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Molecular Endocrinology, doi:10.1210/me.2007-0323
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Molecular Endocrinology 21 (12): 3050-3061
Copyright © 2007 by The Endocrine Society

Homodimerization of Ror2 Tyrosine Kinase Receptor Induces 14-3-3β Phosphorylation and Promotes Osteoblast Differentiation and Bone Formation

Yan Liu, John F. Ross, Peter V. N. Bodine and Julia Billiard

Women’s Health and Musculoskeletal Biology (Y.L., P.V.N.B., J.B.), Wyeth Research, Collegeville, Pennsylvania 19426; and Biological Technologies (J.F.R.), Wyeth Research, Cambridge, Massachusetts 02140

Address all correspondence and requests for reprints to: Julia Billiard, Ph.D., Women’s Health and Musculoskeletal Biology, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426. E-mail: billiaj{at}wyeth.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ror2 receptor plays a key role in bone formation, but its signaling pathway is not completely understood. We demonstrate that Ror2 homodimerizes at the cell surface, and that dimerization can be induced by a bivalent antibody. Antibody-mediated dimerization causes receptor autophosphorylation and induces functional consequences of its signaling, including osteogenesis in mesenchymal stem cells and bone formation in organ culture. We further show that Ror2 associates with and phosphorylates 14-3-3β scaffold protein. Endogenous Ror2 binds 14-3-3β in U2OS osteosarcoma cells, and purified intracellular domain of Ror2 interacts with 14-3-3β in vitro. 14-3-3β Is tyrosine phosphorylated in U2OS cells, and this phosphorylation is inhibited by down-regulating Ror2 and enhanced by overexpressing the kinase. Purified Ror2 phosphorylates 14-3-3β in vitro, confirming 14-3-3β as the first identified Ror2 substrate. Down-regulating 14-3-3β potentiates osteoblastogenesis in mesenchymal stem cells and increases bone formation in calvarial cultures, indicating that 14-3-3β exerts a negative effect on osteogenesis. This raises a possibility that Ror2 induces osteogenic differentiation, at least in part, through a release of the 14-3-3β-mediated inhibition. Our research forms a foundation for several new areas of investigation, including the molecular regulation of 14-3-3 by tyrosine phosphorylation and the role of this scaffold in osteogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ROR2 BELONGS TO a family of receptor tyrosine kinases (RTKs) that in mammals consists of two members, Ror1 and Ror2. Ror receptors are most closely related to Trk neurotrophin receptors and muscle-specific kinase (1). Ror2 plays key roles in developmental morphogenesis, particularly of the cartilage-derived skeleton. Disruption of mouse Ror2 leads to profound skeletal abnormalities with all endochondrally derived bones foreshortened or misshapen (2, 3, 4), and mutations within Ror2 gene in humans are responsible for short stature, limb bone shortening, and segmental defects of the spine (5, 6, 7, 8). In addition to its function during development, Ror2 has been identified as a prosurvival kinase in HeLa cervical carcinoma cells (9) and shown to modulate neurite extension in central neurons (10). We recently demonstrated that Ror2 is expressed in human osteoblasts and in mouse calvarial bones (11), and that it initiates commitment of mesenchymal stem cells (MSCs) to osteoblastic lineage and promotes differentiation at early and late stages of osteoblastogenesis (12). MSCs are pluripotent cells that can differentiate into several distinct lineages, including osteoblasts. The transition from stem cells to mature osteoblasts is characterized by the formation of mineralized extracellular matrix. The molecular mechanisms underlying Ror2-induced osteogenesis remain unknown.

It has been well documented that ligand binding to RTKs triggers receptor dimerization (13). The epidermal growth factor receptor (EGF-R) was the first RTK found to dimerize after ligand binding (14). Furthermore, it was shown that bivalent EGF-R antibody can induce EGF-R dimerization, and that is sufficient for activation of the EGF-R tyrosine kinase (15). We therefore tested whether Ror2 is activated by homodimerization, and whether dimerization of the receptor is sufficient to promote osteoblastogenesis.

The molecular mechanisms of Ror2 signaling have just recently begun to be elucidated. Ror2 was shown to bind the Wnt family of glycoproteins and to modulate both canonical and noncanonical Wnt signaling (11, 16, 17, 18). Ror2 was also found to functionally interact with BRI-b, a bone morphogenetic protein receptor that modulates chondrogenesis (19). Ror2 binds to and regulates cellular distribution of a transcriptional regulator Dlxin-1 (20). Kani et al. (21) demonstrated that Ror2 associates with and is phosphorylated by casein kinase I{epsilon}. Furthermore, coexpression of casein kinase I{epsilon} and Ror2, but not the kinase-inactive Ror2 mutant, results in tyrosine phosphorylation of G protein-coupled receptor kinase 2 (21). However, to our knowledge, no direct substrate for the Ror2 tyrosine kinase has been identified to date.

14-3-3 Is a highly conserved protein family ubiquitously expressed in all eukaryotes, from plants to mammals, with an approximately 30-kDa molecular mass. Seven isoforms (β, {gamma}, {epsilon}, {varsigma}, {zeta}, {tau}, and {eta}) have been identified in mammals (22), and the crystal structure for each of them in ligand-bound or -unbound state has been solved (23). 14-3-3 Molecules reside in the cytoplasm as homo- or heterodimers (23, 24, 25) and interact with more than 100 different proteins. Binding of 14-3-3 can alter the localization, stability, activity and/or molecular interactions of a target protein. Consequently, 14-3-3 scaffolds regulate many crucial cellular processes including differentiation, apoptosis, cell-cycle control, protein trafficking, signal transduction, and malignant transformation. The proapoptotic protein Bax interferes with the antiapoptotic function of Bcl in the mitochondria unless it is bound to 14-3-3 in the cytoplasm (26). The protein tyrosine phosphatase Cdc25 that regulates G2/M transition of the mitotic cell cycle is held in the cytosol by 14-3-3, and mutant Cdc25 that cannot bind 14-3-3 accumulates in the nucleus leading to disruption of mitotic- and G2 checkpoint control (27, 28). 14-3-3 Regulates gene expression through sequestering transcription factors in the cytoplasm, including FKHRL1 (29), glucocorticoid receptor (30), and Nur77 (31). The binding of 14-3-3 increases catalytic activity of tryptophan and tyrosine hydroxylases (32) and of protein kinase C (33). 14-3-3 Binding protects Raf-1 from dephosphorylation (34, 35) and protects plant nitrate reductase from proteolysis (36). 14-3-3 Proteins also serve as scaffolds to bridge two targets, as for example Raf and protein kinase C (37), or Raf and Bcr (38).

