Wnt3a induces osteoblastogenesis coupled with PKC activation in ST2 cells
To investigate the molecular mechanism underlying Wnt signaling during osteoblast differentiation, we established a Wnt-responsive osteoblastogenesis system. The murine bone marrow-derived stromal cell line ST2 (Ogawa et al., 1988
), upon incubation with a conditioned medium containing Wnt3a (hereafter Wnt3a medium), expressed a markedly higher alkaline phosphatase (AP) activity than cells cultured in a control conditioned medium (hereafter L medium) (). Real-time PCR revealed that AP
mRNA levels steadily increased during the first 3 days of Wnt3a treatment before reaching a plateau (). Similarly, expression of bone sialoprotein
) was activated within the first 24 hours, and reached a plateau by 72 hours of Wnt3a treatment (). On the other hand, osteocalcin
), a marker for mature osteoblasts, was induced only after 96 hours of Wnt3a stimulation (). Interestingly, Wnt3a did not stimulate Runx2
expression, but significantly induced Osterix
) after 96 hours (). Moreover, in the presence of ascorbic acid and β-glycerophosphate, Wnt3a induced widespread formation of mineralized nodules (). Finally, purified recombinant Wnt3a dose-dependently induced AP in ST2 cells in a serum-free medium (). Thus, Wnt3a is sufficient to induce osteoblast differentiation in ST2 cells.
Wnt3a induces osteoblast differentiation and MARCKS phosphorylation via PKCδ in ST2 cells
To identify downstream molecules responsible for Wnt-induced osteoblastogenesis, a proteomics approach was taken to compare protein profiles in ST2 cells cultured in Wnt3a versus L medium. Meristoylated alanine-rich C kinase substrate (MARCKS), a prototypic substrate for protein kinase C (PKC) (Blackshear, 1993
) was detected at a increased level after 24 hours of Wnt3a stimulation (, arrows), a result confirmed by Western analyses of total cell lysates (). As MARCKS is known to redistribute from the plasma membrane to the cytosol following phosphorylation (Arbuzova et al., 2002
), the cytosolic fractions of cells were therefore analyzed for the levels of phosphorylated MARCKS, using a phospho-specific antibody. These studies revealed that MARCKS phosphorylation was markedly enhanced after 1 hour of incubation in Wnt3a medium, and that upregulation was sustained for at least 24 hours (). In fact, when cells were stimulated with purified Wnt3a without serum, phospho-MARCKS was readily detectable at 10 minutes post-stimulation, and steadily rose within the first hour (). Thus, Wnt3a robustly induces MARCKS phosphorylation in ST2 cells.
PKCδ mediates Wnt3a-induced osteoblastogenesis, independent of β-catenin
Induction of MARCKS phosphorylation prompted us to examine the role of PKC in Wnt3a-induced osteoblastogenesis. The PKC family of serine and threonine protein kinases consists of at least 11 members, including the classic PKC isoforms (α, β1, β2,γ) activated by diacylglycerol (DAG), phosphatidylserine and Ca++
, the novel PKC subfamily (δ, ε, η, θ) by DAG and phosphatidylserine, and the atypical PKC isoforms (λ, ι, ζ) only by phosphatidylserine (Newton, 1997
). Ro-31-8220, an inhibitor for all PKC isoforms, significantly impaired AP induction by Wnt3a (). However, Gö 6976, an inhibitor specific for classic PKC, had no effect even at 10 μM (IC50
2.3 nM for PKCα, 6.7 nM for PKCβ) (). Similarly, the intracellular Ca++
chelator BAPTA/AM did not inhibit Wnt3a-induced AP expression (). In addition, a peptide inhibitor specific for atypical PKC (PKCζ pseudosubstrate) also failed to inhibit Wnt3a-induced osteoblastogenesis (data not shown). In contrast, rottlerin, a selective inhibitor for PKCδ and PKCθ, significantly reduced AP induction in a dose-dependent manner (), but a PKCθ pseudosubstrate had no effect (data not shown). The inhibition of osteoblastogenesis by rottlerin was confirmed by real-time PCR of osteoblast markers (Supplementary data, Fig. S1
). Similarly, rottlerin inhibited Wnt3a-induced AP activity in primary cultures of limb primordial cells, isolated from E13.5 mouse embryos and containing osteoprogenitors but no mature osteoblasts (). Moreover, rottlerin abolished Wnt3a-induced MARCKS phosphorylation in ST2 cells (). Thus, PKCδ is required for Wnt3a-induced osteoblast differentiation and MARCKS phosphorylation.
