Bone 75 (2015) 201–209

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Bone journal homepage: www.elsevier.com/locate/bone

Original Full Length Article

Cbl-b and c-Cbl negatively regulate osteoblast differentiation by enhancing ubiquitination and degradation of Osterix☆ You Hee Choi a, Younho Han a, Sung Ho Lee a, Yun-Hye Jin a, Minjin Bahn b, Kyu Chung Hur b, Chang-Yeol Yeo b,⁎, Kwang Youl Lee a,⁎⁎ a b

College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju 500-757, Republic of Korea Department of Life Science and Global Top5 Research Program, Ewha Womans University, Seoul 120-750, Republic of Korea

a r t i c l e

i n f o

Article history: Received 11 October 2014 Revised 23 February 2015 Accepted 24 February 2015 Available online 3 March 2015 Edited by: Sakae Tanaka Keywords: Cbl Osterix Ubiquitination Osteoblast differentiation

a b s t r a c t E3 ubiquitin ligase Cbl-b and c-Cbl play important roles in bone formation and maintenance. Cbl-b and c-Cbl regulate the activity of various receptor tyrosine kinases and intracellular protein tyrosine kinases mainly by regulating the degradation of target proteins. However, the precise mechanisms of how Cbl-b and c-Cbl regulate osteoblast differentiation are not well known. In this study, we investigated potential targets of Cbl-b and c-Cbl. We found that Cbl-b and c-Cbl inhibit BMP2-induced osteoblast differentiation in mesenchymal cells. Among various osteogenic transcription factors, we identified that Cbl-b and c-Cbl suppress the protein stability and transcriptional activity of Osterix. Our results suggest that Cbl-b and c-Cbl inhibit the function of Osterix by enhancing the ubiquitin–proteasome-mediated degradation of Osterix. Taken together, we propose novel regulatory roles of Cbl-b and c-Cbl during osteoblast differentiation in which Cbl-b and c-Cbl regulate the degradation of Osterix through the ubiquitin–proteasome pathway. © 2015 Elsevier Inc. All rights reserved.

Introduction Bone is a highly dynamic tissue that is actively maintained by the coordinated balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption [1,2]. The differentiation and activity of osteoblasts are regulated by various anabolic factors such as insulin, members of the TGF-β family (including BMPs) and Wnt proteins, and by intracellular kinases such as Akt [3]. In particular, bone morphogenetic proteins (BMPs) initiate osteoblast differentiation through the induction of expression and post-translational modification of various osteogenic transcription factors including Runx2 (Cbfa1/AML3), Osterix and several homeodomain-containing Dlx proteins [4–9]. Subsequently, these osteogenic transcription factors regulate the differentiation of osteoblasts [9–15]. ☆ Author contributions: Y.H.C.: collection and analysis of data, data interpretation, and manuscript drafting; Y.H.: data interpretation and analysis of data; S.H.L.: data interpretation and analysis of data; Y.H.J.: data interpretation and analysis of data; M.B.: data interpretation and analysis of data; K.C.H.: manuscript drafting, and final approval of manuscript; C.Y.Y.: conception and design, manuscript drafting, financial support, and final approval of manuscript; K.Y.L.: conception and design, manuscript drafting, financial support, and final approval of manuscript. ⁎ Correspondence to: C. Yeo, Department of Life Science, Ewha Womans University, Seoul 120-750, Republic of Korea. Fax: +82 2 3277 2385. ⁎⁎ Correspondence to: K. Lee, College of Pharmacy, Chonnam National University, Gwangju 500-757, Republic of Korea. Fax: +82 62 530 2949. E-mail addresses: [email protected] (C.-Y. Yeo), [email protected] (K.Y. Lee).

http://dx.doi.org/10.1016/j.bone.2015.02.026 8756-3282/© 2015 Elsevier Inc. All rights reserved.

Osterix (also known as Sp7) is a zinc finger-containing osteoblastspecific transcription factor and it is essential for the differentiation and proliferation of osteoblast [16–19]. The DNA-binding domain of Osterix is located at the C-terminus and it contains three C2H2-type zinc finger domains that share a high degree of identity with similar motives in Sp1, Sp3, and Sp4. N-terminal proline-rich region (PRR) mediates the protein–protein interaction. Osterix acts downstream of Runx2 and it, in turn, regulates the expression of many osteoblast differentiation markers including Alkaline phosphatase, Osteocalcin, Osteonectin, Osteopontin and Runx2 [14,16,20]. Cbl-b and c-Cbl proteins, members of mammalian Cbl (Casitas B-lineage lymphoma) family, act as cytoplasmic adaptors and E3 ubiquitin ligases, and they regulate bone formation both positively and negatively [21–28]. Other members of Cbl family have not been shown to possess ubiquitination-related function. Cbl-b and c-Cbl share a high degree of sequence identity, particularly within the N-terminal half (84% identity) but the C-terminal part is much less conserved among Cbl proteins. Cbl-b and c-Cbl proteins each consist of an N-terminal tyrosine kinase-binding (TKB) domain which mediates binding to specific phospho-tyrosine motives, a Zn-coordinating RING finger domain that interacts with E2 ubiquitin-conjugating enzymes. However, Cbl-b and c-Cbl exhibit structural differences in the C-terminal parts. c-Cbl has a presence of Y731 that acts as a docking site for the Src homology 2 (SH2) domain of the p85 subunit of phosphorylated phosphatidylinositol- kinase (PI3K). Also, the UBA