Here we address the molecular mechanism underlying Ror2-induced osteogenesis. We find that Ror2 forms homodimers at the cellular membrane, and that the extent of dimerization is greatly enhanced by treatment with a bivalent Ror2 antibody. Antibody-induced dimerization causes receptor autophosphorylation and increases osteogenesis in human MSCs (hMSCs) and bone formation in mouse calvarial bones ex vivo. Thus, homodimerization of the Ror2 receptor is sufficient to cause the functional consequences of its signaling. Using immunoprecipitation followed by mass spectrometric analysis of U2OS osteosarcoma cells expressing Ror2, we find that Ror2 associates with 14-3-3β scaffold protein. We confirm this association in cells and in vitro and further demonstrate that 14–3-3β is directly phosphorylated by the Ror2 tyrosine kinase. Down-regulating 14-3-3β expression with a specific short hairpin RNA (shRNA) potentiates osteogenesis in MSCs and increases bone formation in calvarial cultures, suggesting that 14-3-3β exerts a negative effect on osteogenesis. We propose that Ror2 may induce osteogenic differentiation, at least in part, through a release of the 14-3-3β-mediated inhibition.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We studied the molecular mechanisms of the Ror2 receptor activation and signaling. We hypothesized that the Ror2 receptor can form homodimers similar to EGF-R and platelet-derived growth factor receptor. To test this hypothesis, we expressed flag-tagged and his-tagged Ror2 receptor constructs in U2OS osteosarcoma cells and treated the cells for 1 h at 37 C with a bivalent Ror2-specific polyclonal antibody raised against the entire extracellular domain of Ror2 or with a nonspecific IgG control. High degree of specificity of the anti-Ror2 antibody was verified by demonstrating no binding to Ror1 at all concentrations tested (data not shown). Upon incubation, total cellular proteins were precipitated on anti-flag affinity agarose and subjected to immunoblotting with anti-his antibody. As shown in Fig. 1AGo, flag-tagged Ror2 precipitated the his-tagged protein, demonstrating that Ror2 does form homodimers in this overexpression system. The homodimer formation was enhanced more than 5-fold upon treatment with the bivalent anti-Ror2 antibody (densitometric analysis of Fig. 1AGo). The specificity of interactions was demonstrated by the fact that anti-flag antibody failed to immunoprecipitate Ror2-his in the absence of Ror2-flag, and that anti-his antibody did not recognize the Ror2-flag protein (Fig. 1AGo).


Figure 1
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  		Fig. 1. Antibody-Induced Dimerization Stimulates Ror2 Autophosphorylation and Promotes Receptor Activation

A, U2OS cells were transiently transfected with the indicated Ror2 constructs and treated with anti-Ror2 antibody or nonspecific IgG (50 µg/ml each) for 1 h at 37 C. Cell lysates were precipitated on anti-flag agarose and subjected to immunoblotting with anti-his antibody (top panel). In the bottom panel, 10% of the precipitated material was immunoblotted with anti-flag antibody to verify equal expression and precipitation. B, U2OS cells were transiently transfected with or without Ror2 plasmid and treated with IgG (100 µg/ml) or anti-Ror2 antibody (100 µg/ml or indicated concentrations) for 1 h at 37 C. Cell lysates were precipitated on antiphosphotyrosine agarose and subjected to immunoblotting with anti-Ror2 antibody. C, U2OS cells were transiently transfected with Ror2 plasmid and treated with 100 µg/ml IgG or anti-Ror2 antibody for 1 h at 37 C. Cell lysates were precipitated on anti-flag agarose and subjected to immunoblotting with antiphosphotyrosine antibody (top panel). In the bottom panel, 10% of the precipitate was immunoblotted with antiflag antibody. D (left), Schematic representation of the Ror2-TrkB chimera; EC, extracellular domain; M, transmembrane domain; IC, intracellular domain. D (right), HEK293 cells stably transfected with 3xCRE-luciferase and Ror2-TrkB chimera (HEK293 CRE-luc Ror2-TrkB) were treated with anti-Ror2 antibody or nonspecific IgG at indicated concentrations for 24 h. Luciferase activity was assessed as described in Materials and Methods, and the signal obtained in IgG-treated sample was set at 1. The results are means ± SE of n = 8 (*, P < 0.05). ab, Antibody; IP, immunoprecipitation; WB, Western blot.

 
Next, we asked whether Ror2 dimerization results in receptor activation as assessed by autophosphorylation. U2OS cells were treated with IgG or anti-Ror2 antibody with or without Ror2 overexpression, and the lysates were precipitated with antiphosphotyrosine agarose and probed for Ror2. Ror2-specific antibody induced tyrosine phosphorylation of the endogenous Ror2 receptor in a dose-dependent manner (Fig. 1BGo, left panel). Ror2 overexpression also resulted in an increased level of background phosphorylation that was further enhanced by treatment with the Ror2 antibody (Fig. 1BGo, right panel). This observation was confirmed in a reverse experiment (Fig. 1CGo), in which overexpressed Ror2-flag was precipitated on anti-flag agarose and blotted for phosphotyrosine. Because anti-Ror2 antibody used for cell treatment remained bound to Ror2 after cell lysis, we could not use protein A sepharose to pull down endogenous Ror2 and confirm its increased phosphorylation status. These data provide strong evidence that Ror2 dimerization induced by overexpression or by treatment with a bivalent Ror2-specific antibody activates the Ror2 tyrosine kinase receptor.

Next, we tested whether Ror2 dimerization can result in activation of intracellular signaling. Because the Ror2 signaling mechanism is not completely understood, we used a different RTK as a signaling read-out. To this end, the extracellular domain of Ror2 was fused to the transmembrane and cytoplasmic domains of the TrkB receptor tyrosine kinase (Fig. 1DGo, left panel). The TrkB signaling is activated by receptor homodimerization and includes phosphorylation of ERK and stimulation of the cAMP response element (CRE)-mediated transcription. Human embryonic kidney (HEK)293 cells stably overexpressing 3xCRE (cAMP responsive element) promoter-luciferase reporter gene and the Ror2-TrkB chimeric receptor showed a dose-dependent increase in luciferase activity in response to treatment with the Ror2-specific antibody (Fig. 1DGo, right panel), confirming that dimerization of Ror2 results in activation of intracellular signaling.