PKCδ is required for Wnt3a-induced osteoblastogenesis but not for canonical Wnt signaling in ST2 cells
To determine whether inhibition of PKC interfered with canonical Wnt signaling, we examined the potential effect of Ro-31-8220 or rottlerin on Wnt3a-induced transcriptional activation of a Lef1-luciferase reporter, and β-catenin stabilization. Not only did Ro-31-8220 not impair Wnt3a-induced luciferase expression or β-catenin stabilization, it synergized with Wnt3a (). The fact that Ro-31-8220 also inhibits GSK3β, a known negative regulator of canonical Wnt signaling, may explain this observation. Similarly, rottlerin did not impair Wnt3a-induced β-catenin stabilization (). Thus, PKCδ mediates Wnt-induced osteoblastogenesis independent of canonical Wnt signaling.
To corroborate the role of PKCδ, we knocked down its expression with siRNA. Western analyses confirmed that PKCδ siRNA reduced the protein level of PKCδ by ~66% (). Importantly, the knockdown decreased Wnt3a-induced AP induction by approximately 50% (). Similarly, overexpression of a dominant negative form of PKCδ (PKCδ-ΔC) using a retroviral vector severely impaired Wnt3a-induced AP expression (data not shown). Moreover, when cultured in a mineralization medium, cells expressing PKCδ-ΔC formed significantly fewer bone nodules than control cells expressing GFP (). These results support the conclusion that PKCδ activity is required in Wnt3a-induced osteoblast differentiation.
PKCδ activation via Gq signaling promotes Wnt3a-induced osteoblastogenesis and requires Dvl in ST2 cells
Gq-activated phosphatidylinositol signaling, independent of β-catenin, mediates Wnt3a-induced osteoblastogenesis
We next set out to unravel the signaling cascade leading to PKCδ activation in response to Wnt3a. PKCδ is activated by DAG, which is in turn produced through hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC). Since the PLC-βisoenzymes are activated by both the Gq
subfamily of α subunits and the βγ subunits of heterotrimeric G proteins (Morris and Malbon, 1999
), we examined whether G protein-linked phosphatidylinositol signaling was responsible for Wnt3a-induced PKCδ activation and subsequent osteoblastogenesis. Pertussis toxin, which catalyzes ADP-ribosylation of the Gi
family of the α-subunits thus uncoupling them from their activating receptors, is known to inhibit PLC-β activation by the βγ-subunits (Morris and Malbon, 1999
). The toxin, however, did not inhibit Wnt3a-induced osteoblast differentiation in ST2 cells (data not shown). We therefore focused subsequent studies on Gq
To examine the role of Gq
signaling in Wnt-induced osteoblastogenesis, we took advantage of a dominant negative reagent Gq
I, which is a COOH-terminal peptide (a.a. 305–359) of Gαq
previously shown to partially inhibit Gq
signaling (Akhter et al., 1998
I expression significantly reduced Wnt3a-induced AP expression (), as well as bone nodule formation (). Thus, Gq
signaling likely mediates Wnt3a-induced osteoblastogenesis.