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domains have different sequences that differ in their ability to bind ubiquitylated proteins and polyubiquitin chains [29–32]. One mechanism by which Cbl-b and c-Cbl regulate osteoblast differentiation is by acting as E3 ubiquitin ligases through their RING finger domains [22–24,31,33]. E3 ubiquitin ligases control the ubiquitination and proteasome-mediated degradation of target proteins. Ubiquitin ligase activity of Cbl proteins has been shown to regulate bone formation both positively and negatively [25,26,34]. In addition, down-regulation of Cbl-b or c-Cbl has been shown to affect osteoblast proliferation through the regulation of PI3K-Akt signaling [23,24]. However, the precise molecular mechanisms of how Cbl-b and c-Cbl regulate osteoblast differentiation are not well understood. In this study, we attempted to identify potential targets of Cbl-b and c-Cbl during osteoblast differentiation. We found that Cbl-b/c-Cbl reduce the protein stability and transcriptional activity of Osterix during BMP2-induced osteoblast differentiation in C2C12 cells. In addition, we found evidences that Cbl-b/c-Cbl down-regulate the function of Osterix through ubiquitin–proteasome-mediated degradation of Osterix. Taken together, our results suggest a novel regulatory mechanism of Osterix function controlled by E3 ubiquitin ligases Cbl-b and c-Cbl during osteoblast differentiation. Materials and methods Plasmids, antibodies and reagents Plasmids for Myc-Osterix, HA-Msx2, HA or Myc-Smurf2, GFP-Dlx3, GFP-Dlx5, Flag-Smurf1 and Flag-Ub were constructed in a CMV promoter-derived expression vector (pCS4 +). HA-Cbl-b, HA-c-Cbl, N-terminal SH2 domain deletion mutant of Cbl-b ΔN and a ubiquitindeficient RING finger mutant of c-Cbl ΔC3AHN were generously provided by Dr. N. Kim (Chonnam National University, Korea). For knockdown of mouse c-Cbl, oligonucleotides targeting a 19-base sequence (5′-GGAGACACTTTCCGGATTA-3′) were synthesized. Sense and antisense oligonucleotides were annealed and ligated into pSuperretro (Oligoengine). Antibodies for the following epitopes were used: HA (12CA5) and Myc (9E10) from Roche Applied Science, Flag (M2) and α-Tubulin (B-5-1-2) from Sigma-Aldrich; Cbl-b (G-1), GFP (FL) and Osterix (A-13) from Santa Cruz Biotechnology; and c-Cbl (17/c-Cbl) from BD Biosciences. Cycloheximide (Sigma), MG132 (Calbiochem) and recombinant human BMP2 protein (355-BM, R&D Systems) were used. Cell culture and transient transfection 293 Human embryonic kidney epithelial cells, C2C12 mouse myoblasts and MC3T3-El cells, a clonal osteoblast-like mouse calvarial cell were cultured at 37 °C, 5% CO2 in Dulbecco Modified Eagle Medium (DMEM) or α-Modified Eagle's Minimal Essential Medium (α-MEM) supplemented with 5% (for 293 cells) or 10% (for C2C12 cells and MC3T3-E1) FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. DMEM, α-MEM, FBS and antibiotics were purchased from Life Technologies. Transient transfection was performed using polyethyleneimine (Polysciences, Inc.) or the calcium phosphate-mediated method. GFP expression plasmid is included as a transfection control. Total amounts of transfected plasmids in each group were equalized by adding empty vector. For treatment with cycloheximide (CHX), cells were incubated in fresh growth medium (DMEM with 5% FBS and antibiotics) 24 h after transfection and treated with CHX (40 μM) for indicated amounts of time. Immunoblotting (IB) and immunoprecipitation (IP) 293 Cells or C2C12 cells were lysed in an ice-cold lysis buffer [25 mM HEPES (pH 7.5), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol, 25 mM NaF, 1 mM EDTA, 1 mM Na3VO4, 250 μM PMSF,