To further elucidate the Ror2 signaling pathway, we identified potential Ror2 binding factors by mass spectrometric analysis of Ror2 protein complexes. U2OS cells were transiently transfected with Ror2-flag or pcDNA3.1 expression plasmids, and the whole-cell extracts were immunoprecipitated with anti-flag agarose. The resulting products were separated on a one-dimensional polyacrylamide gel, and the proteins from each entire lane were analyzed by mass spectrometry as described in more detail in Materials and Methods. After comparing the results from the analysis of the Ror2 immunoprecipitation with the negative control sample, the scaffold protein 14-3-3β was identified as a potential Ror2 binding partner.

The interaction between endogenous Ror2 and 14–3-3β in U2OS cells was confirmed by precipitating Ror2 with anti-14-3-3β antibody (Fig. 2AGo). Because Ror2 is not autophosphorylated in the absence of overexpression or anti-Ror2 antibody treatment (Fig. 1BGo), 14-3-3β must bind to the inactive form of the receptor. Furthermore, bacterially expressed glutathione-S-transferase (GST)-tagged cytosolic domain of Ror2 (GST-R2C), but not GST alone, interacted with in vitro translated 14-3-3β, indicating that the interaction is direct and does not require other binding partners (Fig. 2BGo). Finally, U2OS cells were infected with adenoviruses encoding the flag-tagged wild-type Ror2, the flag-tagged point mutant lacking most of the tyrosine kinase activity [Ror2 kinase domain mutant (Ror2KD), described in Ref. 11 ], or β-galactosidase (β-gal), and the interaction was confirmed by immunoprecipitation on both anti-flag and anti-14-3-3β agarose (Fig. 2Go, C and D). Equal levels of protein expression and precipitation were verified by immunoblotting (Fig. 2Go, C and D). We noticed that the complex formation was stronger with the Ror2KD mutant, suggesting that the wild-type kinase may phosphorylate 14-3-3β leading to substrate release.


Figure 2
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  		Fig. 2. Ror2 Binds 14-3-3β Scaffold Protein in Cells and in Vitro

A, U2OS whole-cell protein extracts were precipitated on anti-14-3-3β antibody-conjugated agarose or unconjugated agarose (control) and subjected to immunoblotting with anti-Ror2 antibody. B (top panel), Schematic representation of GST-R2C. B (bottom panel), GST-R2C or GST alone was expressed in bacterial cells, precipitated on glutathione sepharose, and incubated with in vitro translated 14-3-3β for 4 h at 4 C. Proteins bound to glutathione sepharose at the end of this incubation were resolved by SDS-PAGE and probed with anti-14-3-3β antibody. C, U2OS cells were infected with β-Gal, Ror2, or Ror2KD adenoviruses for 48 h, and the whole-cell extracts were immunoprecipitated on anti-flag agarose and subjected to immunoblotting analysis with anti-14-3-3β antibody (top panel) and anti-flag antibody (middle panel, control for equal precipitation of Ror2 and Ror2KD). The bottom panel shows Western blot analysis with anti-14-3-3β antibody of the U2OS extracts (50 µg/lane, control for equal loading in the immunoprecipitation reactions). D, U2OS cells were infected with β-Gal, Ror2, or Ror2KD adenoviruses, and the whole-cell extracts were immunoprecipitated on anti-14-3-3β antibody and analyzed by immunoblotting with anti-flag (top panel) or anti-14-3-3β antibodies (bottom panel, control for equal loading and precipitation of 14-3-3β). AA, Ascorbic acid; IP, immunoprecipitation; WB, Western blot.

 
To test this hypothesis, β-gal, Ror2, or Ror2KD were overexpressed in U2OS cells, and the phosphorylation state of 14-3-3β was examined by anti-14-3-3β immunoprecipitation followed by antiphosphotyrosine immunoblotting. As shown in Fig. 3AGo (left panels), the background level of 14-3-3β phosphorylation observed in β-gal-infected cells was enhanced by Ror2 expression, but not by Ror2KD, indicating that the kinase activity of Ror2 is required for tyrosine phosphorylation of 14-3-3β. The reverse approach, i.e. precipitation on phosphotyrosine antibody followed by detection with anti-14-3-3β, confirmed this observation (Fig. 3AGo, right panel). We frequently observed 14–3-3β as a doublet (e.g. Fig. 3Go, A and B), which may represent a degradation product or reflect differential states of phosphorylation on both tyrosine as well as serine-threonine. To test whether endogenous Ror2 mediates the background 14-3-3β phosphorylation, we transfected U2OS cells with the Ror2-specific short interfering RNA (siRNA), which reduced Ror2 protein levels by approximately 90% (data not shown). The specificity of the siRNA was confirmed by demonstrating no change in the levels of Ror1 mRNA upon transfection (data not shown). The siRNA-mediated reduction in Ror2 levels led to a significant down-regulation of 14-3-3β phosphorylation as assessed by two alternative immunoprecipitation approaches (Fig. 3BGo). This is especially obvious when precipitation is done on 14-3-3β antibody. The higher level of background phosphorylation observed upon phosphotyrosine precipitation may be related to the fact that phosphotyrosine antibody brings down all phosphorylated proteins in the cell and if any of these proteins are bound to 14-3-3β, it will be precipitated even when it is not tyrosine phosphorylated itself. The precipitation with 14-3-3β antibody is therefore a more direct way to assess changes in the 14-3-3β phosphorylation status.


Figure 3
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  		Fig. 3. Ror2 Tyrosine Kinase Phosphorylates 14-3-3β in Cells and in Vitro

A, U2OS cells were infected with β-Gal, Ror2, or Ror2KD adenoviruses for 48 h, and the whole-cell extracts were immunoprecipitated on anti-14-3-3β or antiphosphotyrosine agarose and subjected to immunoblotting with the indicated antibodies (top panels). In the bottom panel, 10% of the precipitate was used for immunoblotting with anti-14-3-3β antibody to demonstrate equal expression and precipitation. B, U2OS cells were transfected with Ror2 siRNA or nonspecific siRNA, and 48 h later whole-cell extracts were immunoprecipitated on anti-14-3-3β or antiphosphotyrosine agarose and subjected to immunoblotting analysis with the indicated antibodies (top panels). The bottom panels show Western blot analysis with anti-14-3-3β antibody of 10% of the immunoprecipitate or 50 µg/lane of the total U2OS extracts. C, Purified GST-tagged 14-3-3β was used as a substrate in the in vitro kinase assay with GST-R2C or GST alone. At the end of the kinase assay, the proteins were separated by SDS-PAGE, and the extent of phosphorylation was assessed by immunoblotting with antiphosphotyrosine antibody. D, U2OS cells were treated with IgG or anti-Ror2 antibody (50 µg/ml) for 1 h at 37 C. Cell lysates were immunoprecipitated on anti-14-3-3β or antiphosphotyrosine agarose and subjected to immunoblotting analysis with the indicated antibodies (top panels). In the bottom panel, 10% of the precipitate was used for immunoblotting with anti-14-3-3β antibody. IP, Immunoprecipitation; WB, Western blot.

