FGF2 stimulates osteogenic differentiation through ERK induced TAZ expression

Bone 58 (2014) 72–80

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Original Full Length Article

FGF2 stimulates osteogenic differentiation through ERK induced TAZ expression Mi Ran Byun a, A Rum Kim a, Jun-Ha Hwang a, Kyung Min Kim a, Eun Sook Hwang b, Jeong-Ho Hong a,⁎ a b

Department of Life Sciences, Korea University, Seoul 136-701, Republic of Korea College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea

a r t i c l e

i n f o

Article history: Received 19 June 2013 Revised 17 September 2013 Accepted 21 September 2013 Available online 11 October 2013 Edited by: J. Aubin Keywords: FGF2 Osteogenesis Runx2 TAZ

a b s t r a c t TAZ (transcriptional coactivator with PDZ-binding motif) is a transcriptional modulator that regulates mesenchymal stem cell differentiation. It stimulates osteogenic differentiation while inhibiting adipocyte differentiation. FGFs (fibroblast growth factors) stimulate several signaling proteins to regulate their target genes, which are involved in cell proliferation, differentiation, and cell survival. Within this family, FGF2 stimulates osteoblast differentiation though a mechanism that is largely unknown. In this report, we show that TAZ mediates FGF2 signaling in osteogenesis. We observed that FGF2 increases TAZ expression by stimulating its mRNA expression. Depletion of TAZ using small hairpin RNA blocked FGF2-mediated osteogenic differentiation. FGF2 induced TAZ expression was stimulated by ERK (extracellular signal-regulated kinase) activation and the inhibition of ERK blocked TAZ expression. FGF2 increased nuclear localization of TAZ and, thus, facilitated the interaction of TAZ and Runx2, activating Runx2-mediated gene transcription. Taken together, these results suggest that TAZ is an important mediator of FGF2 signaling in osteoblast differentiation. © 2013 Elsevier Inc. All rights reserved.

Introduction TAZ is a transcriptional co-regulator and is identified as a 14-3-3binding protein [1]. For transcriptional regulation, TAZ interacts with many transcription factors, including Runx2, PPARγ, TEADs, TTF-1/ Nkx2.1, Tbx5, Pax3, Smad2/3–4 complexes, and MyoD [2–10]. Through these interactions, several target genes can be activated or suppressed in a context-dependent manner, producing diverse biological functions. Recently, along with Yes-associated protein (YAP), TAZ was characterized as the effector protein of the Hippo signaling pathway, which plays an important role in cell proliferation and tumorigenesis [11,12]. Moreover, TAZ modulates mesenchymal stem cell differentiation by activating osteoblast and myoblast differentiation and inhibiting adipocyte differentiation [2,10]. TAZ stimulates Runx2-mediated gene transcription, but its interaction with PPARγ inhibits PPARγ-mediated gene transcription [2]. Even though the role of TAZ has been revealed in mesenchymal stem cell differentiation, the regulatory signal of the differentiation has not yet been characterized. Fibroblast growth factors (FGFs) are secreted glycoproteins that regulate embryonic development and are involved in cellular proliferation, differentiation, and cell survival. Thus far, 22 ligands of

Abbreviations: TAZ, transcriptional co-activator with PDZ binding domain; YAP, Yes-associated protein; FGF2, fibroblast growth factor 2; ERK, extracellular signal-regulated kinase; MAPK, mitogen activated protein kinases; PKC, protein kinase C; MEK, MAPK kinase. ⁎ Corresponding author at: Department of Life Sciences, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-701, Republic of Korea. Fax: +82 2 927 9028. E-mail address: [email protected] (J.-H. Hong). 8756-3282/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bone.2013.09.024

