Prolyl isomerase Pin1 regulates the osteogenic activity of Osterix

Molecular and Cellular Endocrinology 400 (2015) 32–40

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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

Prolyl isomerase Pin1 regulates the osteogenic activity of Osterix Sung Ho Lee a, Hyung Min Jeong a, Younho Han a, Heesun Cheong b, Bok Yun Kang a, Kwang Youl Lee a,* a b

College of Pharmacy & Research Institute of Drug Development, Chonnam National University, Gwangju 500-757, Republic of Korea Research Institute, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do 410-769, Republic of Korea

A R T I C L E

I N F O

Article history: Received 31 July 2014 Received in revised form 27 October 2014 Accepted 20 November 2014 Available online 24 November 2014 Keywords: Pin1 Osterix Osteoblast differentiation

A B S T R A C T

Osterix is an essential transcription factor for osteoblast differentiation and bone formation. The mechanism of regulation of Osterix by post-translational modification remains unknown. Peptidyl-prolyl isomerase 1 (Pin1) catalyzes the isomerization of pSer/Thr-Pro bonds and induces a conformational change in its substrates, subsequently regulating diverse cellular processes. In this study, we demonstrated that Pin1 interacts with Osterix and influences its protein stability and transcriptional activity. This regulation is likely due to the suppression of poly-ubiquitination-mediated proteasomal degradation of Osterix. Collectively, our data demonstrate that Pin1 is a novel regulator of Osterix and may play an essential role in the regulation of osteogenic differentiation. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Osterix, a zinc finger-containing transcription factor, plays a critical role in osteoblast differentiation and bone formation. No bone formation occurs in Osterix-knockout mice (Baek et al., 2009, 2010; Nakashima et al., 2002; Zhou et al., 2010). Osterix is required for bone morphogenetic protein 2 (BMP2) and Runx2-induced osteoblast differentiation and bone growth in both postnatal and adult mice (Lee et al., 2003; Matsubara et al., 2008). Runx2 expression level in osteogenic cells of Osterix-null mutants is comparable to that in wild-type osteoblasts. In contrast, Runx2-deficienct embryos do not express Osterix, suggesting that Osterix acts downstream of Runx2, which in turn regulates the expression of many osteoblastspecific differentiation markers, including alkaline phosphatase, osteocalcin, osteonectin, osteopontin, and Runx2 (Fu et al., 2007; Nakashima et al., 2002; Zhang et al., 2008). The functional roles of Osterix in bone and cartilage development have only recently been investigated. Osterix is thought to regulate the transcription of several osteoblast marker genes containing GC-rich and Sp-binding sites on their promoters. Forced expression of Osterix in vitro has been reported to induce the expression of osteocalcin; collagen, type 1, alpha 1 (Nakashima et al., 2002); collagen, type 11, alpha 2 (Goto et al., 2006); and osteopontin (Kim et al., 2006). Osterix can also inhibit osteoblast proliferation through the inhibition of the Wnt signaling pathway (Zhang et al., 2008) and act as a negative regu-

* Corresponding author. College of Pharmacy & Research Institute of Drug Development, Chonnam National University, Gwangju 500-757, Republic of Korea. Tel.: +82-62-530-2939; fax: +82-62-530-2949. E-mail address: [email protected] (K.Y. Lee). http://dx.doi.org/10.1016/j.mce.2014.11.017 0303-7207/© 2014 Elsevier Ireland Ltd. All rights reserved.