 
GST-R2C purified from Escherichia coli phosphorylated purified GST-tagged 14-3-3β in vitro (Fig. 3CGo) confirming 14-3-3β as a direct substrate for the Ror2 tyrosine kinase. Finally, treatment of U2OS cells with the Ror2-specific antibody increased 14-3-3β phosphorylation level compared with IgG control (Fig. 3DGo), indicating that activation of the Ror2 kinase by the antibody causes tyrosine phosphorylation of a downstream substrate.

Next, we addressed the functional consequences of antibody-induced dimerization and activation of Ror2. We have previously shown that Ror2 initiates commitment of hMSCs to osteoblastic lineage and promotes differentiation at early and late stages of osteoblastogenesis (12). Because hMSCs do not express Ror2 unless differentiated toward the osteogenic phenotype (11), we induced Ror2 expression by treatment with osteogenic cocktail [mesenchymal stem cell growth medium (MSCGM) supplemented with 0.05 mM ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone] and added increasing amounts of anti-Ror2 antibody, anti-Ror1 antibody, or nonspecific goat IgG. After 9 d of incubation, the degree of mineralized matrix formation was assessed with alizarin red-S histochemical staining. As shown in Fig. 4AGo, anti-Ror2 antibody increased the extent of calcified matrix formation in hMSCs in a dose-dependent manner. Neither nonspecific IgG nor a polyclonal antibody raised against the entire extracellular domain of human Ror1 had any effect on matrix mineralization at all concentrations tested (Fig. 4AGo). The anti-Ror2 antibody effect was mediated through the Ror2 receptor, because it disappeared when the Ror2 expression in hMSC was inhibited by the Ror2-specific shRNA (Fig. 4BGo). Furthermore, anti-Ror2 antibody induced calcified matrix formation even in the absence of dexamethasone if the cells were induced to express Ror2 through adenovirus infection (Fig. 4CGo). Thus, Ror2-specific antibody can dimerize and activate the Ror2 receptor and promote Ror2-mediated calcified matrix formation in hMSCs.


Figure 4
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  		Fig. 4. Antibody-Induced Ror2 Dimerization Promotes Mineralized Matrix Formation in hMSCs

A, hMSCs were cultured in MSCGM supplemented with 0.05 mM ascorbic acid (AA), 10 mM β-GP, and 100 nM dexamethasone (Dex) in the presence of indicated concentrations of nonspecific IgG, anti-Ror2, or anti-Ror1 antibody. After 9 d, cells were subjected to alizarin red-S staining for matrix mineralization. B, hMSCs were infected with adenoviruses containing shRNA specific for Ror2 or EGFP (control) and incubated in MSCGM supplemented with 50 µg/ml anti-Ror2 antibody, 0.05 mM ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone. After 12 d, cells were subjected to alizarin red-S staining for matrix mineralization. C, hMSCs were infected with Ror2 adenovirus and cultured in MSCGM supplemented with 0.05 mM ascorbic acid, 10 mM β-glycerophosphate in the presence of 50 µg/ml of nonspecific IgG, or anti-Ror2 antibody. After 19 d, cells were subjected to alizarin red-S staining for matrix mineralization. ab, Antibody; cont, control.

 
To test whether the Ror2 substrate 14-3-3β plays a role in osteogenic differentiation, we inhibited 14-3-3β expression in the absence and presence of Ror2 overexpression. hMSCs were infected with the 14-3-3β-specific shRNA, which down-regulated the endogenous protein expression by 60% when compared with scrambled control shRNA (Fig. 5AGo). The specificity of the shRNA was confirmed by demonstrating no down-regulation of the two closely related 14-3-3 isoforms, 14-3-3{gamma} and 14-3-3{epsilon} (Fig. 5BGo). Down-regulation of 14-3-3β greatly promoted the formation of mineralized matrix (Fig. 5CGo) in the absence or presence of Ror2, suggesting that 14-3-3β scaffold protein exerts inhibitory effects on osteogenic differentiation of hMSCs. Down-regulation of 14-3-3β did not affect proliferation or apoptosis of hMSCs (data not shown). Figure 5CGo also demonstrates that the effects of Ror2 activation and 14-3-3β inhibition are not additive, suggesting that the two may be part of the same pathway.


Figure 5
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  		Fig. 5. Down-Regulation of 14-3-3β Promotes Mineralized Matrix Formation in hMSCs

A, hMSCs were infected with adenoviruses encoding 14-3-3β shRNA or scrambled shRNA and after 9 d of incubation, 50 µg of total cellular protein was subjected to Western immunoblotting for the endogenous 14-3-3β protein. B, U2OS osteosarcoma cells were infected with adenoviruses encoding 14-3-3β shRNA or scrambled shRNA and 24 h later, total cellular RNA was isolated and real-time RT-PCR analysis was performed to quantify expression of the indicated isoforms of 14-3-3. The levels of mRNA expression for each isoform were normalized to the expression of a housekeeping gene, cyclophilin B, and expression in the presence of scrambled shRNA was set at 100%. Only 14-3-3β mRNA was significantly down-regulated by 14-3-3β-specific shRNA. C, hMSCs were infected with adenoviruses encoding β-Gal, Ror2, 14-3-3β shRNA, or scrambled shRNA, as indicated. After 12 d of incubation in MSCGM supplemented with 0.05 mM ascorbic acid and 10 mM β-glycerophosphate, alizarin red-S staining was performed to assess matrix mineralization. The graph on the right provides quantification of the alizarin red-S staining from three independent experiments (*, P < 0.05). scr, Scrambled; WB, Western blot.