the FGF family have been identified, and they bind to FGF receptors (FGFRs) and activate downstream signaling pathways, including the RAS-MAPK signal. Within FGF family members, FGF2 signaling plays an important role in self-renewal of mesenchymal stem cells. Human MSCs are proliferated rapidly and retained multilineage differentiation potential in the presence of FGF2 [13–16]. The role of FGF2 in osteogenic differentiation was previously investigated. FGF2-null mice have decreased bone mass and bone formation [17]. Suppressed osteogenic differentiation and increased adipogenesis were observed in FGF2-null mouse-derived mesenchymal stem cells [18]. FGF2 promotes osteoblast differentiation in bone marrowderived mesenchymal cells [19,20], though the inhibitory effect of FGF2 was also reported [21]. FGF2 induces osteogenic differentiation through Runx2 activation in vascular smooth muscle cells [21]. Recently, it was shown that FGF2 stimulation of osteoblast differentiation and bone formation is mediated by modulation of the WNT pathway [22]. Connexin43 plays a critical role in FGF2 mediated osteogenesis by impacting PKC delta and Runx2 function [23]. The importance of FGFR in bone formation was observed in previous studies (for review, [24,25]). Conditional inactivation of FGFR2 in mice resulted in skeletal dwarfism and decreased bone density [26]. Loss of function mutations in FGFR2 reduced Runx2 expression [27]. In contrast, gain of function mutations of FGFR2 in mice also increased osteoblast gene expression and bone formation [28–30]. The effects of FGFR1 in bone development are dependent on developmental stage. Conditional deletion of FGFR1 in osteoprogenitor cells resulted in delayed osteoblast differentiation whereas FGFR1 inactivation in differentiated osteoblasts accelerated osteoblast differentiation [31].

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So, many reports indicated the positive role of FGF2 in bone formation, but a detailed regulatory mechanism of FGF2 signaling has not been clearly addressed. In the present study, we investigated the role of TAZ in FGF2 mediated osteogenic differentiation. Our results show that FGF2 significantly increases TAZ expression, and the induced TAZ facilitates osteogenic differentiation through osteoblastic marker gene induction. Thus, we suggest that TAZ is a mediator of FGF2 signaling.

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Biology, Singapore). Twenty-four hours later, cells were lysed for 20 min at 4 °C with 150 mM NaCl, 50 mM Tris–HCl pH 7.5, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM NaF, 1 mM Na-orthovanadate and protease inhibitors. Total cell proteins were immunoprecipitated for 2 h at 4 °C using anti-FLAG M2 agarose beads (Sigma-Aldrich). Protein-bead complexes were washed three times with lysis buffer and denatured with 2× SDS sample buffer. Denatured proteins were detected by immunoblot analysis.

Materials and methods Quantitative real-time PCR analysis Cell culture and osteoblast differentiation C3H10T1/2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone) and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin). To induce osteoblast differentiation, C3H10T1/2 cells were seeded on 6 well culture plates at a density of 2 × 105 cells/cm2 and 48 h later the culture media were changed with DMEM containing 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate and 10% FBS (Differentiation media) for 4 days. The media were replaced every 2 days. FGF2 treatment C3H10T1/2 cells were seeded on 6-well culture plates (2 × 105 cells/ well). Twenty-four hours later (80% confluency), cells were washed with PBS, and culture media were changed to DMEM containing 0.1% FBS for 16 h (serum free condition). Then, indicated amounts of FGF2 were used to treat cells for 6 h in DMEM containing 0.1% FBS. TAZ expression levels were analyzed by western blot analysis and quantitative real-time PCR analysis. Stable cell lines Phoenix cells were cultured in 100-mm dishes and transfected using the calcium phosphate method with pSRP (Vector control), pSRP-TAZ (Ti) and pBabe puro-TAZ (TAZ), which have been described previously [2]. Viral supernatants were harvested 24 h after transfection and applied to C3H10T1/2 cells in DMEM containing 10% FBS and 4 μg/ml polybrene. Twenty-four hours later, the cells were incubated with 2μg/ml puromycin to eliminate uninfected cells. After one week of selection, cells were used in differentiation studies. Depleted and overexpressed TAZ levels were verified by western blot analysis with a TAZ antibody. Cell fractionation For the fractionation assay, cells were harvested and incubated in hypotonic buffer (20 mM HEPES, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA and protease inhibitors) for 5 min. After incubation, they were passed through a 20G needle 20 times using a 1 ml syringe and centrifuged at 8000 rpm for 10 min (supernatant-cytosolic fraction, pellet-nucleus fraction), then, the pellet was lysed in RIPA buffer. Fractionated proteins were analyzed by immunoblot analysis. Immunoblot analysis Cells were harvested and lysed in TNE lysis buffer and protease inhibitors. Total cell lysates were denatured in 4× SDS sample buffer and resolved in 8% SDS-PAGE gel. After transfer, the membranes were incubated with TAZ [1] or Runx2 (MBL) antibodies. Immunoprecipitation First, 293T cells were transfected with FLAG tagged TAZ and EF-Runx2 expression vector using calcium phosphate. EF-Runx2 plasmid was a gift from Yoshiaki Ito (Institute of Molecular and Cell

Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA. Real-time PCR reactions were performed using a LightCycler 480 real-Time PCR system (Roche Applied Science). The relative transcript levels were calculated by normalizing the threshold cycle (Ct) values to that of GAPDH. Following primers were used for analyzing the expression levels of osteopontin (OPN), TAZ, Runx2, Dlx5, osteocalcin (OC), osterix (OSX), type collagen alpha 2 (Col1A2) and GAPDH. The forward (F) and reverse (R) primer sequences were as follows: TAZ-F, 5′-GTCACCAACAGTAGCTCAGATC-3′; TAZ-R, 5′-AGTGATTAC AGCCAGGTTAGAAAG-3′; OPN-F, 5′-GATTTGCTTTTGCCTGTTTGG-3′; OPN-R, 5′-TGAGCTGCCAGAATCAGTCACT-3′; Runx2-F, 5′-CGGCCCT CCCTGAACTCT-3′; Runx2-R, 5′-CGGTGGGGAAGACTGTGCCTG-3′; O C-F, 5′-CTGACCTCACAGATGCCAAGC-3′; OC-R, 5′-TGGTCTGATAGCT CGTCACAAG-3′; OSX-F, 5′-CCCCTTGTCGTCATGGTTACAG-3′; OSX-R, 5′-AGAGAAAGCCTTTGCCCACCTA-3′; Col1A2-F, 5′-GTGTTCAAGGTGGC AAGGT-3′; Col1A2-R, 5′-GAGAC CGAA TTCACCAGGAA-3′; Dlx5-F, 5′-CTAGGACTGACGCAAACACAG3′; Dlx5-R, 5′-GGAGCTGGGACTGTGCTCC-3′; GAPDH-F, 5′-CTTCACC ACCTTCTTGATGTC-3′; GAPDH-R, 5′-CCAAAAGGGTCATCATCTCTG-3′. Luciferase reporter activity assay For the luciferase reporter activity assay, 293T cells were plated at a density of 1×105 cells/well in 24-well culture plates. After 24h, 6XOSE2luciferase reporter, Myc tagged Runx2 and FLAG tagged TAZ expression plasmids were transfected using X-tremeGENE 9 DNA Transfection Reagent (Roche Applied Science). 6XOSE2-luciferase reporter and Myc tagged Runx2 plasmids were a gift from R. Derynck (UCSF, San Francisco, CA) and Kwang Youl Lee (Chunnam Univ, Korea), respectively. Six hours after transfection, media containing DMEM, 10% FBS, and FGF2 were exchanged. Then, 24 h later, cells were lysed using passive lysis buffer (Promega) for 20min, and luciferase reporter activity was assessed with reagents of Luciferase Assay System (Promega).

Chromatin immunoprecipitation Cells were crosslinked with 0.75% formaldehyde in PBS and the harvested cells were lysed with ChIP lysis buffer. Cell lysates were incubated for 2h with anti-FLAG M2 agarose beads. DNA-bead complexes were washed three times with 1 ml of wash buffer containing 0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 150 mM NaCl, 20 mM Tris–HCl pH 8.0, and two times with 1 ml of final wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 500 mM NaCl, 20 mM Tris–HCl pH 8.0). After immunoprecipitation, DNA-bead complexes were eluted with 1% SDS, 100mM NaHCO3 and incubated at 65°C, for 4–5h or overnight to reverse formaldehyde cross-link. DNA was purified using the RBC PCR purification kit (Real Biotech Corporation) and used for PCR. PCR primer sequences were as follows: osteocalcin promoter F; 5′-CTGAACTGGG CAAATGAGGACA-3′, osteocalcin promoter R; and 5′-AGGGGATGCTGCC AGGACTAAT-3′.