lator of chondrogenesis. Therefore, understanding the regulation of Osterix gene expression will be beneficial in modulating osteoblast differentiation and bone formation. Pin1 is a highly conserved and relatively smaller enzyme (18 kDa) (Lu et al., 1996), which contains an N-terminal WW domain that acts as a phosphoprotein-binding module (Lu et al., 1999) and a C-terminal catalytic domain that is distinct from other conventional peptidyl–prolyl cis–trans isomerases (PPIases) (Ranganathan et al., 1997; Yaffe et al., 1997). By virtue of its unique WW and catalytic domains, Pin1 isomerizes specific phosphorylated Ser/Thr-Pro bonds and regulates the function of a defined subset of phosphoproteins (Yaffe et al., 1997). The isomerization of Ser/Thr-Pro motifs is especially important because kinases and phosphatases can specifically recognize the cis or trans conformation of the prolyl peptide bond of their substrates (Werner-Allen et al., 2011; Zhou et al., 2000), and phosphorylation further slows down the isomerization rate of proline (Lu and Zhou, 2007; Yaffe et al., 1997). Pin1 activity controls a subset of protein functions in diverse cellular processes such as cell cycle and cell growth (Liou et al., 2002, 2003; Lu and Zhou, 2007; Lu et al., 1999, 2007; Ryo et al., 2002, 2003; Wulf et al., 2002, 2003; Zhou et al., 2000). Importantly, Pin1 is tightly regulated by several mechanisms (Lu et al., 2002; Ryo et al., 2002), and its deregulation can contribute to a plethora of human diseases, including aging, cancer, neurological disorders, and autoimmune and inflammatory diseases (Lee et al., 2011; Liou et al., 2002; Lu and Zhou, 2007). However, the underlying mechanism of how Pin1 is involved in bone metabolism, particularly osteogenesis, remains unknown. In this study, we investigated the functional roles of Pin1 during osteoblast differentiation. We found that Pin1 increases BMP2induced osteoblast differentiation in C2C12 cells, and induces the protein stability and transcriptional activity of Osterix. This

S.H. Lee et al./Molecular and Cellular Endocrinology 400 (2015) 32–40

up-regulation of Osterix function is likely due to the reduction of ubiquitin proteasome-mediated degradation of Osterix. Taken together, our results suggest a novel regulatory mechanism of Pin1-mediated Osterix function that can subsequently induce osteoblast differentiation.

2. Materials and methods 2.1. Plasmids, antibodies, and reagents Plasmids for the expression of Myc-Osterix, HA-Osterix, and GFPOsterix were constructed in a CMV promoter-derived expression vector (pCS4+). The Xpress-tagged Pin1 wild-type, S16A, K63A, and M130F mutant plasmids were generously provided by Dr. Hong Seok Choi (Chosun University, Gwangju, Korea). For Pin1-knockdown, the following oligonucleotides targeting the mouse Pin1 (accession number: NP_006221) sequence (in capital letters) were annealed and inserted into pSUPER. Puro: sense, 5′-gat ccc cGC CGG GTG TAC TTC AAT tca aga gAT TGA AGT ACA CCC GGC ttt ttg gaa a-3′ and antisense, 5′-agc ttt tcc aaa aaG CCG GGT GTA CTA CTT CAA Tct ctt gaA TTG AAG TAG TAC ACC CGG Cgg g-3′. For inhibition of MEK activity, 10 mM stock solution of the MEK inhibitor, U0126 (Calbiochem, Cat#662005), was used at 5 μM final concentration. Antibodies against the following epitopes were used: Myc (9E10) and HA (12CA5) from Roche Applied Science; Flag (M2) and α-Tubulin (B5-1-2) from Sigma-Aldrich; Pin1 (SC-46660), GFP (FL), and Osterix (A-13) from Santa Cruz Biotechnology; Anti-Xpress (R91025) antibody from Invitrogen. Pin1 inhibitor Juglone (420120) and MG132 (Calbiochem), cycloheximide (Sigma), calf intestinal alkaline phosphatase (CIAP; Invitrogen), and recombinant human BMP2 (335-BM, R&D Systems) were used.