10 μg/ml leupeptin, and 10 μg/ml aprotinin]. After centrifugation, supernatants were used as cell lysates. For immunoprecipitation, cell lysates were incubated with appropriate antibodies overnight at 4 °C and then bound to protein A or G-Sepharose beads for 5 h. Cell lysates containing 30 μg of total protein or immunoprecipitated proteins were subjected to SDS–PAGE and proteins were transferred to PVDF membrane. Proteins were detected using appropriate primary antibodies, horseradish peroxidase-coupled secondary antibodies (GE Healthcare Life Sciences), and enhanced chemiluminescence (ECL) reagent (Millipore). Signals were detected and analyzed by an LAS4000 luminescent image analyzer (Fuji Photo Film Co.). Luciferase reporter assay C2C12 cells were seeded on 24-well plates the day before transfection. ALP-Luc and BSP-Luc luciferase reporters contain the regulatory sequence of osteoblast differentiation marker Alkaline phosphatase or Bone sialoprotein respectively. Cells were transfected with CMV promoter-driven β-galactosidase reporter (pCMV-β-gal), luciferase reporter and indicated combinations of expression plasmids. Thirtysix hours later, luciferase activities were measured using Luciferase Reporter Assay Kit (Promega, E1501), using a luminometer and normalized with corresponding β-galactosidase activities for transfection efficiency. Experiments were performed in triplicate and repeated at least three times. Average and SD of representative experiments are shown. RNA preparation and semi-quantitative RT-PCR Total cellular RNA was prepared using TRIzol reagent (Life Technologies) according to the manufacturer's instruction. Random hexamer-primed cDNAs were synthesized from 1 μg of total RNA using SuperScript III First-Strand Synthesis System (Life Technologies). The following conditions were used for amplification by PCR: initial denaturation at 94 °C for 1 min; followed by 23–30 cycles of denaturation at 94 °C for 30 s, annealing at a temperature optimized for each primer pair for 30 s, and extension at 72 °C for 30 s; and final extension at 72 °C for 5 min. The following PCR primers were used: ALP forward 5′-GGG TGG ACT ACC TCT TAG GTC-3′ and reverse 5′- ATG ATG TCC GTG GTC AAT CCT G − 3′ (30 cycles); BSP forward 5′-CAG AAG TGG ATG AAA ACG AG-3′ and reverse 5′-CGG TGG CGA GGT GGT CCC AT-3′ (25 cycles); ColIα1 forward 5′-TCT CCA CTC TTC TAG GTT CCT-3′ and reverse 5′-TTG GGT CAT TTC CAC ATG C-3′ (23 cycles); GAPDH forward 5′-ACC ACA GTC CAT GCC ATC AC-3′ and reverse 5′-TCC ACC ACC CTG TTG CTG TA-3′ (25 cycles). Alkaline phosphatase (ALP) staining, von Kossa staining and activity assay For ALP staining, C2C12 cells were treated with BMP2 (10 ng/ml) for 3 days, fixed in 4% paraformaldehyde for 10 min at room temperature (RT), rinsed with phosphate buffered saline (PBS) and stained with BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) solution (Sigma-Aldrich, 300 μg/ml) for 15 min at RT. To determine alkaline phosphatase enzymatic activity, C2C12 cells were treated with BMP2 (10 ng/ml) for 3 days and transfected with indicated plasmids. ALP enzymatic activity was measured using SensoLyte pNPP Alkaline Phosphates Assay Kit (AnaSpec) according to the manufacturer's protocol. The absorbance of alkaline phosphatase activity was measured at 405 nm using a microplate reader, Bio-Tek Instruments. For the von Kossa staining, C2C12 cells were treated with BMP2 (10 ng/ml) for 14 days, fixed in 4% paraformaldehyde for 10 min at room temperature (RT), rinsed with phosphate buffered saline (PBS), stained with 5% silver nitrate solution with exposure to ultraviolet light for 30 to 60 min, incubated with 5% sodium thiosulfate solution for 2 to 3 min, and incubated with nuclear fast red solution for 5 min (Millipore).

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and incubated with cell lysates for 5 h at 4 °C. Bound proteins were analyzed by SDS-PAGE and immunoblotting (IB).

Alizarin Red S staining The alkaline phosphatase-positive cells were stained blue/purple. For the Alizarin Red S staining, MC3T3-El cells, a clonal osteoblastlike mouse calvarial cell line were cultured at 37 °C, 5% CO 2 in α-Modified Eagle's Minimal Essential Medium supplemented with10% FBS (growth medium) and antibiotics. For the induction of osteoblast differentiation, the growth medium was supplemented with 50 μM ascorbic acid and 10 mM β-glycerophosphate (osteoblast differentiation medium). DMEM, FBS and antibiotics were purchased from Life Technologies. MC3T3-E1 cells in 24-well plates were transfected using polyethyleneimine (PEI; Polysciences) mediated method. MC3T3-E1 cells were induced by stimulating the cells with BMP2 (20 ng/ml). Cells were pretreated with BMP2 for 14 days. These cells were cultured at 5% CO2, 37 °C. Transfected MC3T3-E1 cells were fixed in 4% paraformaldehyde for 10 min at room temperature (RT) and washed with PBS. They were exposed to Alizarin Red S solution (A5533, Sigma-Aldrich) and were adjusted to 4.1–4.3 using 0.5% ammonium hydroxide for 30 min at RT. The mineralization-positive cells were stained red.

Statistical analysis All experiments were performed with triplicate independent samples and were repeated at least twice, giving qualitatively identical results. Results are expressed as mean ± standard error of the mean. Data were analyzed using Student's t-test, with p b 0.05 indicating significance. Results Cbl-b and c-Cbl inhibit BMP2-induced osteoblast differentiation

Glutathione-S-transferase (GST) pull-down assay Recombinant GST-tagged Osterix protein was expressed in E. coli and purified using glutathione–Sepharose bead. For each GST-pull down assay, glutathione–Sepharose beads carrying 10 μg of GSTfusion protein (GST-Osterix) were equilibrated with cell lysis buffer

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BMP2 stimulation induces osteoblast differentiation in C2C12 myoblasts and MC3T3-El osteoblastic cells. We examined whether Cbl-b and c-Cbl affect BMP2-induced osteoblast differentiation in C2C12 and MC3T3-El cells. Osteoblast differentiation was measured by ALP staining and Alizarin Red S-indicated mineralization. Cbl-b and c-Cbl decreased BMP2-induced expression of an osteoblast marker alkaline phosphatase (ALP) when examined by ALP staining or ALP activity assay (Fig. 1A–B). Similarly, Cbl-b and c-Cbl decreased the mineralization by Alizarin Red S staining (Fig. 1C). We confirmed overexpression and endogenous Cbl-b and c-Cbl in C2C12 cells (Fig. S1). In addition, Cbl-b and c-Cbl decreased BMP2-induced expression of osteoblast-specific marker genes ALP, Bone sialoprotein (BSP), and Collagen type I α1 (ColIα1) (Fig. 1D). Taken together, these results