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Alkaline phosphatase staining and enzyme activity assay

Results

Differentiated osteoblast cells were fixed with 3.7% formaldehyde and stained with 0.1% mg/ml naphthol AS-MX phosphate, 0.5% N, N-dimethylformamide, 2 mM MgCl2, 0.6 mg/ml fast blue BB salt and 0.1 M Tris–HCl (pH 8.5) for 30 min. For the ALP enzyme activity assay, cells were lysed in 25 mM HEPES (pH7.6), 0.1% Triton X-100 and 0.9% NaCl, and incubated with p-nitrophenylphosphate substrate solution for 1 h at 37 °C. And then, 3 M NaOH solution was added to the incubation solution. The ALP activity was measured at 405 nm by microplate reader.

FGF2 increases TAZ expression through induction of TAZ mRNA To study the role of TAZ in FGF2 mediated cellular signaling, the expression of TAZ was analyzed in C3H10T1/2 cells after FGF2 treatment. As shown in Fig. 1A, TAZ is induced by FGF2 in a dose-dependent manner. Its expression was significantly induced 6 h after FGF2 treatment in C3H10T1/2 cells (Fig. 1B). Next, to study the regulatory mechanism for the TAZ expression, TAZ mRNA expression was analyzed. As shown in Fig. 1C, we observed that TAZ mRNA was induced 3 h after FGF2 treatment in C3H10T1/2 cells, which suggests that the induction of TAZ is due to increased expression of TAZ mRNA. To further study the mechanism of increased TAZ mRNA expression after FGF2 treatment, cultured cells were treated with actinomycin D, an RNA synthesis inhibitor. In this experiment, we observed that the inhibitor significantly decreased TAZ protein levels 6 h after treatment (Fig. 1D), indicating again that increased TAZ mRNA

Statistical analysis All data are given as means ± SDs. Results were analyzed for statistically significant differences using Student's t-test, and statistical significance was set at p b 0.05. *; p b 0.05. **; and p b 0.01.

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Fig. 1. FGF2 increases TAZ expression and nuclear localization of TAZ. (A) FGF2 increases TAZ expression. Proliferating C3H10T1/2 cells were serum-deprived for 16 h and incubated with indicated amounts of FGF2. After 24 h, cell lysates were prepared, and the expression of TAZ was analyzed by immunoblot analysis. The level of β-actin was analyzed as a loading control. (B) Serum-deprived C3H10T1/2 cells were incubated with 100 ng/ml FGF2 and, at indicated time points, cell lysates were prepared and the expression of TAZ was analyzed by immunoblot analysis. (C) The mRNA in (B) was prepared, and TAZ mRNA expression was analyzed by quantitative RT-PCR (qRT-PCR) analysis. Their relative expression was calculated after normalization to the GAPDH level. (D) Serum-deprived C3H10T1/2 cells were incubated with 100 ng/ml FGF2 in the absence or presence of actinomycin D (0.2 μg/ml) and, after 6 h, the cell lysates were prepared and levels of TAZ were analyzed by immunoblot analysis. (E) FGF2 increases nuclear localization of TAZ. Serum-deprived C3H10T1/2 cells were incubated with 100 ng/ml of FGF2 and 6 h after, the cells were fixed and cellular location of TAZ was analyzed by immunocytochemistry. FITC-conjugated secondary antibody was used for green fluorescence signal. DAPI staining indicates nuclei of cells. (F) The cellular distribution of TAZ proteins in (E) was quantitatively analyzed according to whether it was higher in the nucleus (N N C), higher in the cytoplasm (N b C) or evenly distributed between the nucleus and cytoplasm (N = C). The percentage of cells was scored after observing cells in five different fields of (E). (G) C3H10T1/2 cells were treated with 100 ng/ml FGF2, and the cell lysates were fractionated into cytosolic and nuclear extracts according to Experimental Methods. The level of TAZ was analyzed by immunoblot analysis. Lamin B1 was used as a marker of nuclear proteins, and α-tubulin was used as a marker of cytosol proteins. Nuc and Cyt indicate nuclear and cytosolic fraction, respectively.