To confirm the role of Gq subfamily of α-subunits, we reduced the levels of Gαq and Gα11, the two widely expressed members, with siRNA. A combination of siRNA oligonucleotides against Gαq or Gα11, reduced their combined protein levels by ~43%, as detected by an antibody recognizing both molecules (). Importantly, these oligonucleotides reduced Wnt3a-induced AP expression by >50% (). Similarly, single knockdowns of either Gαq or Gα11 also partially inhibited AP induction (data not shown). Thus, both Gαq and Gα11 are likely to mediate Wnt3a-induced osteoblast differentiation.
To further test the function of phosphatidylinositol signaling, we examined the effect of U73122, a PLC inhibitor, on Wnt3a-induced osteoblastogenesis. U73122 not only inhibited AP induction by ~50% (), but also reduced bone nodule formation (). Thus, PLC activity is important for Wnt-induced osteoblast differentiation.
Next we examined whether inhibition of Gq signaling or PLC activity affected Wnt3a-induced PKCδ activation or β-catenin stabilization. Both U73122 () and GqI () markedly reduced phospho-MARCKS, without significantly altering β-catenin stabilization. These results indicate that Wnt3a activates a Gαq/11→PLCβ→PKCδ pathway independent of β-catenin signaling to promote osteoblast differentiation.
PKCδ activation by Wnt3a requires Dvl but is insensitive to Dkk1
We next examined whether PKCδ activation involves the Dishevelled (Dvl) family of molecules. Immunostaining revealed that in unstimulated ST2 cells, both PKCδ and Dvl-2 were present diffusely in the cytosol (). However, within 30 minutes of Wnt3a stimulation, both molecules were translocated to the plasma membrane in a punctate pattern, although some signal remained in the perinuclear region (). Remarkably, PKCδ and Dvl-2 co-localized at the plasma membrane (). Similar results were observed with Dvl-1 and Dvl-3 (Supplementary data, Fig. S2
). Thus, Wnt3a signaling translocates PKCδ and Dvl to common domains within the plasma membrane.
PKCδ activation by Wnt3a correlates with Dvl-2 translocation to the plasma membrane and is insensitive to Dkk1 in ST2 cells
To determine the kinetics of membrane translocation for PKCδ and Dvl-2, we used Western analyses to quantify the levels of these proteins in the cytosol following Wnt3a stimulation. At 10 minutes post-stimulation, both PKCδ and Dvl-2 were markedly reduced in the cytosol (, lane 2). At 30 minutes, the cytosolic content of PKCδ or Dvl-2 was partially recovered but remained significantly lower than the pre-stimulation level (, lane 3). Interestingly, at 60 minutes both proteins were present in the cytosol at a higher than pre-stimulation level (, lane 4). Consistent with activation of PKCδ at the cell membrane, the levels of phospho-MARCKS in the cytosol steadily rose within the first hour of stimulation (; ). Thus, concurrent with PKCδ activation, Wnt3a signaling acutely and transiently translocates PKCδ and Dvl-2 to the plasma membrane with similar kinetics.
We next examined whether Dvl signaling is required for PKCδ activation. Since the DIX, PDZ and DEP domains were reported to preferentially mediate distinct Wnt pathways, we set out to evaluate whether overexpression of Dvl-2 variants lacking one of the three conserved domains (ΔDIX, ΔPDZ and ΔDEP) (Habas et al., 2001
) differentially affects Wnt3a-induced PKCδ activation versus β-catenin stabilization. Cells expressing any of the Dvl-2 variants showed a marked reduction in MARCKS phosphorylation (). Similarly and unexpectedly, all three Dvl-2 variants also inhibited β-catenin accumulation (). These results support that Dvl proteins are required for both PKCδ activation and β-catenin stabilization in response to Wnt3a, and that all three conserved domains may participate in both pathways.
To determine whether the Wnt-PKCδ pathway requires LRP5/6 signaling, we examined whether the membrane translocation of PKCδ in response to Wnt3a is sensitive to Dkk1 inhibition. Here, cells infected with a retrovirus co-expressing Dkk1 and nuclear GFP were immunostained for endogenous PKCδ, with or without Wnt3a stimulation. As in control cells (), Wnt3a induced characteristic translocation of PKCδ from the cytosol () to the plasma membrane () in cells overexpressing Dkk1. Thus, Dkk1 does not inhibit Wnt3a-induced membrane translocation of PKCδ.
