lineage, expanding the osteoprogenitor cell pool and promoting its differentiation with a final goal of forming a mineralized bone tissue (
Lian et al., 2006).
Multipotent mesenchymal stem cells are able to differentiate into osteoblasts, adipocytes, chondrocytes, and myoblasts. The differentiation of mesenchymal stem cells is regulated by interaction with specific extracellular mediators. Both Wnt/β-catenin and BMP pathways play pivotal roles in triggering the differentiation of mesenchymal stem cells to osteoblast (
Takada et al., 2009;
Vukicevic and Grgurevic, 2009). Therefore, we extended to examine osteoblast differentiation from human mesenchymal stem cells (hMSCs). As shown in Fig. 6A, transient BIM treatment cooperatively accelerated bone formation together with following BMP stimulation as visualized by mineralization detection (Fig. 6A). Collectively, through suppressing GSK3β kinase activity, BIM activates Wnt/β signaling and enhances BMP/Smad responses, therefore promoting osteogenesis (Fig. 6B).
DISCUSSION
Bone resorption is balanced by activated osteoclasts and-bone forming osteoblasts. While targeted inhibition of osteoclasts with bisphosphonates has achieved success in preventing bone loss, searching factors to stimulate bone formation has been challenging (
Rosen, 2003;
Zhao and Su, 2011).
Osteogenic differentiation of mesenchymal stem cells toward osteoprogenitor and osteoblastic cells is tightly regulated by several growth and transcription factors at the molecular level, in which Wnt/β-catenin and BMP signaling is essentially involved. Therefore, searching factors/compounds/drugs that can induce Wnt/β-catenin and BMP signaling would be promising to identify potential candidates of promoting bone formation. GSK3β is a central component of β-catenin destruction complex. Blocking GSK3β kinase activity prevents the phosphorylation and ubiquitination-mediated degradation of β-catenin thus leading to the accumulation of cytosolic β-catenin and subsequent activation of Wnt signaling. The intensity of BMP signals was determined by BMP receptors via Smad1 C-terminal phosphorylations. GSK3β sequentially phosphorylates Smad1 linker region, causing polyubiquitinylation of C-terminal phosphorylated-Smad1. Therefore, antagonizing GSK3β kinase activity prevents not only β-catenin degradation but also the degradation of activated Smad1, leading to the prolonged duration of BMP signals. Here in the present study, we screened up to 40 kinds of kinase inhibitors and identified BIM as an efficient inducer of Wnt/β-catenin activation by suppressing GSK3β kinase activity. Thus this study supported previous reports (
Hers et al., 1999;
Cho et al., 2008) and confirmed BIM as an agonist for Wnt/β-catenin cascades in osteogenic cells. We further proved its role of extending BMP signaling and explored its application as an osteogenic inducer through multiple assays. ALP activity is an early marker for bone formation; we further confirmed read out of signaling and ALP assay by analyzing calcium deposition and the formation of mineralized matrix, occurring late in differentiation.
Another aspect that deserves discussion is the role of BIM as an inhibitor for protein kinase C (
Pajak et al., 2008), a multifunctional protein kinase family whose members exhibit distinct properties (
Dempsey et al., 2000). It was therefore of considerable interest to identify whether the observed effects are related to consequences of PKC family or their individual isozyme’s action. Of note, we observed a growth arrest effect in hMSCs by even suboptimal doses of BIM when it was long-time treated to cells, which was not considerably detected in preosteoblast. Whether this effect is due to suppression of PKC family members requires further investigations.
The fact that BIM enhances osteogenic differentiation emphasizes its physiological relevance of regulating Wnt/β-catenin and BMP signals as a GSK3β inhibitor. Although much more work is needed to elucidate the possible application of BIM in bone disease, the present study provides a potential therapeutic method.
MATERIALS AND METHODS
Reagents and plasmids
β-catenin antibody sample kit (#2951), GSK3β (#9315) and phospho-GSK-3â (Ser9) (#9336) antibodies were purchased from Cell Signaling Technology, Inc., USA. Actin (ab8227) was purchased from Abcam. Control and β-catenin siRNA were purchased from Thermo (Dharmacon RNAi Technologies), TopFlash-luciferase, FopFlash-luciferase, LEF-luciferase, β-catenin SA mutant, Wnt3a, GSK3β wt, S9A, K85M and dnLEF-1 constructs were as described previously (
Zhang et al., 2006;
Zhou et al., 2008a,
2008b,
2011a).
Cell culture
C2C12, KS483, L cells and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Hyclone), nonessential amino acids, L-glutamine, and penicillin/streptomycin in a 5% CO2-containing atmosphere at 37°C.
Luciferase reporter assay
Cells were transfected and lysed as described (
Zhang et al., 2006) and luciferase activities were measured by a luminometer (Berthold Technologies). Reporter activity was normalized to β-Gal activity, resulting from a cotransfected internal control plasmid. Experiments were performed in triplicate.
Transfection and immunoblotting
As previously described (
Nie et al., 2010;
Zhang et al., 2011;
Zhou et al., 2011a,
2011b), cells were transiently transfected using Lipofectamine (Invitrogen). 40 h post-transfection, cells were lysed and immunoblotting was performed with specific antibody and secondary anti-mouse or anti-rabbit antibodies that were conjugated to horseradish peroxidase (Amersham Biosciences). Proteins were visualized by chemiluminescence.
ALP staining and mineralization assays
Histochemical examination of ALP activity in cells was performed using naphtol AS-MX phosphate (Sigma) and fast blue RR salt (Sigma) (
van Dinther et al., 2010). For mineralization assay cells were washed with PBS, fixed with 3.7% formaldehyde, again washed with PBS and then incubated with 2% alizarin red S solution (pH 4.2) for 2 min and washed with distilled water (
van Dinther et al., 2010).
QRT-PCR (quantitative real-time-PCR)
Total RNA was isolated using NucleoSpin® RNA II kit (BIOKÉ, Netherlands) reagent. 1 μg RNA was reverse-transcribed using the RevertAid™ First Strand cDNA Synthesis Kits (Fermentas). Quantitative real-time-PCR was accomplished with SYBR Green incorporation (Applied Bioscience) using a StepOne Plus real-time PCR system (Applied Bioscience). Results were normalized to those obtained with GAPDH. Primers used for QRT-PCR were: mGAPDH forward, 5'-AACTTTGGCATTGTGGAAGG-3'; mGAPDH reverse, 5'-ACACATTGGGGGTAGGAACA-3'; mRunx2 forward, 5'-GAATGCTTCATTCGCCTCAC-3'; mRunx2 reverse, 5'-GTGACCTGCAGAGATTAACC-3'; mOpg (osteoprotegerin) forward, 5'-CGCAAAAGTGTGGAATAGATGTCA-3'; mOpg (osteoprotegerin) reverse, 5'-GGTAGGAACAGCAAACCTGAAGA-3'.
Preparation of cytosolic fractions
Cytosolic and membrane fractions were prepared using the Proteo-Extract kit (Calbiochem) according to the manufacturers’ standard procedures.
Statistical analysis
Statistical analyses were performed with a two-tailed unpaired t test. P < 0.05 was considered statistically significant.
Higher Education Press and Springer-Verlag Berlin Heidelberg 2012
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