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Fig. 1. Cbl-b and c-Cbl inhibit BMP2-induced osteoblast differentiation. C2C12 myoblasts and MC3T3-El, a clonal osteoblast-like mouse calvarial cell were transfected with HA-Cbl-b (indicated as b) or HA-c-Cbl (indicated as c) (0.5 or 1 μg for panel A, 1 μg for panels B, C and D) and then treated with BMP2 (10 ng/ml) for 3 days (ALP staining) or with BMP2 (20 ng/ml) for 14 days (Alizarin Red S staining). (A) The extent of osteoblast differentiation was examined by alkaline phosphatase (ALP) staining. (B) ALP activities were measured by ALP assay. Averages and SD of triplicate samples are shown. * and ** indicate that the differences are significant compared to BMP2 non-treated or BMP2 treated cells without any transfected DNA respectively (p b 0.05, by Student's t-test). (C) The mineralization of osteoblast differentiation was examined by Alizarin Red S staining. (D) Left panel: the expression levels of osteoblast-specific markers ALP, Bone sialoprotein (BSP), and collagen type I α1 (ColIα1) were compared by RT-PCR. GAPDH was used as a loading control. Right panel: the relative expression levels of osteoblast-specific markers in the left panel were determined by densitometry. Averages and SD of triplicate samples are shown.

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Cbl-b and c-Cbl suppress the transcriptional activity of Osterix

indicate that Cbl-b and c-Cbl inhibit BMP2-induced osteoblast differentiation in C2C12 cells.

We examined whether Cbl-b/c-Cbl affects the transcriptional activity of Osterix using osteoblast-specific luciferase reporters ALPLuc and BSP-Luc. Cbl-b and c-Cbl significantly reduced Osterixinduced expression of the reporters (Fig. 3A). However, MG132 relieved the suppression of Osterix transcriptional activity by Cbl-b/c-Cbl. These results suggest that Cbl-b and c-Cbl reduce the transcriptional activity of Osterix possibly by inducing the proteasomal degradation of Osterix. Next, we examined whether the E3 ubiquitin ligase activity of Cbl-b or c-Cbl is important for its ability to suppress the transcriptional activity of Osterix. Cbl-b ΔN mutant lacks the N-terminal SH2 domain and c-Cbl ΔC3AHN mutant lacks a region in the RING finger domain. These mutants are ubiquitin ligase-deficient and act as dominant negative mutants (see Figs. 4 and 5). Cbl-b ΔN, unlike wild type Cbl-b, failed to reduce the transcriptional activity of Osterix (Fig. 3B), and c-Cbl ΔC3AHN acted similarly (Fig. 3C). These results suggest that the E3 ubiquitin ligase activity of Cbl-b/c-Cbl is important for their ability to suppress the transcriptional activity of Osterix.

Cbl-b and c-Cbl reduce the protein level of Osterix specifically BMP2 induces osteoblast differentiation by inducing the expression of several key osteogenic transcription factors including Dlx3, Dlx5, Msx2, and Osterix [4–8]. In order to investigate the mechanism of how Cbl proteins regulate osteoblast differentiation, we examined whether they affect the protein levels of osteogenic transcription factors since Cbl proteins can act as E3 ubiquitin ligases. Cbl-b and c-Cbl reduced the level of endogenous Osterix protein in BMP2-stimulated C2C12 cells (Fig. 2A). However, Cbl-b and c-Cbl did not affect the level of Osterix mRNA significantly (data not shown). We also examined whether Cbl-b and c-Cbl reduce the level of ectopically expressed Osterix protein in non-osteogenic 293 cells. When co-expressed with Osterix in 293 cells, Cbl-b and c-Cbl significantly reduced the level of overexpressed Osterix protein in dose-dependent manners (Fig. 2B–C). Cbl-b/c-Cbl-induced decrease of Osterix protein level was abolished when cells were treated with a proteasome inhibitor MG132 (Fig. 2D). However, Cbl-b/c-Cbl did not affect the levels of overexpressed Dlx3, Dlx5 and Msx2 proteins (Fig. 2E–G). These results indicate that Cbl-b and c-Cbl reduce the translation and/or protein stability, but not the transcription, of Osterix specifically. Next, we examined whether the protein level of Osterix is modulated specifically by Cbl proteins. When co-expressed with Osterix in 293 cells, WW-HECT type E3 ubiquitin ligases Smurf2 and NEDD4, unlike RING finger type E3 ubiquitin ligases Cbl-b and c-Cbl, did not affect the protein level of Osterix significantly (Fig. 2H). These results suggest that the protein level of Osterix is modulated specifically by RING finger type E3 ubiquitin ligases Cbl-b and c-Cbl.

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Cbl-b has been shown to increase the protein stability and transcriptional activity of Runx2 in osteoblasts [35]. Our results suggest that the E3 ubiquitin ligase activity of Cbl-b/c-Cbl is important for their ability to modulate the function of Osterix through proteasomal degradation. Therefore, we examined whether Cbl-b/c-Cbl affects the stability of Osterix protein. Wild type Cbl-b significantly shortened the half-life of Osterix protein whereas Cbl-b ΔN mutant significantly prolonged the half-life of Osterix protein (Fig. 4A–B). Similarly, wild type c-Cbl significantly shortened the half-life of Osterix whereas c-Cbl ΔC3AHN failed to do so (Fig. 4C–D). These results suggest that Cbl-b and c-Cbl reduce the protein stability of Osterix.