We next examined whether Dkk1 inhibits Wnt3a-induced PKCδ activation. To this end, cytosolic proteins from virally infected cells with or without Wnt3a stimulation were assayed for phospho-MARCKS by Western analyses. As a control for the efficacy of the Dkk1 virus, β-catenin levels were also analyzed. In addition, the virally infected cells were transfected with the Lef1-luciferase reporter and assayed for response to Wnt3a. As expected, in Dkk1-overexpressing cells, Wnt3a failed to either stabilize β-catenin (), or activate transcription of the reporter (). On the other hand, Wnt3a induced phosphorylation of MARCKS in these cells, similar to that in the control cells (). Thus, in contrast to the canonical Wnt pathway, Wnt-PKCδ signaling does not appear to engage the LRP5/6 co-receptors.
Genetic deletion of PKCδ results in a reduction in embryonic bone formation
To determine the role of PKCδ in bone formation in vivo
, we analyzed the skeleton of PKCδ
knockout mice (PKCδ−/−
). The PKCδ −/−
animals are viable and fertile but were reported to exhibit hyperactivation of B-cell proliferation and auto-immunity (Miyamoto et al., 2002
), as well as deficiency in stress-induced apoptosis of blood vessel smooth muscle cells (Leitges et al., 2001
). We reasoned that removal of PKCδ might lead to quantitative defects more evident during early phases of bone formation, and therefore focused our analyses on early embryos. At E15.5, the wild type embryos showed obvious ossification in the maxilla, the mandible, the ribs as well as the limbs (). In contrast, PKCδ −/−
littermates exhibited much less ossification (). In particular, the maxilla and the mandible of mutant embryos showed only minimal mineralization compared with wild type littermates (, compare C and D). In limbs, bone collars of ossifying skeletal elements were notably shorter in the mutant embryo (). Accordingly, von Kossa
staining on sections of long bones showed that bone collars were shortened in mutants at both E14.5 and E15.5 (). Indeed, the relative bone collar length normalized to the total skeletal element length was significantly reduced in the mutant (). Thus, removal of PKCδ
results in less bone in the early embryonic skeleton.
Removal of PKCδ results in a deficit in embryonic bone formation
As bone collar formation in the embryo is coupled with cartilage development, we examined the status of chondrocyte maturation in PKCδ −/− versus wild type littermates. At E14.5, in mutant embryos, the hypertrophic zone expressing Colα1(X) was shortened; the domains expressing parathyroid hormone related peptide-receptor (PTHrP-R), and Indian hedgehog (Ihh) were less well separated, and the terminal hypertrophic zone expressing matrix metalloproteinase 13 (MMP 13) was reduced (). Thus loss of PKCδ delays chondrocyte maturation in long bones.
We next examined whether progression of osteoblast differentiation was perturbed in PKCδ −/−embryos. To this end, we took advantage of the fact that osteoblastogenesis in the perichondrium of long bones progresses linearly along the epiphysis to diaphysis axis. In particular, the onset of expression for later osteoblast markers is coupled with chondrocyte maturation, resulting in characteristic positioning of the leading edge in relation to the hypertrophic cartilage. Thus we examined by in situ hybridization the expression of a panel of osteoblast markers in the perichondrium, and evaluated whether their positioning against the Colα1(X)-expressing hypertrophic chondrocytes on adjacent sections was altered in PKCδ −/−versus wild type littermates at E15.5. In the wild type embryo, the early markers AP (, arrow), Colα1(I) and Runx2 (data not shown) were detected throughout much of the metaphyseal perichondrium. On the other hand, Osx and Bsp were activated in perichondrial cells immediately preceding the hypertrophic region, with their leading edges (orange vertical lines) positioned at a characteristic distance from the first row of Colα1(X)-positive cells (purple vertical line) (). In the PKCδ −/− embryo, the expression patterns of AP (), Colα1(I) and Runx2 (data not shown) were similar to those in the wild type littermate. However, the leading edges of Osx and Bsp (green vertical lines) were significantly closer to the boundary of the hypertrophic zone (purple vertical line) (). Thus, removal of PKCδ appears to delay the onset of Osx expression in the osteoblast lineage, which may in turn impede subsequent differentiation.