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2.4. Luciferase reporter assay C2C12 cells were seeded on 24-well plates 1 day before transfection. The cells were transfected with a luciferase reporter plasmid [containing the regulatory sequences of osteoblast differentiation marker ALP, BSP, or OC genes], CMV promoter-driven β-galactosidase reporter (pCMV-β-gal), and the indicated combinations of the expression plasmids. Luciferase activity was measured after 36 h, using the Luciferase Reporter Assay Kit (Promega) and normalized with the corresponding β-galactosidase activity for transfection efficiency. All the experiments were performed in triplicate and repeated at least three times. The average and S.D. of the representative experiments are shown. 2.5. RT-PCR analysis C2C12 cellular RNA was prepared using TRIzol reagent (Life Technologies), according to the manufacturer’s instructions. 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 PCR amplification: initial denaturation at 94 °C for 5 min; 28–30 cycles of denaturation at 94 °C for 1 min, annealing at a temperature optimized for each primer pair for 1 min, and extension at 72 °C for 1 min; and final extension at 72 °C for 10 min. The following PCR primers were used: mouse ALP (accession number: NP_031457) forward 5′-ATT GCC CTG AAA CTC CAA AAC C-3′ and reverse 5′-CCT CTG GTG GCA TCT CGT TAT C-3′; mouse BSP (accession number: NP_032344) forward 5′-CAG AAG TGG ATG AAA ACG AG-3′ and reverse 5′-CGG TGG CGA GGT CCC AT-3′; mouse COL1A1 (accession number: NP_031768) forward 5′-TCT CCA CTC TTC TAG GTT CCT-3′ and reverse 5′-TTG GGT CAT TTC CAC ATG C-3′; and mouse GAPDH (accession number: XP_001476757) forward 5′-ACC ACA GTC CAT GCC ATC AC3′ and reverse 5′-TCC ACC CTG TTG CTG TA-3′.

2.2. Cell culture and transient transfections 2.6. Alkaline phosphatase (ALP) staining Human embryonic kidney (HEK) 293 cells, C2C12 mouse myoblast cells, and Pin1−/− mouse embryonic fibroblasts (MEFs), which were kindly provided by Dr. Kun Ping Lu (Beth Israel Deaconess Medical Center, Harvard Medical School), were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. All these cell lines were cultured and maintained at 37 °C in humidified air containing 5% CO2. DMEM, FBS, and the antibiotics were purchased from Life Technologies. Transient transfection was performed using polyethyleneimine (PEI; Polysciences, Inc.). The total amount of transfected plasmids in each group was normalized by adding an empty vector.

2.3. Immunoblotting (IB) and immunoprecipitation (IP) HEK293 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 Na 3 VO 4 , 250 μM PMSF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin). After centrifugation, the supernatants were used as cell lysates. For immunoprecipitation, the cell lysates were incubated with the appropriate antibodies and protein A- or G-Sepharose beads. Cell lysates containing 30 μg of total protein or immunoprecipitated proteins were subjected to SDS–PAGE and the proteins were transferred to a PVDF membrane. Proteins were detected using the relevant primary antibodies, horseradish peroxidase-coupled secondary antibodies (GE Healthcare Life Sciences), and enhanced chemiluminescence (ECL) reagent (Millipore). The signals were detected and analyzed by an LAS4000 luminescent image analyzer (Fuji Photo Film Co.).

For ALP staining, C2C12 cells were seeded in 24-well plates, transfected, and then incubated in the differentiation medium (2% FBS/ DMEM) with BMP2 (10 ng/mL) for 72 h. The cells were then fixed in 4% paraformaldehyde for 10 min at room temperature (RT), rinsed with PBS, and then incubated with 300 μg/mL BCIP/NBT (5-bromo4-chloro-3-indolyl phosphate/nitro blue tetrazolium) solution (Sigma-Aldrich) for 20 min at RT. ALP-positive cells were stained blue/purple. 2.7. Glutathione-S-transferase (GST) pull-down assay Recombinant GST-tagged empty vector and Pin1 (WT, Y23A, Y24A, and Y94A) proteins were expressed in E. coli and purified using glutathione–sepharose beads. For each GST-pull down assay, beads carrying 10 μg of GST-Osterix protein were equilibrated with cell lysis buffer and incubated with the cell lysates. The retained proteins were subjected to SDS–PAGE and IB. 2.8. Immunocytochemical analysis C2C12 cells grown on microscope coverslips were transfected with the GFP-Osterix plasmids. The cells were fixed with 4% paraformaldehyde for 10 min at RT and then washed with PBS. The washed slides were treated with a blocking solution (0.2% Triton X-100 in PBS) for 1 h at RT. The cells were then incubated with primary antibodies at 4 °C overnight, followed by secondary antibodies for 1 h at RT. To visualize the nuclei, the cells were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Calbiochem). The cells were washed three times with PBS, mounted