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Fig. 2. Cbl-b and c-Cbl reduce the level of Osterix protein specifically. (A) C2C12 cells were transfected with HA-Cbl-b (indicated as b) or HA-c-Cbl (indicated as c) (each 0.5 μg). Cells were then treated with BMP2 (10 ng/ml) and MG132 (1 μM) or DMSO as a vehicle control for 15 h. The levels of endogenous Osterix protein were compared by immunoblotting (IB). Tubulin was used as a loading control. (B–C) 293 Cells were transfected for GFP (0.5 μg) and Myc-Osterix (0.5 μg) along with increasing amounts of HA-Cbl-b or HA-c-Cbl (0.5 or 1 μg). The levels of overexpressed Osterix protein [Myc (Osterix)] and Cbl proteins [HA (Cbl-b) and HA (c-Cbl)] were compared by anti-Myc and anti-HA IB. Numbers indicate relative densities of Osterix bands when that of Osterix transfected alone is considered as 1. GFP was used as a transfection control. (D) 293 Cells were transfected for Myc-Osterix (0.5 μg) along with HA-Cbl-b or HA-c-Cbl (0.5 μg each). Cells were then treated with MG132 (1 μM) or DMSO for 15 h. The levels of overexpressed proteins were compared by IB. (E–G) 293 Cells were transfected for indicated combinations of HA-Cbl-b, HA-c-Cbl, GFP-Dlx3, GFP-Dlx5 and HA-Msx2 (0.5 μg each). The levels of overexpressed Dlx3 [GFP (Dlx3)], Dlx5 [GFP (Dlx5)] and Msx2 [HA (Msx2)] were compared by IB. (H) 293 Cells were transfected for Myc-Osterix (0.5 μg) along with HA-Cbl-b, HA-c-Cbl, Flag-Smurf2 or NEDD4 (0.5 μg each). The levels of overexpressed proteins were compared by IB.

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Fig. 3. Cbl-b and c-Cbl suppress the transcriptional activity of Osterix. (A) C2C12 cells were transfected for pCMV-β-gal (0.05 μg), a luciferase reporter [ALP-Luc (left panel) or BSP-Luc (right panel)] (0.2 μg) along with indicated combinations of Osterix, Cbl-b and c-Cbl. For panel A, cells were then treated with MG132 (1 μM) or DMSO for 15 h. (B and C) Cells were transfected with indicated combinations of Osterix, Cbl-b ΔN, and c-Cbl ΔC3AHN. Luciferase activities were measured, and average and SD of triplicate samples are shown. * and ** respectively indicate that the difference is significant compared with control (first bar from the left) or Osterix transfection alone (third bar) (p b 0.05, by t-test). # indicates that the difference is significant compared to MG132 non-treated cells (p b 0.05, by t-test).

Cbl-b and c-Cbl induce ubiquitination of Osterix Our results suggest that Cbl-b/c-Cbl induce proteasomal degradation of Osterix. In general, proteins are targeted for proteasomal degradation by ubiquitination [36]. Therefore, we examined whether Cbl-b/c-Cbl promote the ubiquitination of Osterix. In BMP2-stimulated C2C12 cells, transfection of ubiquitin reduced the levels of endogenous and exogenous Osterix proteins, and co-transfection of Cbl-b or c-Cbl reduce the levels of Osterix proteins further (Fig. 5A). However, MG132 relieved the reduction of Osterix proteins by Cbl-b/c-Cbl (Fig. 5A). Next, we examined whether Cbl-b and c-Cbl induce ubiquitination of Osterix. When co-expressed in 293 cells, Cbl-b and c-Cbl enhanced ubiquitination of Osterix and simultaneous reduction of Osterix protein level (Fig. 5B, left panel). However, when cells were treated with MG132, the ubiquitination and protein level of Osterix were not affected by Cbl-b or c-Cbl (Fig. 5B, right panel). Next, we examined whether the E3 ubiquitin ligase activity of Cbl-b or c-Cbl is important for its ability to induce the ubiquitination of Osterix. Wild type Cbl-b significantly

enhanced the ubiquitination of Osterix whereas Cbl-b ΔN mutant markedly reduced it (Fig. 5C). Similarly, wild type c-Cbl significantly enhanced the ubiquitination of Osterix whereas c-Cbl ΔC3AHN mutant failed to do so (Fig. 5D). These results suggest that Cbl-b and c-Cbl reduce the protein stability of Osterix by enhancing ubiquitin–proteasome-mediated degradation of Osterix. Cbl-b and c-Cbl interact with Osterix We examined whether Cbl-b/c-Cbl interacts with Osterix by co-immunoprecipitation. Wild type and ΔN mutant Cbl-b both interacted with Osterix (Fig. 6A). Similarly, wild type and ΔC3AHN mutant c-Cbl interacted with Osterix (Fig. 6B). These results suggest that the E3 ubiquitin ligase activity Cbl-b/c-Cbl is not required for interaction with Osterix although it is required for the modulation of Osterix function. We then examined whether Cbl proteins interact with purified Osterix protein by GST pull-down assay. Cbl-b and c-Cbl interacted with purified GST-Osterix (Fig. 6C), suggesting a direct

Y.H. Choi et al. / Bone 75 (2015) 201–209

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Fig. 4. Cbl-b and c-Cbl decrease the protein stability of Osterix. (A, C) 293 Cells were transfected for Myc-Osterix along with HA-tagged wild type (Cbl-b WT) or mutant (Cbl-b ΔN) Cbl-b (panel A), or along with HA-tagged wild type (c-Cbl WT) or mutant (c-Cbl ΔC3AHN) c-Cbl (panel B). Cells were then treated with cycloheximide (CHX, 40 μg/ml) for indicated amounts of time. The levels of Osterix protein [Myc (Osterix)] were compared by IB. (B, D) The intensities of Osterix bands in panel A and C were determined by densitometry. The level of Osterix protein in cells transfected with Osterix alone and not treated with CHX (first lanes in panel A and C) were considered as 100%. Experiment was performed in triplicates, and average and SD are shown. * indicates that the difference is significant compared to Osterix transfection alone (p b 0.05, by t-test).