To confirm that loss of PKCδ results in intrinsic deficits in osteoblast differentiation, we performed in vitro osteoblastogenesis assays using primary cell cultures. We first assayed for bone nodule formation by E13.5 limb primordial cells. PKC −/− cells produced significantly fewer bone nodules than normal cells (, compare K and L), but instead generated more cartilage nodules (, compare M and N). Secondly, we assayed for AP production by primary bone marrow stromal cells (BMSC) in culture. Here, PKC −/− BMSC showed a significantly lower level than wild type cells (). These results therefore support the conclusion that PKCδ in skeletal progenitor cells promotes osteoblast differentiation.
Lastly, we examined MARCKS phosphorylation levels in the cytosol of E14.5 limb primordial cells. The level of phospho-MARCKS was markedly lower in PKC −/− cells than in wild type cells (). Thus, MARCKS is likely an endogenous substrate of PKCδin vivo.
Wnt7b induces osteoblastogenesis via a noncanonical, PKCδ-dependent mechanism
To assess the physiological relevance of Wnt-PKCδ signaling in bone formation, we investigated whether Wnt7b, a ligand expressed by osteogenic cells in vivo
and able to induce osteoblast differentiation in vitro
(Hu et al., 2005
), signals through this pathway. In keeping with the previous finding, overexpression of Wnt7b, either by transient transfection (), or by viral infection (), induced AP expression in the multipotent mouse embryonic mesenchymal cell line C3H10T1/2 (Taylor and Jones, 1979
). Moreover, Wnt7b overexpression induced formation of bone nodules in both C3H10T1/2 and ST2 cells in mineralization medium (). Finally, Wnt7b also induced osteoblast differentiation in primary cultures of E13.5 limb primordial cells (). Interestingly, in the limb cells, Wnt7b induced more robust osteoblastogenesis than a dominant active form of β-catenin (daβcat) (), even though >90% cells expressed daβcat as judged by co-expression of GFP (data not shown). Thus, Wnt7b activates the osteogenic program in multiple cell systems, and activation may include alternative pathways to that mediated by β-catenin.
Wnt7b induces osteoblastogenesis and PKCδ activity but does not activate canonical Wnt signaling
We next examined whether Wnt7b activates the canonical or the PKCδ pathway in the cell cultures. Wnt7b failed to activate Lef1-Luciferase expression in either ST2 or C3H10T1/2 cells, even though Wnt3a and daβcat greatly stimulated expression (). Accordingly, Wnt7b failed to stabilize β-catenin in C3H10T1/2 cells (, top panel). On the other hand, Wnt7b induced MARCKS phosphorylation in both ST2 () and C3H10T1/2 cells (). Thus Wnt7b does not stimulate canonical Wnt signaling in either ST2 or C3H10T1/2 cells, but activates PKCδ in both cell types.
We then evaluated the potential role of canonical versus PKCδ signaling in Wnt7b-induced osteoblastogenesis. Co-expression of Dkk1 did not impair AP induction by Wnt7b in either C3H10T1/2 () or the primary limb primordial cells (data not shown). However, rottlerin strongly inhibited Wnt7b-induced AP activity in both cell types ( and data not shown). Thus, Wnt7b induces osteoblast differentiation in multiple cell systems via the PKCδ-mediated noncanonical mechanism.