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using the mounting fluid (DakoCytomation), and observed with a Nikon fluorescent microscope.

the endogenous and epitope-tagged overexpressed Pin1 interacted with overexpressed Osterix in HEK293 and C2C12 cells (Fig. 1A–C). We then examined whether Pin1 can directly bind to Osterix using the GST pull-down assay. Purified GST-Pin1 interacted with Osterix (Fig. 1D), suggesting a direct interaction between Pin1 and Osterix. Consequently, we examined whether Pin1 and Osterix are co-localized in C2C12 cells. GFP-Osterix was co-expressed with Pin1 (Fig. 1E). These results indicate that Pin1 interacts and co-localizes with Pin1.

2.9. Protein stability assay HEK293 cells were transfected with combinations of Osterix and Pin1 expression plasmids. For the cycloheximide (CHX) treatment, the cells were incubated in fresh growth medium 48 h after transfection and treated with CHX (40 μM) for the indicated amounts of time (0, 2, 4, and 8 h).

3.2. Pin1 WW domain is essential for binding to Osterix 2.10. Statistical analysis Next, we sought to determine the region of Pin1 responsible for the Pin1–Osterix interaction. In Pin1, the WW domain functions as the protein interaction domain that specifically recognizes pSer/ Thr-Pro motifs in target proteins (Lu et al., 1999). Since Pin1 interacts with Osterix (Fig. 1A–D), we examined whether the Pin1 WW domain is involved in Osterix binding. Phosphorylation of Ser 16 residue of Pin1 is important for its pSer/Thr-Pro motif recognition. We tested Ser-to-Tyr (including Ser16) substitution mutants of Pin1 (Fig. 2A) for their interaction with Osterix. The substitution of Ser16 to Ala abolished the binding of Pin1 to Osterix; however, the substitution of Lys63 to Ala and Met 130 to Phe did not (Fig. 2B). This is because Lys63 and Met130 residues are located in the PPI domain that is involved in the catalytic activity and not for binding.

All the experiments were performed in triplicate and repeated at least twice, to obtain reproducible results. Results are expressed as mean ± standard error of the mean. Data were analyzed using Student’s t-test, with p < 0.05 indicating statistical significance. 3. Results 3.1. Pin1 interacts and co-localizes with Osterix We examined whether Pin1 interacts with Osterix, which is also a key transcription factor involved in osteoblast differentiation. Both

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Fig. 1. Pin1 interacts and co-localizes with Osterix. (A) Pin1 interacts with Osterix. HEK293 cells were transfected with the indicated combinations of Xpress-tagged Pin1 and Myc-tagged Osterix. Anti-Pin1 immunoprecipitates (IP: Pin1) were analyzed by immunoblotting for Myc-Osterix (IB: Myc) (top panel). The expression level of the proteins in the lysates was also compared (bottom 2 panels). (B) Pin1 interacts with Osterix. HEK293 cells were transfected with the indicated combinations of Xpress-tagged Pin1 and Myc-tagged Osterix. Anti-Myc immunoprecipitates (IP: Myc) were analyzed by immunoblotting for Pin1 (IB: Pin1) (top panel). The expression level of the proteins in the lysates was also compared (bottom 2 panels). (C) Endogenous Pin1 interacts with Osterix. Anti-Pin1 immunoprecipitates (IP: Pin1) from mock or Myc-Osterixtransfected HEK293 cells were analyzed by immunoblotting for Myc-Osterix (IB: Myc). (D) Purified Pin1 interacts with Osterix. Lysates from Myc-Osterix-transfected HEK293 cells were incubated with GST or GST-Pin1-bound agarose beads, and analyzed by IB for Myc-Osterix. (E) Co-localization of Osterix and Pin1. HEK293 cells were transfected with a plasmid expressing GFP-fused Osterix and Pin1. The same samples were stained with anti-Pin1 antibody and Alexa Fluor 594 anti-mouse antibody. GFP-Osterix and Pin1 were double-immunostained and visualized with green or red dye; the nucleus is counterstained by DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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IB: Pin1 IB: Tubulin Fig. 2. Pin1 WW domain is important for binding to Osterix. (A) Schematic diagram of Pin1 mutants. (B) HEK293 cells were transfected with the indicated combinations of Xpress-Pin1 (wild type or S16A or K63A, or M130F mutants) and HA-Osterix. HA-Osterix IPs were analyzed by IB for Xpress-Pin1. (C) The lysates from Myc-Osterixtransfected HEK293 cells were subjected to the GST pull-down assay with the indicated Pin1 proteins.