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It was shown that FGF2 stimulates osteoblast differentiation of mesenchymal cells [18]. TAZ also stimulates osteoblast differentiation and is suggested as an important mediator of cell signaling [2,32]. Thus, to study the role of TAZ in FGF2-mediated osteoblast differentiation, we analyzed the expression of TAZ during this process. C3H10T1/2 cells were cultivated in the absence or presence of FGF2 in osteogenic differentiation media. Similar to previous observations, FGF2 increased osteoblast differentiation as evidenced by increased alkaline phosphatase activity and osteogenic marker gene expression (Fig. 2). Our previous

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FGF2 stimulates the interaction of TAZ and Runx2 and stimulates Runx2-mediated gene transcription TAZ stimulates Runx2-mediated gene transcription during osteoblast differentiation [2]. We thus investigated the effects of FGF2 on the stimulation of Runx2-mediated gene transcription by TAZ. Ectopic Runx2 expression in 293T cells increased reporter gene activity, which

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FGF2 increases TAZ expression during osteoblast differentiation of C3H10T1/2 cells

study showed that overexpression of TAZ stimulates osteoblast differentiation [2]. Thus, these results suggested that increased expression of TAZ may play a role in FGF2-mediated osteogenic stimulation in C3H10T1/2 cells. To understand whether TAZ is important for FGF2-mediated osteogenic stimulation, TAZ-depleted cells were made using TAZ shRNA-producing retrovirus. As shown in Fig. 3A, TAZ-depleted cells showed decreased levels of TAZ compared to control cells. The cells were incubated with osteogenic differentiation media in the presence of FGF2. The potential of osteogenic differentiation was assessed with the control and TAZ-depleted cells. Osteogenic differentiation of control cells was significantly increased in the presence of FGF2, but TAZ-depleted cells showed decreased osteogenic potential in the presence of FGF2, as indicated by alkaline phosphatase activity (Figs. 3B and C). Additionally, decrease in the expression of osteogenic marker genes was observed in FGF2-treated TAZ-depleted cells (Fig. 3D). The results show that signal activation in FGF2 requires TAZ. These results suggest that TAZ is an important component of FGF2mediated osteogenic gene activation.

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precedes protein up-regulation. Taken together, the results show that FGF2 significantly induces TAZ expression at the transcriptional level. Next, to analyze the cellular distribution of TAZ after FGF2 treatment, immunocytochemical analysis was assessed. In Fig. 1E, TAZ expression and localization were revealed by green fluorescence signal. Compared to control cells, FGF2-treated cells showed increased fluorescence signal and increased nuclear localization of TAZ. Approximately 20% of cells showed increased nuclear localization of TAZ after FGF2 treatment (Fig. 1F). To verify the results biochemically, FGF2-treated cells were fractionated into either cytosol or nuclei, and the amount of TAZ was analyzed by immunoblot analysis (Fig. 1G). Indeed, TAZ was significantly expressed in both cytosol and nuclear compartments, which suggests that FGF2 increases TAZ expression and also facilitates nuclear localization.

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Fig. 2. FGF2 increases TAZ mRNA during osteogenic differentiation of C3H10T1/2 cells and stimulates osteogenic differentiation. (A) FGF2 stimulates osteogenic differentiation of C3H10T1/ 2 cells. C3H10T1/2 cells were grown to confluence and differentiated into osteoblast in the presence of the indicated amounts of FGF2 for 8 days. Alkaline phosphatase activity in the differentiated cells was stained according to experimental method. (B) For quantifying alkaline phosphatase activity in (A), enzymatic assay was assessed. (C) Total RNA in (A) was harvested at 2 days after differentiation and subjected to reverse transcription and qRT-PCR analysis. The relative expression levels of osteopontin (OPN), Runx2, Dlx5, and TAZ were determined after normalization to the GAPDH level. (D) 14 days after differentiation, total RNA was harvested and analyzed by qRT-PCR. mRNA expression of type collagen alpha 2 (Col1A2), osteocalcin (OC) and osterix (OSX) was calculated after normalization to the GAPDH level. * indicates p-value; * for p b 0.05, ** for p b 0.01, t-test.