 
We next tested whether the in vitro effects of Ror2 activation and 14-3-3β inhibition translate into increased bone formation in ex vivo organ cultures. Calvarial bones of 4-d-old mouse littermates were infected with adenoviruses containing scrambled shRNA or 14-3-3β-specific shRNA; 48 h later, the calvariae were treated with 12 µg/ml of anti-Ror2 antibody or nonspecific IgG. After 7 d of culture with adenoviruses and antibodies, the bones were stained with hematoxylin-eosin and subjected to histomorphometric analysis. Under control conditions of scrambled shRNA infection and IgG treatment, 600 µm length of calvaria contained 9394 ± 1333 µm2 of bone area and 39.5 ± 7.4 osteoblasts. Inhibition of 14-3-3β by the specific shRNA caused 60% increase in the total bone area and 50% increase in the osteoblast number, indicating that 14-3-3β protein inhibits osteoblast counts and/or activity (Fig. 6Go). Treatment of calvarial bones with anti-Ror2 antibody resulted in 85% increase in osteoblast number and 50% increase in total bone area, suggesting that activating Ror2 antibody promotes osteogenesis. Consistent with the hMSC experiment in Fig. 5CGo, combining the two treatments did not produce an additive effect, resulting in even a slightly smaller response than either treatment alone (Fig. 6Go). Thus inhibition of 14-3-3β and antibody-induced dimerization of Ror2 receptor promote osteogenesis in MSC and in calvarial organ cultures.


Figure 6
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  		Fig. 6. Activation of Ror2 Receptor and Down-Regulation of 14-3-3β Promote New Bone Formation

Calvariae from 4-d-old mouse pups were excised and infected with scrambled shRNA or 14-3-3β shRNA (5 x 107 viruses/ml) for 48 h before addition of nonspecific IgG or anti-Ror2 antibody (12 µg/ml). After 7 d of incubation in the presence of adenovirus and antibody, the total bone area (open bars) and osteoblast number (solid bars) were assessed as described in Materials and Methods. Values obtained in nonspecific shRNA-infected and IgG-treated cultures were set at 100%. The results are means ± SE of four to five calvariae per condition (*, P < 0.05). The results are representative of three independent experiments. scr, Scrambled; WB, Western blot.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our results demonstrate that Ror2 receptor tyrosine kinase is activated by homodimerization and directly phosphorylates 14-3-3β scaffold protein. We show that Ror2 dimerization is sufficient to induce osteogenesis in hMSCs and to promote bone formation in calvarial organ culture. Furthermore, we identify 14–3-3β as a negative regulator of osteogenesis.

Since the first report that ligand binding induces dimerization of the EGF receptor tyrosine kinase resulting in autophosphorylation and activation (14), many other RTKs have been shown to be activated by dimerization (13). In addition to homodimer formation, heterodimers between different RTKs as well as heteromeric complexes with other types of membrane receptors, for example G protein-coupled receptors, have been reported (39, 40). Formation of different dimerization complexes can account for signal diversification of some RTKs (41). Ror2 has been previously shown to form a heteromeric complex with a bone morphogenetic protein family serine/threonine kinase receptor BRI-b and to become phosphorylated by this kinase (19). This complex formation was independent of the BRI-b ligand, GDF5 (19). Taken together with our current observation of homodimer formation, these results indicate that Ror2 is capable of both homo- and heterodimerization. Whether Ror2 binds BRI-b as a dimer remains to be determined.

Here we show direct interaction between cytoplasmic domain of Ror2 and 14-3-3β in vitro and between endogenous proteins in U2OS cells. In cells, 14-3-3β bound endogenous Ror2 in the absence of dimerization agents under conditions of no detectable tyrosine phosphorylation of the Ror2 receptor (cf. Figs. 1BGo and Fig. 2AGo). These data indicate that 14-3-3 binding does not require Ror2 activation. The binding of 14-3-3β to Ror2KD (Fig. 2Go, C and D) seems to support this conclusion; however, Ror2KD may become transphosphorylated upon dimerization with endogenous Ror2 protein. Our attempts to test whether Ror2 activation by the antibody altered 14-3-3 binding failed due to very high background in Western immunoblotting, presumably because traces of anti-Ror2 antibody and nonspecific IgG in the whole-cell extracts bound protein A sepharose used for IP (data not shown).

Most of 14-3-3 binding proteins require serine phosphorylation for interaction, and two high-affinity binding motifs have been identified: RSXpSXP and RXXXpSXP, where pS represents phosphoserine (42, 43). Ror2 does not possess either of these sequences. On the other hand, phosphorylation-dependent sites that diverge significantly from these motifs have been described (23). Furthermore, several proteins have been identified that do not require phosphorylation for binding, including Bax (26), p190RhoGEF (44), and exoenzyme S (45). A recent crystallography study by Ottmann et al. (46) found that exoenzyme S binds 14-3-3 through hydrophobic residue contacts. Unlike the phosphoserine-dependent binding motifs that are recognized by all 14-3-3 isoforms, phosphorylation-independent binding may provide isoform specificity (47, 48). Investigating the mechanism of Ror2–14-3–3β interactions will undoubtedly further our understanding of both these signaling pathways.

We found that Ror2 directly phosphorylates 14-3-3β on tyrosine residue(s). There are 11 tyrosines within the 14-3-3β sequence, of which nine are conserved not only between all human 14-3-3 isoforms, but evolutionary from yeast to humans. It is not yet known which particular tyrosine(s) are phosphorylated by Ror2. Previously, tyrosine phosphorylation of 14-3-3 has been demonstrated for several family members, but the specific phosphorylation sites have not been identified. Both 14-3-3β and {zeta} were found to be tyrosine phosphorylated by IGF receptor 1 in yeast and in vitro (49), and 14-3-3{tau} was shown to be phosphorylated on tyrosine in vitro and in mammalian cells by a Bcr-Abl chimeric kinase produced in Ph-positive leukemias (50). However, functional consequences of the tyrosine phosphorylation of 14-3-3 have only been demonstrated in plants where phosphorylation of Tyr137 in the maize 14-3-3 isoform GF14–6 by IGF receptor 1 inhibited interaction with the plasma membrane H+-ATPase (51). Unlike tyrosine phosphorylation, regulation of 14-3-3 proteins by serine/threonine phosphorylation has been well established. Jnk-dependent phosphorylation of 14-3-3{zeta} on Ser184 leads to the dissociation of 14-3-3 from Bax and initiation of apoptosis (52). MAPKAPK2 (53), Akt (54), and sphingosine-dependent kinase (55) phosphorylate 14-3-3{zeta} on Ser58 and that phosphorylation compromises the ability of 14-3-3{zeta} to dimerize (55), which should have profound consequences on all its binding partners. 14-3-3{zeta} And {tau} are phosphorylated by casein kinase I{alpha}, and phosphorylation of 14-3-3{zeta} on Thr233 in HEK293 cells correlates with loss of c-Raf binding (56). Whether tyrosine phosphorylation of 14-3-3β by Ror2 has functional consequences for any of its binding partners remains to be elucidated. Interestingly, infection of mouse calvarial cultures with the Ror2 virus increases total bone area by 72%, whereas the Ror2 KD virus has no effect (12), suggesting that a tyrosine phosphorylation event is required for the osteogenic function of the Ror2 receptor ex vivo.