interaction between Osterix and Cbl-b/c-Cbl. We also examined whether Osterix interacts with endogenous Cbl-b/c-Cbl. In 293 cells, endogenous Cbl-b and c-Cbl interacted with overexpressed Osterix (Fig. 6D). The interaction between overexpressed Osterix and endogenous Cbl-b/c-Cbl was specific as control IgG, in contrast to Cbl-b/c-Cbl-specific antibodies, did not co-immunoprecipitate Osterix (Fig. 6E). Taken together, these results suggest that Cbl-b and c-Cbl interact with Osterix possibly through a direct binding. c-Cbl knockdown relieves the suppression of Osterix function and osteoblast differentiation by c-Cbl Next, we examined the specificity of c-Cbl action by shRNAmediated knockdown. The osteoblast differentiation was decreased by c-Cbl. In contrast, c-Cbl shRNA (sh c-Cbl) relieved the decrease of osteoblast differentiation. Osteoblast differentiation was measured by alkaline phosphatase (ALP) staining and von Kossa staining (Fig. 7A–B). c-Cbl shRNA (sh c-Cbl) significantly reduced the expression of endogenous and exogenous c-Cbl proteins in C2C12 cells (Fig. 7C). sh c-Cbl alleviated the reduction of endogenous as well as overexpressed Osterix protein by c-Cbl (Fig. 7C). Similarly, sh c-Cbl relieved c-Cbl-mediated inhibition of osteoblast marker expression in C2C12 cells (Fig. 7D). Finally, sh c-Cbl reverted the inhibitory effect of c-Cbl on the transcriptional activity of Osterix (Fig. 7E). Taken together, these results suggest that the E3 ubiquitin ligase c-Cbl negatively regulates osteoblast differentiation, at least in part, by enhancing the ubiquitin–proteasome-mediated degradation of Osterix. Discussion In this study, we investigated the potential targets of Cbl-b and c-Cbl during osteoblast differentiation, and found that Cbl-b/c-Cbl modulates the protein stability and transcriptional activity of Osterix by enhancing the ubiquitination and proteasomal degradation of Osterix. RING-finger type E3 ubiquitin ligases Cbl-b and c-Cbl, but not WW-HECT type E3 ubiquitin ligases Smurf2 and NEDD, affected Osterix function. However, Cbl-b/c-Cbl did not affect the protein levels of other osteogenic transcription factors such as Dlx3, Dlx5, and Msx2. These results suggest a specific regulation of Osterix function by Cbl-b and c-Cbl during osteoblast differentiation.

Cbl-b and c-Cbl function as important regulators of cellular signaling, acting as both adapter proteins and E3 ubiquitin ligases [33,37–40]. Cbl-b/c-Cbl can modulate signaling from multiple receptor tyrosine kinases (RTKs) during osteoblast differentiation. Cbl-b enhances the ubiquitination and degradation of insulin-like growth factor receptor (IGFR) in osteoblasts and, in turn, inhibits bone formation through the suppression of IGF signaling [26]. Inhibition of c-Cbl interaction with RTKs with a dominant negative c-Cbl mutant (c-Cbl G306E) promoted the expression of osteoblast markers and increased osteogenic differentiation in human mesenchymal stromal cells (hMSCs) [22]. Inhibition of c-Cbl-RTK interaction in hMSCs also decreased the ubiquitination and degradation of fibroblast growth factor receptor 2 (FGFR2) and platelet-derived growth factor receptor α (PDGFRα), resulting in increased expression of these receptors and the activation of downstream ERK1/2 and PI3K signaling pathways [23,24,41]. RTKs such as IGFR, FGFR, epidermal growth factor receptor (EGFR) and PDGFR play crucial roles in the control of osteoblast differentiation and bone formation [42]. Therefore, Cbl-b/c-Cbl-mediated degradation of these RTKs modulates the differentiation and function of osteoblasts. Another mechanism by which Cbl-b/c-Cbl controls osteoblast differentiation is through the regulation of osteogenic transcription factors. Recently, c-Cbl-mediated ubiquitination and degradation of the transcription factor STAT5 have been shown to decrease the interaction between STAT5 and Runx2 and to reduce osteoblast differentiation of MSCs [25]. Cbl-b also increased the protein stability and transcriptional activity of Runx2 in osteoblast cell lines [35]. But it is unknown in previous results reported by other groups whether another transcription factor, Osterix can be regulated by E3 ubiquitin ligases, Cbl-b/c-Cbl. Cbl-b and c-Cbl also regulate the activity of c-Src in bone tissue [43]. The functional interaction between Src and Cbl proteins plays important roles in bone formation [37,44–46]. Src kinase activity can be downregulated by ubiquitin-dependent degradation [47,48], and Cbl proteins regulate Src activity [49,50]. c-Cbl-mediated ubiquitination of Src, EGFR and other target proteins is required for Src-catalyzed phosphorylation of c-Cbl [37,51]. In addition, phosphorylation of c-Cbl by Src is necessary for the ubiquitination of both Src and c-Cbl [37]. The interplay between Cbl-b/c-Cbl and c-Src may affect the ability of Cbl-b/c-Cbl to regulate Osterix function. We investigated that Cbl-b and c-Cbl interact with Osterix possibly through a direct binding using the co-immunoprecipitation and GST pull-down assay (Fig. 6). If Cbl