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Fig. 3. TAZ knockdown decreases FGF2 induced osteogenic differentiation. (A) Depletion of TAZ in C3H10T1/2 cells was assessed with retrovirus producing a TAZ shRNA. The expression of TAZ was analyzed by immunoblot analysis. Con and Ti indicate vector control cells and TAZ depleted cells, respectively. (B) The Con and Ti cells were differentiated into osteoblasts in the presence of 20 ng/ml FGF2 for 6 days. Alkaline phosphatase activity in the differentiated cells was stained according to experimental method. (C) The relative alkaline phosphatase activity in (B) was shown. (D) Total RNAs were harvested from the cells in (B) and subjected to reverse transcription and qRT-PCR analysis. The relative expression levels of Runx2, Dlx5, and OPN were determined after normalization to the GAPDH level. ** for p b 0.01, t-test.

contains 6 copy of Runx2 binding site. Further increased activity was observed in the presence of FGF2 (Fig. 4A), indicating that FGF2 stimulates Runx2-mediated gene transcription. At the same condition, the effect of TAZ was analyzed. Ectopic introduction of TAZ increased Runx2-mediated reporter activity and increased TAZ level showed further increased reporter activity (Fig. 4A). In the presence of FGF2, the TAZ and Runx2-mediated reporter activity was increased in a dose-dependent manner (Fig. 4A). Thus, the results showed that TAZ could intensify the osteogenic stimulation of FGF2 via activation of Runx2-mediated gene transcription. It is shown that TAZ interacts with Runx2 binding site of endogenous osteocalcin promoter for stimulation of osteocalcin gene expression [2]. Next, to study whether TAZ stimulates endogenous osteocalcin gene expression in the presence of FGF2, chromatin immunoprecipitation analysis was assessed with FLAG-tagged TAZ-overexpressing cells. As shown in Fig. 4B, FLAG-TAZ was recruited into the Runx2-binding site of the osteocalcin gene promoter, and about 34% increase in the recruitment of FLAG-TAZ was observed in the presence of FGF2. The results showed that FGF2 stimulates osteocalcin gene expression through the recruitment of TAZ into the osteocalcin promoter in differentiating cells. In Figs. 1E and F, we observed that FGF2 increases nuclear localization of TAZ. This result suggested that increased nuclear localization of TAZ can activate the FGF2 target genes through facilitation of binding with transcription factors. Thus, we tested whether FGF2 facilitates the interaction between TAZ and Runx2. For the experiment, TAZ and Runx2 expression plasmids were introduced into 293T cells and the cells were incubated in the presence or absence of FGF2. Interestingly, the physical interaction was increased in the presence of FGF2 by about 1.8 fold, suggesting that increased nuclear localization of TAZ by FGF2 facilitates the physical interaction between TAZ and Runx2 (Fig. 4C). Taken