Tyr-to-Ala substitution (Y23A or Y24A) in the WW domain also significantly reduced the interaction of Pin1 with Osterix, whereas a similar substitution in the PPIase catalytic domain (Y94A) did not have any apparent effect (Fig. 2C). Collectively, these results suggest that the WW domain of Pin1 is important for its binding to Osterix.

ined whether Pin1 affects Osterix poly-ubiquitination, which is required for its proteasomal degradation. Pin1 was found to reduce the ubiquitination of Osterix (Fig. 4H). These results suggest that Pin1 enhances the stability of the Osterix protein by inhibiting its ubiquitin-mediated proteasomal degradation.

3.3. Pin1 enhances the osteogenic activity of Osterix and PPIase domain is important for activation of Osterix

3.5. Pin1 knockdown decreases the protein stability and transcriptional activity of Osterix

To analyze whether Pin1 can modulate the osteogenic activity of Osterix, we examined the effects of Pin1 on Osterix-induced osteoblast differentiation. BMP2 induced ALP staining and mRNA level of ALP were further increased by Pin1 (Fig. 3A and B). We then examined whether Pin1 influences the transcriptional activity of Osterix. Osterix induced the expression of osteoblast-specific luciferase reporters (ALP, BSP, and OC), and Pin1 further enhanced their expression in a dose-dependent manner (Fig. 3C–E). These results indicate that Pin1 positively regulates the osteogenic activity of Osterix. Next we examined whether PPIase domain is essential for the activation of Osterix. Pin1-WW (ΔPPIase domain) decreased the level of overexpressed Osterix (Fig. 3F) and did not make a significant change of transcriptional activity of Osterix (Fig. 3G).

Next, we examined the effects of Pin1-knockdown on the stability and function of Osterix. Pin1-knockdown reduced the Osterix protein level (Fig. 5A) and decreased Osterix-induced expression of osteoblast-specific luciferase reporters (Fig. 5B). Overexpression of Osterix recovered the expression of these reporters in Pin1deficient cells (Fig. 5C). Furthermore, MG-132 also rescued the Osterix-induced expression of the osteoblast-specific luciferase reporters in Pin1-deficient cells (Fig. 5D). Collectively, these results indicate that Pin1 activity is important for the protein stability and transcriptional activity of Osterix.

3.4. Pin1 suppresses the proteasomal degradation and increases the protein stability of Osterix

As Pin1 recognizes the pSer/Thr-Pro motif, we examined whether the phosphorylation status of Osterix affects its interaction with Pin1. Lysates of Osterix-transfected cells were incubated with or without CIAP and subjected to GST-Pin1 pull-down assay. Dephosphorylation of Osterix abolished the binding between Osterix and Pin1 (Fig. 6B). Interestingly, MEK activity modulates both Pin1 and Osterix protein levels at the same time. MEK-EE increases Pin1 protein level while MEK-AA decreased it (Fig. 6C). Other research supports our result (Lim et al., 2011). It implies that MEK activity is important for Pin1-dependent modulation. To further confirm it, we used MAPK inhibitor (U0126) and we found that MAKP inhibitor decreases Pin1induced protein level of Osterix (Fig. 6D). To determine the precise mechanism of phosphorylation and the site of interaction, we used the SCANSITE program to identify the possible phosphorylation sites of Osterix by activated Pro-directed kinases in http://scansite.mit.edu (Obenauer et al., 2003). SCANSITE analysis indicated that Ser76 and