Genetic ablation of Wnt7b results in deficiency in embryonic bone formation
To assess the physiological role of Wnt7b in bone formation, we genetically removed Wnt7b
from the skeletal progenitors by using the Cre-loxP
technique. An initial report examining E18.5 embryos devoid of Wnt7b failed to show any clear skeletal phenotype (Rodda and McMahon, 2006
). Here, we generated Wnt7b
mutant mice (Dermo1-Cre; Wnt7bn/c3
) carrying a Wnt7b
null allele (Wnt7bn
) (Parr et al., 2001
), a Wnt7b
conditional allele (Wnt7bc3
, JR, TJC and APM in preparation), and also a Dermo1-Cre
allele (Yu et al., 2003
). The Wnt7bc3
allele had loxP
sites flanking the essential exon 3 and functioned as a null allele upon recombination by Cre (to be reported elsewhere). Wnt7b
mutant animals were viable after birth with no obvious phenotype. However, whole-mount skeletal staining revealed that, at E15.5 when wild type embryos showed obvious ossification, Wnt7b
mutant littermates exhibited a diminution in ossification, while some mutant embryos also appeared to be slightly smaller (). Regardless of the overall size, the bone collar of long bones was consistently shorter in mutant littermates (), as confirmed by quantitation of the relative bone collar length over total length of the element (). At E18.5, mutant skulls exhibited less alizarin red staining and widened sutures (). Thus, Wnt7b deficiency results in less bone in mouse embryos.
Removal of Wnt7b in skeletal cells results in defects in bone formation
We next examined whether chondrocyte maturation was perturbed in Wnt7b mutants. At E14.5, the overall length of the hypertrophic zone expressing Colα1(X) was similar between mutant and wild type littermates (). However, in mutants the domains expressing PTHrP-R or Ihh were less well separated, and the terminal hypertrophic region expressing MMP13 was clearly reduced. At E15.5, the distance between the two Colα1(X)-expressing domains was significantly reduced in the mutant. Thus, loss of Wnt7b appears to delay subsequent maturation of chondrocytes after the initiation of Colα1(X) expression.
To examine potential intrinsic defects in the osteoblast lineage, we assayed the expression of AP, Bsp, Runx2, Osx and PTHrP-R, in relation to Colα1(X) on adjacent sections of E15.5 long bones. Whereas AP and Runx2 expression in the perichondrium appeared indistinguishable between mutant and wild type littermates, the leading edges for Osx, PTHrP-R and Bsp were consistently closer to the boundary of the hypertrophic zone in the mutant (). Thus, removal of Wnt7b, similar to that of PKCδ, results in a deficit in Osx activation and subsequent osteoblast differentiation.
To determine whether removal of Wnt7b disrupted canonical Wnt signaling in long bones, we performed in situ hybridization for Dkk1 and Tcf1, two known target genes of the pathway. Both molecules were expressed normally in Wnt7b mutant embryos (). In contrast, the limb primordial cells from E14.5 mutant embryos contained a significantly lower level of phospho-MARCKS than wild type littermates (), indicating that Wnt7b stimulates MARCKS phosphorylation in vivo. Thus, the bone defect in Wnt7b mutant embryos is unlikely due to disruption of canonical Wnt signaling, but appears to correlate with impairment in PKCδ activation.
To confirm that Wnt7b directly regulates osteoblast differentiation, we evaluated Wnt7b-deficient cells for their ability to differentiate in vitro. We utilized calvarial cells from neonates, as well as BMSC from adult mice, both containing osteoblast precursors. In both cases, Wnt7b-deficient cells produced significantly fewer bone nodules than wild type cells (). These results are consistent with the notion that endogenous Wnt7b promotes osteoblast differentiation from progenitors.
In summary, the present study reveals a novel osteogenic pathway in which Wnt molecules, such as Wnt7b, signal through the Gq family of G protein α-subunits to activate PKCδ that in turn promotes the transition from Runx2- to Osx-expressing cells ().