together, the results suggest that the FGF2 signal can stimulate Runx2mediated gene transcription through TAZ. MAPK signaling is important for FGF2-mediated TAZ expression and osteogenic stimulation FGF2 stimulates several signaling pathways, including phosphoinositol 3-kinase (PI3K) and Ras/mitogen activated protein kinases (MAPK) [33]. In osteoblast differentiation, FGF2 stimulates ERK and protein kinase C (PKC) [34–36]. To study the induction mechanism of TAZ after FGF2 treatment, inhibitors of the kinases were treated in the presence of FGF2. Interestingly, U0126, a MAPK kinase (MEK) inhibitor, significantly blocked the expression of TAZ mRNA and protein 6 h after FGF2 treatment (Figs. 5A and B), and further significant suppression was observed until 24 h after FGF2 treatment (Fig. 5C), suggesting that the ERK/MAPK pathway plays a critical role in FGF2mediated TAZ expression. Gö6976, a calcium-dependent PKC inhibitor, had no effect on TAZ expression and Gö6983, a broad inhibitor of PKC, slightly repressed TAZ expression. To study the role of ERK activation in FGF2 mediated osteogenic stimulation, cells were co-treated with U0126 and FGF2 during osteogenic differentiation. As shown in Figs. 6A and B, FGF2-mediated osteogenic stimulation was inhibited by U0126, as indicated by decreased alkaline phosphatase activity, which suggests that FGF2-mediated osteogenic stimulation requires the activation of ERK signaling pathway. The osteogenic genes were also decreased in the presence of U0126 (Fig. 6C). To further study the increased TAZ expression is important for FGF2mediated osteogenic gene transcription, we investigated whether TAZ overexpression can rescue the expression of osteoblast genes in U0126 treated cells. In Fig. 7A, increased level of TAZ was observed after TAZ overexpression. In Fig. 7B, TAZ-overexpressed cells showed

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Fig. 4. FGF2 stimulates Runx2-mediated gene transcription and increases the physical interaction between TAZ and Runx2. (A) 293T cells were co-transfected with the 6 copy of Runx2 binding site containing a luciferase reporter construct (6XOSE2-luc), Renilla and Runx2 expression plasmids with the indicated amounts of TAZ expression plasmids. Twenty four hours after transfection, luciferase activity was analyzed. * for p b 0.05, t-test. (B) Chromatin immunoprecipitation of TAZ with the endogenous osteocalcin promoter in response to FGF2. Stable FLAG-tagged TAZ-expressing C3H10T1/2 cells (T) were treated for 2 days with osteogenic differentiation media in the presence of 20 ng/ml FGF2, and the immunoprecipitates of anti-FLAG antibodies were analyzed for osteocalcin promoter occupancy by PCR (Up). Bp indicates control cells. For quantifying recruitment of TAZ into osteocalcin gene promoter, qRT-PCR was assessed (Bottom). The numbers indicate the relative fold induction. ** for p b 0.01, t-test. (C) Runx2 was ectopically expressed in 293T cells along with FLAG-tagged TAZ, and the cells were treated with 20 ng/ml FGF2 for 24 h. Whole cell lysates were harvested and incubated with FLAG-M2 agarose beads to immunoprecipitate FLAG-TAZ bound proteins. The immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblotting using an anti-Runx2 or anti-TAZ antibody. Whole cell lysates were separated by SDS-PAGE as an input control.

significantly increased osteogenic marker gene expression and recovered the gene expression which was suppressed by U0126. Thus, the results suggest that TAZ expression via ERK activation plays an important role in FGF2-mediated osteogenic differentiation. Taken together, the results show that FGF2 significantly increases TAZ expression through induction of TAZ mRNA, facilitates the interaction between TAZ and Runx2 and activates Runx2-mediated gene expression in osteogenesis. Thus, we propose that TAZ is a mediator of FGF2 signaling. Discussion In this study, we show that TAZ plays an important role in FGF2 mediated osteogenic gene transcription of C3H10T1/2 cells with the following evidences. First, FGF2 induces TAZ by increasing the expression of TAZ mRNA and nuclear localization of TAZ for TAZ activity. Second, depletion of TAZ decreases FGF2 mediated osteogenic differentiation. Third, TAZ expression is altered by ERK activity induced by FGF2. We and others observed that ERK activation is important for the TAZ expression because inhibition of ERK suppresses TAZ expression [37–39]. These results suggest that ERK activation signals facilitate the function of TAZ. Indeed, it was shown that ERK activation regulates mesenchymal cell differentiation. FGF2 activates the ERK signal, phosphorylates Runx2 and enhances Runx2-mediated gene transcription through its receptor,