Pin1 regulates the function of many of its target proteins by affecting their stability (Hall et al., 2010). Therefore, we examined whether Pin1 affects the stability of the Osterix protein. Pin1 increased the level of overexpressed Osterix in a dose-dependent manner (Fig. 4A), whereas Pin1 S16A or K63A substitution mutants failed to increase the level of Osterix (Fig. 4B). However, Pin1 did not cause any appreciable change in the BMP2-induced transcription of endogenous Osterix (Fig. 4C). We also compared the halflife of Osterix in the control, overexpressed Pin1, and treated juglone. Pin1 and juglone considerably increased and decreased the halflife of Osterix, respectively (Fig. 4D). Furthermore, knockdown of Pin1 decreased the half-life of Osterix (Fig. 4F). These results suggest that Pin1 modulates the protein stability of Osterix. Next, we exam-

3.6. Phosphorylation of Ser76 and Ser80 of Osterix is important for Pin1 interaction and function

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Fig. 3. Pin1 enhances the osteogenic activity of Osterix. (A) Pin1 enhances Osterix-induced ALP activity. C2C12 cells were transfected with an empty vector or the indicated combinations of Osterix and Pin1, stimulated with BMP2, and then stained for ALP activity. (B) Pin1 enhances the expression of ALP gene. C2C12 cells were transfected with the empty vector or the indicated combinations of Osterix and Pin1, and stimulated with BMP2. The expression of ALP and GAPDH was compared by RT-PCR. GAPDH was used as the internal control. (C–E) Pin1 increases the expression of osteoblast-specific reporters. C2C12 cells were transfected with the indicated combination of Osterix and Pin1 and a luciferase reporter containing ALP, BSP, or OC promoter element. Luciferase activity was measured after 36 h. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared to the Osterix-overexpression group. (F) Pin1-WW (ΔPPIase domain) decreases the expression of Osterix. HEK293 cells were transfected with HA-Osterix and the indicated Xpress-Pin1. After 48 h, the cell lysates were analyzed by IB. Tubulin was used as the loading control. (G) Pin1-WW (ΔPPIase domain ) does not affect the expression of osteoblast-specific reporter. C2C12 cells were transfected with the indicated combination of Osterix and Pin1 (WT or WW) and a luciferase reporter containing BSP promoter element. Luciferase activity was measured after 36 h. *, p < 0.05; **, p < 0.01.

Ser80 were the candidate phosphorylation sites (Fig. 6A). To determine whether phosphorylation of Osterix at these sites is essential for Pin1 binding, we generated double Ser-to-Ala substitution mutants (S76A and S80A) of Osterix and examined their interac-

tion with Pin1. We found that mutations diminished the interaction between Pin1 and Osterix (Fig. 6E). We also examined whether Pin1 regulates the protein levels of Osterix S76 and 80A. The protein levels of the overexpressed Osterix S76 and 80A were not affected by Pin1

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Fig. 4. Pin1 increases the protein level of Osterix. (A) Pin1 increases the level of Osterix. HEK293 cells were transfected with expression vector Xpress-Pin1 and increasing amounts of HA-Osterix. After 48 h, the cell lysates were analyzed by IB. Tubulin was used as the loading control. (B) Pin1 mutants fail to increase the level of Osterix protein. HEK293 cells were transfected with HA-Osterix and the indicated Xpress-Pin1. After 48 h, the cell lysates were analyzed by IB. (C) Pin1 does not affect the transcription of Osterix. Mock- or Pin1-transfected C2C12 cells were analyzed by RT-PCR. (D) Pin1 increases the half-life of the Osterix protein. HEK293 cells were transfected with HAOsterix alone or with Xpress-Pin1 or with Juglone (5 μM). After 48 h, cells were treated with CHX (40 μg/mL) for the indicated time period and the cell lysates were analyzed by IB. (E) The intensities of the Osterix bands in panel D were determined by densitometry. The Osterix protein expression level in CHX-untreated cells (0 h) was considered to be 100%. (F) Pin1 knockdown decreases the half-life of the Osterix protein. C2C12 cells were transfected with HA-Osterix alone or with si-Pin1. After 48 h, cells were treated with CHX (40 μg/mL) for the indicated time period and the cell lysates were analyzed by IB. (G) The intensities of the Osterix bands in panel F were determined by densitometry. The Osterix protein expression level in CHX-untreated cells (0 h) was considered to be 100%. (H) Pin1-overexpression reduces the poly-ubiquitination of Osterix. HEK293 cells were transfected with the indicated combination of Flag-ubiquitin (Ub), Myc-Osterix, and Xpress-Pin1 plasmids. Osterix IPs (anti-Myc) was analyzed by ubiquitin IB (anti-flag). The level of Pin1 in the cell lysates (Input) is shown (bottom panels).