Y.H. Choi et al. / Bone 75 (2015) 201–209

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Fig. 5. Cbl-b and c-Cbl induce ubiquitination of Osterix. (A) C2C12 cells were transfected for Myc-Osterix along with indicated combinations of Flag-ubiquitin (Flag-Ub), HA-Cbl-b and HA-c-Cbl (0.5 μg each). Cells were then treated with BMP2 (10 ng/ml) with MG132 (1 μM) or DMSO for 15 h. The levels of endogenous (Endo) and exogenous (Exo) Osterix protein [Osterix] were compared by IB. (B) 293 Cells were transfected for Myc-Osterix along with indicated combinations of Flag-ubiquitin (Flag-Ub), HA-Cbl-b and HA-c-Cbl. Cells were then treated with DMSO (left panel) or MG132 (1 μM, right panel) for 15 h. Ubiquitination of overexpressed Osterix was examined by anti-Myc immunoprecipitation of Osterix [IP: Myc (Osterix)] followed by anti-Flag IB of ubiquitin [Flag (Ub)]. The levels of overexpressed proteins in cell lysates were also compared. (C–D) 293 Cells were transfected for Myc-Osterix along with indicated combinations of Flag-ubiquitin (Ub), HA-Cbl-b WT, HA-Cbl-b ΔN, HA-c-Cbl WT and HA-c-Cbl ΔC3AHN. Ubiquitination of overexpressed Osterix was examined by anti-Myc IP [IP: Myc (Osterix)] followed by anti-Flag IB [Flag (Ub)].

proteins interact with Osterix indirectly, we expect that adapter proteins with SH2 domain or SH3 domain may also be present and mediate them. The members of Src family including the wellknown c-Src are representative candidates among adaptor proteins. We predict that the triple complex between Osterix and Cbl proteins through the regulation of c-Src may be present. Therefore, characterization of regulatory mechanisms and post-translational modification of Osterix by the cooperation between Cbl-b/c-Cbl and c-Src are necessary to understand how Osterix function is regulated during osteoblast differentiation Cbl-mediated ubiquitination of target proteins may be a potential target for therapeutic intervention in osteoporosis and other pathological conditions of bone. Recent results indicate that c-Cbl may play a role in osteosarcoma. In human osteosarcoma tissues, the expression of c-Cbl is decreased with a concomitant increase in EGFR and PDGFRα expression compared to normal bone tissue [32]. Ectopic c-Cbl expression in osteosarcoma cells may have therapeutic implications as it could reduce the expression of EGFR and PDGFRα. This could, in turn,

result in decreased proliferation and survival of osteosarcoma cells, and reduced incidences of metastasis. Therefore, promoting the ubiquitination and degradation of RTKs with c-Cbl may provide an effective mean to reduce tumor growth and metastasis in which excessive expression and activation of RTKs occur [52]. Our results suggest that ectopic c-Cbl may also have therapeutic implications in bone homeostasis. Ectopic c-Cbl expression could inhibit Osterix function and osteoblast differentiation, tilting the balance of bone maintenance toward more bone resorption and, possibly, toward the pathological condition of osteoporosis. Our current results suggest that the function of Osterix can be regulated post-translationally by Cbl-b and c-Cbl in osteogenesis. However, whether Cbl-b and c-cbl control Osterix in vivo is unknown. Identification of Cbl functions in vivo for the regulation of Osterix will enhance our understanding of the regulatory mechanisms of Osterix osteogenic function and how Cbl-b and c-Cbl regulate osteoblast differentiation. Therefore, determination in vivo to understand the significance such regulation between Osterix and Cbl proteins is needed.

Y.H. Choi et al. / Bone 75 (2015) 201–209

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Fig. 6. Cbl-b and c-Cbl interact with Osterix. (A–B) 293 Cells were transfected for Myc-Osterix along with indicated combinations of HA-Cbl-b WT, HA-Cbl-b ΔN, HA-c-Cbl WT and HA-c-Cbl ΔC3AHN. The interaction between Osterix and Cbl proteins was determined by anti-Myc IP of Osterix [IP: Myc (Osterix)] followed by anti-HA IB of Cbl-b [HA (Cbl-b)] (panel A) or anti-HA IB of c-Cbl [HA (c-Cbl)] (panel B). (C) Lysates from 293 cells transfected with HA-Cbl-b (left panels) or HA-c-Cbl (right panels) were incubated with purified GST or GST-Osterix beads for 5 h (GST pull-down). The interaction of Cbl proteins with GST-Osterix was determined by anti-HA IB [HA (Cbl)]. Arrowheads indicate full-length GST-Osterix fusion protein. (D) 293 Cells were transfected for Myc-Osterix. The interaction between overexpressed Osterix and endogenous Cbl-b or c-Cbl was determined by anti-Cbl-b IP (left panels, IP: Cbl-b) or anti-c-Cbl IP (right panels, IP: c-Cbl) followed by anti-Myc IB [Myc (Osterix)]. (E) 293 Cells were transfected for Myc-Osterix. The specificity of interaction between overexpressed Osterix and endogenous Cbl-b or c-Cbl was determined by IP using control IgG, anti-Cbl-b antibody or anti-c-Cbl antibody for 5 h followed by anti-Myc IB [Myc (Osterix)].