We have found that 14-3-3 negatively regulates osteogenesis of pluripotent MSCs. MSCs can differentiate into several distinct lineages, including adipocytes and osteoblasts, and a reciprocal relationship exists between adipogenic and osteogenic differentiation with several pathways promoting one and inhibiting the other (57, 58). The specific mechanism by which 14-3-3 inhibits osteogenesis remains to be elucidated but may involve regulating cellular localization of osteogenic differentiation factors. Indeed, altering subcellular localization of their binding partners is one of the main modes of function for 14-3-3 proteins (22). Furthermore, 14-3-3 has been shown to control cellular localization of TAZ, a transcriptional modulator of MSC differentiation (59, 60). TAZ (transcriptional coactivator with PDZ-binding motif) bound 14-3-3 through a site in TAZ involving Ser89, sequestering this transcriptional modulator in the cytoplasm (60). Replacement of Ser89 with Ala disrupted 14-3-3 binding and relocalized TAZ to the nucleus where it acted as coactivator of Runx2-driven gene expression and as inhibitor of PPAR{gamma}-dependent transcription (59). Because Runx2 drives MSCs to differentiate toward osteoblasts, whereas PPAR{gamma} converts them to adipocytes, TAZ translocation into the nucleus should promote osteogenic differentiation at the expense of adipogenesis. Interestingly, this was also the phenotype observed upon Ror2 overexpression in hMSCs (12).

A proposed model for Ror2/14-3-3β signaling is presented in Fig. 7Go. When inactive, Ror2 remains a monomer bound to a 14-3-3β dimer. This 14-3-3β dimer, in turn, is bound to protein(s) that regulate osteoblast differentiation, possibly TAZ or other osteogenic transcription factor(s). Once dimerized, Ror2 becomes autophosphorylated and tyrosine phosphorylates 14-3-3β, which causes activation of osteogenic factor(s), perhaps through their release and nuclear translocation. Many additional studies will be required to test these hypotheses.


Figure 7
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  		Fig. 7. Ror2 and 14-3-3β Signaling Regulate Osteogenic Differentiation

We hypothesize that in the inactive state, Ror2 exists as a monomer in the cell membrane, and 14-3-3β is bound to osteogenic differentiation factor(s). Association with 14-3-3β prevents these differentiation factors from signaling, possibly by sequestering them in the cytosol or by other inhibitory mechanisms. Upon activation, Ror2 forms homodimers that trigger receptor autophosphorylation and subsequent tyrosine phosphorylation of 14-3-3β. Once 14-3-3β is phosphorylated, osteogenic differentiation factor(s) unbind and become activated. Thus Ror2 promotes osteogenesis through relieving 14-3-3β-mediated inhibition. dif F, Differentiation factor. P, Phosphorylation.

 
In summary, we have demonstrated that the Ror2 receptor tyrosine kinase is activated by dimerization and binds to and phosphorylates 14-3-3β. 14-3-3β Exerts negative effects on osteogenesis raising a possibility that Ror2 may induce osteogenic differentiation in part through a release of the 14-3-3β-mediated inhibition. Our studies identify the first direct substrate of the Ror2 receptor tyrosine kinase and demonstrate a role for 14-3-3 scaffold protein in regulating osteoblastogenesis. These observations provide the foundation for future studies of both Ror2 and 14-3-3 signaling pathways as well as of the novel mechanisms of osteogenic differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials and Tissue Culture
Except where noted, tissue culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA); other reagents and chemicals were purchased from either Sigma Chemical Co. (St. Louis, MO) or Invitrogen. Anti-flag M2 mouse monoclonal antibody and anti-flag M2 affinity agarose were obtained from Sigma; anti-14-3-3β and anti-his rabbit polyclonal antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); antihuman Ror2 or Ror1 goat polyclonal antibodies and control goat IgG were purchased from R&D Systems (Minneapolis, MN). Unconjugated and agarose-conjugated antiphosphotyrosine antibodies (4G10) were obtained from Upstate Cell Signaling Solutions (Charlottesville, VA); immobilized phosphotyrosine antibody P-Tyr-100 was from Cell Signaling Technologies (Beverly, MA). Protein A sepharose and glutathione sepharose were purchased from Amersham Biosciences (Buckinghamshire, UK), and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology.

The hMSCs were purchased from Cambrex, Inc. (Baltimore, MD) and maintained at 37 C in a 5% CO2-95% humidified air incubator using hMSC growth medium (MSCGM, Cambrex). The U2OS human osteosarcoma cells were maintained in McCoy’s 5A Modified Medium, containing 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin, and 2 mM glutaMAX-I. The HEK293 human embryonic kidney cell line was maintained in DMEM, containing 10% heat-inactivated FBS, 1% penicillin-streptomycin, and 2 mM glutaMAX-I.

Plasmids and Adenoviruses
Generation of the human Ror2-flag expression plasmid has been previously described (11). The Ror2-his construct was generated by replacing the flag epitope tag at the COOH terminal of the Ror2-flag with the sequence coding for six histidines. GST-R2C was obtained by inserting the intracellular domain of the human Ror2 (coding for amino acids 428–944) in frame following the GST tag in the pGEX-4T-2 vector (Amersham Biosciences, Buckinghamshire, UK).

Full-length human 14-3-3β cDNA was purchased from Open Biosystems (Huntsville, AL) and subcloned into pET28a bacterial expression vector.

Human Ror2-hTrkB chimera in pcDNA3.1hygro construct was generated by PCR-mediated mutagenesis. It contained the extracellular domain of human Ror2 (amino acids 1–407 of Ror2) followed by the transmembrane and intracellular domains of human TrkB (amino acids 432–822 of TrkB) as shown in Fig. 1CGo.

Generation of adenoviruses expressing flag-tagged Ror2, flag-tagged Ror2 kinase domain mutant (Ror2KD), β-galactosidase (β-gal), and human coxsackie adenovirus receptor (hCAR) was previously described (12). Adenoviruses containing shRNA sequences specific for human Ror2 or for enhanced green fluorescent protein (EGFP) (control) were obtained from Galapagos, Inc. (Mechelen, Belgium). The shRNA target sequences were as follows: 5'-TGTGAGCAACGCCCGCTAC-3' (Ror2) and 5'-GCTGACCCTGAAGTTCATC-3' (EGFP). Adenoviruses containing nonspecific shRNA and shRNA sequence specific for human 14-3-3β were obtained from Invitrogen. The target sequence for 14-3-3β-specific shRNA was 5'-AGGCAGAACTGCAGGACATCT-3'.