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Fig. 5. Pin1-knockdown reduces the transcriptional activity and protein stability of Osterix. (A) Pin1-knockdown decreases the level of Osterix protein. C2C12 cells were transfected with control or increasing amounts of Pin1 shRNA plasmid (si-Pin1), and then stimulated with BMP2. After 48 h, the cell lysates were analyzed by IB for Osterix. (B) Pin1-knockdown reduces the transcriptional activities of Osterix. HEK293 cells were transfected with BSP or OC-Luc reporter alone or with the indicated combinations of Osterix, Pin1, and si-Pin1. Luciferase activity was measured after 36 h. **, p < 0.01 and ***, p < 0.001 compared to cells transfected with Osterix and Pin1. (C) Overexpression of Osterix rescues osteoblast differentiation of Pin1-deficient cells. Pin1-deficient MEFs were transfected with BSP-Luc reporter alone or with the indicated combinations of Osterix and Pin1. Luciferase activity was measured after 36 h. *, p < 0.05. (D) MG-132 increases the transcriptional activities of Osterix in Pin1-deficient cells. Pin1-deficient MEFs were transfected with ALP-Luc reporter alone or with Osterix and then treated with MG-132 (1 or 10 μM). Luciferase activity was measured after 24 h. **, p < 0.01 and ***, p < 0.001 compared to cells transfected with Osterix alone.

in HEK293 cells (Fig. 6F). Taken together, these results indicate that MEK activity is important for Pin1-mediated Osterix regulation and Ser76 and Ser80 in Osterix are the major, if not exclusive, target sites of Pin1 interaction and function. 4. Discussion We have previously shown that Pin1 modulates osteoblast differentiation and is a positive regulator of Runx2 (Lee et al., 2013). In this study, we examined the role of Pin1 in Osterix regulation. Interestingly, in spite of their distinct structures, Osterix interacts with Pin1 and its transcriptional activity and stability are upregulated by Pin1. Our results suggest that Pin1 is a novel factor responsible for osteoblast differentiation. Pin1 activity modulates osteoblast differentiation and induces protein stability and transcriptional activity of Runx2 and Osterix, both of which are markers of the earlier stages of osteoblast differentiation. Moreover, silencing of Pin1 decreases ALP activity and osteogenic gene expression by regulating the stability and transcriptional activity of Runx2 and Osterix, thereby resulting in the suppression of osteoblast differentiation. Osterix is a master transcriptional factor required for osteoblast differentiation and bone formation (Nakashima et al., 2002).