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Fig. 7. c-Cbl knockdown increases BMP2-induced expression of osteoblast markers and the transcriptional activity of Osterix. (A–D) C2C12 cells were transfected with indicated combinations of Myc-Osterix, HA-c-Cbl and c-Cbl shRNA (sh c-Cbl) plasmids (0.5 μg each). Cells were then treated with BMP2 (10 ng/ml) for 3 days (ALP staining) or 14 days (von Kossa staining). For panel A, the extent of osteoblast differentiation was examined by alkaline phosphatase (ALP) staining. For panel B, the deposits of calcium of osteoblast differentiation were examined by von Kossa staining. Positive calcium deposits are black. For panel C, the levels of endogenous and exogenous Osterix protein were examined by anti-Osterix IB [Osterix] and anti-Myc IB [Myc (Osterix)] respectively. For panel D, the expression levels of osteoblast markers were examined by RT-PCR. (E) C2C12 cells were transfected for pCMV-β-gal (0.05 μg), luciferase reporter [ALP-Luc (left panel) or BSP-Luc (right panel)] (0.2 μg) along with indicated combinations of Myc-Osterix, HA-c-Cbl and sh c-Cbl. Luciferase activities were measured, and averages and SD of triplicate samples are shown. * and ** respectively indicate that the difference is significant compared with control or Osterix transfection alone (p b 0.05, by t-test). # indicates that the difference is significant compared to co-transfection of Osterix and c-Cbl (0.3 μg each) (p b 0.05, by t-test).

Y.H. Choi et al. / Bone 75 (2015) 201–209

In conclusion, our work provides a basis for understanding a novel role of E3 ubiquitin ligases Cbl-b/c-Cbl and a novel regulatory mechanism for Osterix function during osteoblast differentiation. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIP) to KYL (NRF-2013R1A2A2A07067609), YHC (2014R1A6A3A01059423) and CYY (2012R1A5A1048236). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bone.2015.02.026. References [1] Katagiri T, Takahashi N. Regulatory mechanisms of osteoblast and osteoclast differentiation. Oral Dis 2002;8:147–59. [2] Heino TJ, Hentunen TA. Differentiation of osteoblasts and osteocytes from mesenchymal stem cells. Curr Stem Cell Res Ther 2008;3:131–45. [3] Kawamura N, Kugimiya F, Oshima Y, Ohba S, Ikeda T, Saito T, et al. Akt1 in osteoblasts and osteoclasts controls bone remodeling. PLoS One 2007;2:e1058. [4] Blum B, Moseley J, Miller L, Richelsoph K, Haggard W. Measurement of bone morphogenetic proteins and other growth factors in demineralized bone matrix. Orthopedics 2004;27:s161–5. [5] Yamaguchi A, Komori T, Suda T. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr Rev 2000;21:393–411. [6] Ohba S, Chung UI, Tei Y. Osteoblast differentiation induced by BMP signaling and Runx2 through Cbfb regulation. Nihon Rinsho 2007;65(Suppl. 9):71–4. [7] Matsubara T, Kida K, Yamaguchi A, Hata K, Ichida F, Meguro H, et al. BMP2 regulates Osterix through Msx2 and Runx2 during osteoblast differentiation. J Biol Chem 2008;283:29119–25. [8] Ulsamer A, Ortuno MJ, Ruiz S, Susperregui AR, Osses N, Rosa JL, et al. BMP-2 induces Osterix expression through up-regulation of Dlx5 and its phosphorylation by p38. J Biol Chem 2008;283:3816–26. [9] Komori T. Regulation of osteoblast differentiation by transcription factors. J Cell Biochem 2006;99:1233–9. [10] Lee MH, Kwon TG, Park HS, Wozney JM, Ryoo HM. BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem Biophys Res Commun 2003;309:689–94. [11] Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997;89:755–64. [12] Bendall AJ, Abate-Shen C. Roles for Msx and Dlx homeoproteins in vertebrate development. Gene 2000;247:17–31. [13] Hassan MQ, Javed A, Morasso MI, Karlin J, Montecino M, van Wijnen AJ, et al. Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene. Mol Cell Biol 2004;24:9248–61. [14] Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002;108:17–29. [15] Li H, Marijanovic I, Kronenberg MS, Erceg I, Stover ML, Velonis D, et al. Expression and function of Dlx genes in the osteoblast lineage. Dev Biol 2008;316:458–70. [16] Fu H, Doll B, McNelis T, Hollinger JO. Osteoblast differentiation in vitro and in vivo promoted by Osterix. J Biomed Mater Res A 2007;83:770–8. [17] Tu Q, Valverde P, Chen J. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells. Biochem Biophys Res Commun 2006;341:1257–65. [18] Kim YJ, Kim HN, Park EK, Lee BH, Ryoo HM, Kim SY, et al. The bone-related Zn finger transcription factor Osterix promotes proliferation of mesenchymal cells. Gene 2006;366:145–51. [19] Hatta M, Yoshimura Y, Deyama Y, Fukamizu A, Suzuki K. Molecular characterization of the zinc finger transcription factor, Osterix. Int J Mol Med 2006;17:425–30. [20] Zhang C, Cho K, Huang Y, Lyons JP, Zhou X, Sinha K, et al. Inhibition of Wnt signaling by the osteoblast-specific transcription factor Osterix. Proc Natl Acad Sci U S A 2008; 105:6936–41. [21] Kaabeche K, Lemonnier J, Le Mee S, Caverzasio J, Marie PJ. Cbl-mediated degradation of Lyn and Fyn induced by constitutive fibroblast growth factor receptor-2 activation supports osteoblast differentiation. J Biol Chem 2004;279:36259–67. [22] Severe N, Miraoui H, Marie PJ. The Casitas B lineage lymphoma (Cbl) mutant G306E enhances osteogenic differentiation in human mesenchymal stromal cells in part by decreased Cbl-mediated platelet-derived growth factor receptor alpha and fibroblast growth factor receptor 2 ubiquitination. J Biol Chem 2011;286:24443–50. [23] Brennan T, Adapala NS, Barbe MF, Yingling V, Sanjay A. Abrogation of Cbl-PI3K interaction increases bone formation and osteoblast proliferation. Calcif Tissue Int 2011; 89:396–410.

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