Transient Transfections
U2OS cells were plated at 75,000 cells/cm2 and transfected 24 h later with 0.3–0.5 µg/cm2 of total plasmid DNA using FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) per manufacturer’s instructions. For siRNA transfections, U2OS cells were plated on 60-mm2 plates at 75,000 cells/cm2 and transfected 24 h later with 25 nM Ror2 siRNA or nonspecific siRNA (both from Dharmacon Inc., Lafayette, CO) using 10 µl of Lipofectamine 2000 reagent per manufacturer’s instructions.

Immunoprecipitation and Western Immunoblotting
Cells were solubilized in lysis buffer (150 mM NaCl; 50 mM Tris-HCl, pH 7.5; 1 mM EDTA; 1% Triton X-100) supplemented with protease and phosphatase inhibitor cocktails (Sigma), and the extracts were clarified by centrifugation. For flag immunoprecipitation, 1 mg of total cell lysates was incubated with 30 µl of M2 flag affinity agarose for 1 h at 4 C. The beads were collected by centrifugation and washed three times in lysis buffer containing 350 mM NaCl and three times in lysis buffer. For 14-3-3β precipitation, 15 µl of 14-3-3β antibody was incubated with 30 µl of protein A sepharose in 1 ml of lysis buffer overnight at 4 C, and the beads were collected by centrifugation and washed in lysis buffer before the addition of 1 mg of total cell lysates. The binding reaction was carried out for 2 h at 4 C. For phosphotyrosine precipitation, 1 mg (for detecting overexpressed proteins) or 1.5 mg (for detecting endogenous proteins) of cellular extract was added to 100 µl of G410 beads and allowed to attach for 3 h at 4 C. At this time, P-Tyr-100-immobilized antibody (100 µl) was added to the mix for an additional 3 h. At the end of immunoprecipitation reactions, the beads were boiled in 30–50 µl of 2x lithium dodecyl sulfate-PAGE buffer with reducing agent, and the solubilized proteins were separated by SDS-PAGE. The gels were transferred onto 0.45 µm nitrocellulose membrane before detection with each specific antibody.

For immunoblotting without precipitation, the indicated amounts of total cellular lysates were resolved by SDS-PAGE under denaturing and reducing conditions before transfer onto 0.45-µm nitrocellulose membranes and detection with each specific antibody.

Mass-Spectroscopy (MS) Analysis
U2OS cells (5 x 107 per plasmid) were transiently transfected with Ror2-flag or pcDNA3.1 as described above and 48 h later solubilized in lysis buffer with protease and phosphatase inhibitors. A total of 14 mg of whole-cell lysates was precipitated on flag affinity agarose as described above and eluted from the resin by incubation in 5 volumes of 150 ng/µl 3xflag peptide (Sigma) for 30 min at 4 C. Sodium dodecyl sulfate was added to the eluates at 0.05% (wt/vol). The samples were then dialyzed against 0.05% sodium dodecyl sulfate and the volumes were reduced by evaporation to about 1/50th the original volume. The samples were applied to a 10% tricine gel (Invitrogen), and the gel was silver stained. Each lane was divided into 22 gel slices and excised using a scalpel. The proteins within each gel slice were reduced with dithiothreitol, alkylated with iodoacetamide, and subjected to an in-gel trypsin digestion using a ProGest Investigator digestion robot (Promega Corp., Madison, WI). After digestion, the volume of each sample was reduced to less than or equal to 10.0 µl using a vacuum concentrator, and the volume was then brought to 20.0 µl with buffer A (0.1 M acetic acid and 1% acetonitrile).

Ten microliters of each sample were applied to a 10 cm x 75 µm C18 reverse-phase column packed with Magic C18AQ (Michrom BioResources, Auburn, CA) in a PicoTip emitter column support (New Objectives, Woburn, MA) that was in line with an LCQ Deca XP plus mass spectrometer (ThermoFinnigan, San Jose, CA). Liquid chromatography was carried out with a flow rate to the column of 0.25 µl/min. The peptides were eluted in buffer A with a gradient of 14–50% buffer B (0.1 M acetic acid, 90% acetonitrile) over 47 min. The peptide masses were recorded by scanning a mass (m)-to-charge (z) ratio range from 375-1200. The fragment ion spectra [tandem mass spectrometry (MS/MS)] were acquired in a data-dependent manner in which each full MS scan was followed by consecutive MS/MS scans on the first three most intense ions from the full MS scan. The resulting MS/MS data were searched against the NCBI nonredundant data base using the Sequest program (ThermoFinnigan). The Sequest search results were summarized and analyzed using the SequestonOracle software developed at Wyeth (Collegeville, PA).

Stable Cell Line Generation
HEK293 cells stably expressing the 3xCRE-luciferase plasmid (HEK293 CRE, Mercury Signal System, CLONTECH Laboratories, Inc., Palo Alto, CA) were used to stably transfect hRor2-hTrkB-pcDNA3.1hygro following a protocol for hTrkB transfection (61). The HEK293 CRE Ror2-TrkB cells were grown at 37 C in a 5% CO2-95% humidified air incubator in DMEM, containing 10% FBS, 1% nonessential amino acids, 1% penicillin-streptomycin, 2 µg/ml puromycin, 250 µg/ml G418, and 350 µg/ml hygromycin.

Reporter Gene Analysis
HEK293 CRE Ror2-TrkB cells were plated at 60,000 cells per well in 96-well plates (Falcon 35-3072) with 150 µl/well of phenol red-free DMEM, containing 5% FBS, 1% nonessential amino acids, and 1% penicillin-streptomycin. After 24 h, 50 µl of the plating medium was replaced with the same medium containing the indicated antibodies at 3-fold the final concentrations. Cells were lysed 24 h later, and extracts were assayed for luciferase activity using Bright-Glo Luciferase Assay System (Promega) as suggested by the manufacturers. Light emission was measured by the TopCount NXT luminometer (PerkinElmer, Shelton, CT).