Osterix is regulated by various post-translational modifications, including phosphorylation, methylation, and ubiquitination (Choi et al., 2011; Jeong et al., 2011; Mandal et al., 2010; Okamura et al., 2009, 2013; Ortuno et al., 2010; Peng et al., 2013; Sinha et al., 2010, 2013; Wang et al., 2007). The phosphorylation of Osterix modifies its transcriptional activities and is mediated by kinases such as p38 (Wang et al., 2007), ERK (Jeong et al., 2011), and Akt (Choi et al., 2011). ERK is the primary kinase that phosphorylates the Ser/Thr-Pro motifs of Pin1 (Schutkowski et al., 1998); thus, we speculated that the Ser76 and Ser80 residues in Osterix could be critical for its binding to Pin1. We generated a Ser-to-Ala substitution mutant, S76, 80A of Osterix and examined its interaction with Pin1. Although the binding between Pin1 and the Osterix mutant was diminished compared to that between Pin1 and the wild-type Osterix (Fig. 6E), we cannot exclude the possibility that other sites are also involved in this interaction. Recent reports demonstrate that Osterix is an unstable protein and that its stability is regulated by the ubiquitin–proteasome system (Peng et al., 2013). Histone demethylases such as NO66 (Sinha et al., 2010, 2013) and retinoblastoma binding protein 2 (RBP2) (Ge et al., 2011) are involved in osteoblast differentiation through the regulation of Osterix. Pin1-mediated conformational changes of Osterix may cause Osterix to associate or dissociate from its binding

S.H. Lee et al./Molecular and Cellular Endocrinology 400 (2015) 32–40

39

B

A 1

431

Input GST-Pull down assay

Myc-Osterix CIAP

S76 S80

T414

Erk1

Cdc2, Cdk5

+ -

+ -

+ -

+ +

IB: Myc (Osterix)

kDa 43

GST-Pin1 HA-Osterix

C

Xpress-Pin1 GST -

MEK-EE MEK-AA

-

25

MEK-EE MEK-AA Ponseau S

IB: HA (Osterix)

D Exo

IB: Pin1 Endo

IB: Tubulin

GST-Pull down assay

GST

HA-Osterix

+

+

+

Xpress-Pin1

-

+

+

U0126

-

-

+

IB: Myc (Osterix)

IB: p-ERK

F

GST-Pin1

+

IB: Xpress (Pin1)

E Myc-Osterix

Myc-Osterix

+

Xpress-Pin1

S76, 80A

WT +

+

IB: ERK IB: Tubulin

+ IB: HA (Osterix) Osterix Myc (Osterix)

IB: Pin1

kDa GST-Pin1

41

IB: Tubulin

27

GST

Input

Ponceau S

Myc (Osterix) Tubulin

Fig. 6. Residues Ser76 and Ser80 of Osterix are important for Pin1 interaction and function. (A) Pin1 interacts with Osterix in a phosphorylation-dependent manner. Lysates of HEK293 cells transfected with Myc-Osterix were treated with or without CIAP for 30 min at 30 °C. The lysates were then incubated with GST or GST-Pin1-bound agarose beads, and analyzed by IB for Myc-Osterix. (B) The map of potential Pin1 binding sites on Osterix is shown. PRR, proline rich region; Z, zinc finger region. (C) MEK enhances Pin1-induced increase of Osterix protein expression. HEK293 cells were transfected with the indicated combination of HA-Osterix, Pin1, and MEK mutants. The cell lysates were analyzed by IB. (D) MAPK inhibitor (U0126) decreases Pin1-induced increase of Osterix protein expression. HEK293 cells were transfected with the indicated combination of HA-Osterix and Pin1 and then treated with U0126 (0 or 5 μM). The cell lysates were analyzed by IB. (E) Osterix Ser76 and Ser80 are critical for interaction with Pin1. Lysates of HEK293 cells transfected with Myc-Osterix WT or Myc-Osterix S76, 80A were incubated with GST or GST-Pin1-bound agarose beads, and analyzed by IB for Myc-Osterix. (F) Osterix S76, 80A is not regulated by Pin1. HEK293 cells were transfected with Myc-Osterix (WT) or (S76, 80A) and Xpress-Pin1. The cell lysates were analyzed by IB after 48 h. Tubulin was used as the loading control.

proteins. Further studies are needed to clarify how Pin1 regulates osteoblast differentiation. In summary, we propose that Pin1 positively regulates Osterix and subsequently induces osteoblast differentiation. Pin1-mediated

conformational changes of Osterix increase its protein stability and transcriptional activity. Our data suggest that the interaction between Pin1 and Osterix plays an important role in osteoblastogenesis. The identification of Pin1 as a novel regulator of Osterix during bone

40

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