Viral Infection and Histochemical Staining
hMSCs were seeded at 6,000/cm2 in 12- or six-well plates and allowed to adhere and proliferate overnight. For adenoviruses containing expression cassettes for Ror2, β-gal, scrambled shRNA, or 14-3-3β-specific shRNA, cells were infected for 24 h in 0.4 ml/cm2 MSCGM with indicated combinations of adenoviruses at multiplicity of infection (MOI) = 750 for each in the presence of hCAR (MOI = 750) to improve infection efficiency. After 24 h, cells were washed once in PBS and placed in MSCGM or MSCGM supplemented with 0.05 mM ascorbic acid and 10 mM β-glycerophosphate (β-GP). For adenoviruses containing Ror2-specific and EGFP-specific shRNA, hMSCs were infected for 72 h in 0.4 ml/cm2 MSCGM at MOI = 4,000 in the presence of hCAR (MOI = 750). After 72 h, cells were washed once in PBS and placed in MSCGM supplemented with 0.05 mM ascorbic acid, 10 mM β-GP, 100 nM dexamethasone, and 50 µg/ml Ror2 antibody. Medium was changed every 5 d. Formation of mineralized nodules was determined on 12-well plates by alizarin red-S histochemical staining (62). To quantify the level of alizarin red-S staining, the dye was eluted with 1 ml/well of 10% (wt/vol) Cetylpyridinium Chloride. Alizarin red-S in the eluted samples was quantified (vs. a standard curve of 0–800 µM dye) at 562 nm with a microplate reader.

U2OS cells were seeded at 75,000/cm2 in 100-mm plates and infected 24 h later with β-gal, Ror2, or Ror2KD adenoviruses at MOI = 100. Infection was allowed to proceed for 24 h, and cell extracts were collected another 24 h later.

GST Pool-Down and in Vitro Kinase Assay
14-3-3β Was in vitro translated from 14-3-3β-pET28a using Expressway in vitro protein synthesis system (Invitrogen) per manufacturer’s instructions in a 50-µl reaction. GST-R2C in pGEX-4T-2 or pGEX-4T-2 (coding for GST alone) was transformed into BL21(DE3) strain of Escherichia coli. Cultures were grown to an A600 of 0.7 and induced to express recombinant proteins by addition of isopropyl-1-thio-β-D-galactopyranoside (Sigma; final concentration of 1 mM) and incubation for 4 h. Bacterial pellets were harvested by centrifugation, washed in PBS, and resuspended in 30 ml of PBS supplemented with protease and phosphatase inhibitor cocktails (Sigma). Cells were lysed by passaging twice through a French Pressure Cell Press (Spectronic Instruments, Rochester, NY) at 16,000 pounds/in.2, and bacterial debris was removed by centrifugation. The produced GST-Ror2c or GST proteins were incubated with glutathione sepharose for 4 h at 4 C. The beads were washed, resuspended in 1 ml PBS, and the entire 50 µl of 14-3-3β in vitro translation reaction was added for 4 h at 4 C. At the end of this incubation, the beads were washed three times in PBS, boiled in 2x SDS-PAGE buffer with reducing agent (Invitrogen), and the solubilized proteins were separated by SDS-PAGE. The gels were transferred onto a 0.45-µm nitrocellulose membrane before detection with each specific antibody.

For the in vitro kinase assay, 6.5 µg of purified recombinant human GST-14-3-3β (Biomol International, LP, Plymouth Meeting, PA) was resuspended in 25 µl of kinase reaction buffer (10 mM MgCl2; 50 mM Tris-HCl, pH 7.5; 1 mM dithiothreitol, 1 mM ATP) with or without addition of 0.9 µg of purified recombinant human GST-Ror2C (Invitrogen). The kinase reaction was allowed to proceed for 30 min at 37 C and stopped by boiling in 1x lithium dodecyl sulfate buffer with reducing agent. Proteins were resolved by SDS-PAGE, transferred onto 0.45-µm nitrocellulose membranes, and detected with antiphosphotyrosine antibody. Subsequently, the membrane was stripped and reprobed with anti-14-3-3β antibody to verify equal loading.

Calvarial Organ Culture and Infection
The mice were used in accordance with the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals. Calvariae were excised from 4-d-old mouse littermates, cut along the sagittal suture, and incubated for 24 h in serum-free BGJ medium containing 0.1% BSA. Each half of the calvaria was then placed with the concave surface downward on a stainless steel grid (Small Parts, Inc., Miami, FL) in a well of a 12-well plate. Calvariae were first infected with adenoviruses containing 14-3-3β-specific shRNA or nonspecific control shRNA (5 x 107 viruses per well) and incubated in a 5% CO2-95% humidified air incubator. After 2 d, the medium was replaced with fresh medium containing 15 µg/ml calcein and 12 µg/ml of nonspecific goat IgG or anti-Ror2 antibody. The medium, calcein, and antibodies were changed after 4 d.

After 7 d of culture, the bones were fixed in 10% neutral phosphate-buffered formaldehyde and stained with hematoxylin-eosin. Consistent 300-µm stretches (450 µm away from frontal sutures) were subjected to histomorphometric analysis using Bioquant-osteo (Bioquant Image Analysis Corp., Nashville, TN).

Statistical Analysis
Data are presented as means ± SE. Statistical significance was determined using two-tail Student’s t test. Results were considered statistically different when P < 0.05.


   ACKNOWLEDGMENTS
 
We thank Dr. Seongeun Cho for providing the hTrkB construct and Mr. Boris Rubin for generating the HEK293 CRE-luc Ror-Trk cell lines. We also thank Ioannis Moutsatsos and Patrick Cody for bioinformatics support with the proteomics data.


   FOOTNOTES
 
Disclosure Summary: Y.L., J.F.R., P.V.N.B., and J.B. are full-time employees of Wyeth Research.

First Published Online August 23, 2007

Abbreviations: CAR, Coxsackie adenovirus receptor; CRE, cAMP responsive element; EGFP, enhanced green fluorescent protein; EGF-R, epidermal growth factor receptor; FBS, fetal bovine serum; β-gal, β-galactosidase; β-GP, β-glycerophosphate; GST, glutathione-S-transferase; GST-R2C, GST-tagged cytosolic domain of Ror2; HEK, human embryonic kidney; MOI, multiplicity of infection; MS, mass spectrometry; MSCGM, mesenchymal stem cell growth medium; MS/MS, tandem mass spectrometry; MSC, mesenchymal stem cell; Ror2KD, Ror2 kinase domain mutant; RTK, receptor tyrosine kinase; shRNA, short hairpin RNA; siRNA, short interfering RNA; TAZr, transcriptional coactivator with PDZ-binding motif.

Received for publication June 26, 2007. Accepted for publication August 15, 2007.


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 DISCUSSION
 MATERIALS AND METHODS
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