FGFR [34,40]. Increased skeletal size and calvarial mineralization were observed in mice that have constitutively active MAPK/ERK in their osteoblasts [41]. FGFR2 promotes osteogenic differentiation in mesenchymal cells through ERK and PKC activation [36]. In the present study, FGF2 signal increased a transcriptional co-regulator, TAZ expression for osteogenic stimulation (Figs. 2 and 3). Thus, increased TAZ and Runx2 stimulated FGF2-mediated osteogenic differentiation. Indeed, increased level of TAZ was observed in the Runx2 binding site of osteocalcin promoter from ChIP analysis (Fig. 4). FGF2 increases TAZ and Runx2 interaction, facilitating Runx2mediated gene transcription (Fig. 4). At this moment, the mechanisms for the increased interaction are not clearly addressed, but increased nuclear localization of TAZ might provide locally increased concentration of TAZ and Runx2 for the interaction. Another possibility is that ERK induced phosphorylation of TAZ increases the interaction with Runx2. Phosphorylation of TAZ by ERK and its functional role should be addressed to understand the mechanisms. It was shown that PKCδ activation by FGF2 induces Runx2 expression and Runx2 mediated gene transcription [42]. To analyze the role of PKC in TAZ expression, PKC inhibitors were used. In Figs. 5A and B, though a broad PKC inhibitor, Gö6976, slightly decreased TAZ expression, but significant reduction of TAZ was not observed after the treatment of PKC inhibitors compare to the effect of MEK inhibitor (Fig. 5B). Thus, it seems that FGF2 induced PKCδ activation is not a major role in TAZ expression.

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Fig. 5. FGF2 induced MAPK/ERK plays an important role in TAZ expression. (A) Serum-deprived C3H10T1/2 cells were incubated with 10 μM of U0126, 2 μM of Gö 6983, and 2 μM of Gö 6976 in the absence or presence of 100 ng/ml of FGF2. After 6 h, total RNAs were prepared, and the level of TAZ mRNA was analyzed by qRT-PCR. Their relative expression was calculated after normalization to the GAPDH level. ** for p b 0.01, t-test. (B) The cell lysates in (A) were prepared, and TAZ expression was analyzed by immunoblot analysis. The level of β-actin was analyzed as a loading control. Inhibitor indicates U0126 or Gö 6983, or Gö6976. (C) Serum-deprived C3H10T1/2 cells were incubated with 10 μM of U0126 in the absence or presence of FGF2, and then the cell lysates were prepared at the indicated time points. TAZ expression was analyzed by immunoblot analysis.

To understand the transcriptional regulatory mechanism, we tried to find transcriptional regulatory regions within the promoter of TAZ. A 1.8 kilobase pair of TAZ promoter regions was joined with luciferase reporter constructs. Using this construct, the transcriptional activity of luciferase was analyzed in the presence of FGF2. However, we could not see a significant increase in luciferase activity with this promoter, which suggests that further upstream or downstream enhancer regions are required for TAZ transcriptional regulation (data not shown). It was previously reported that FGF2 down-regulates TAZ in MC3T3E1 cells, though its transcription was significantly induced after FGF2 treatment [43]. In our experiments, we observed that FGF2 stimulates the induction of both TAZ mRNA and protein in C3H10T1/2 cells. Currently, it is unclear why different expression patterns of TAZ are observed, though transcriptional activation occurred in two different cell lines after FGF2 treatment. Nevertheless, the different cellular components including posttranslational processing proteins in the two cell lines may be responsible for this difference. Further study should be addressed to clarify these mechanisms. Recent studies have shown that TAZ is phosphorylated by Casein Kinase 1 and Lats kinase through the Hippo pathway, and this triggers ubiquitination of TAZ that is recognized by β-TrCP E3 ubiquitin ligase [11]. The phosphorylation of TAZ at serine 89 by Lats kinase induces 14-3-3 binding and cytosolic sequestration [11]. Therefore, the phosphorylation status of TAZ is important for protein stability and cellular localization. Interestingly, we observed that significant amounts

of TAZ were localized in the nucleus after FGF2 treatment (Fig. 1), though the level of TAZ in the cytosol was also increased, thus suggesting that FGF-2 induced TAZ may escape 14-3-3 binding and cytosolic sequestration. Thus, it is intriguing to study whether FGF2 signals block the Hippo pathway to facilitate nuclear localization of TAZ. YAP, a paralog of TAZ, requires for self-renewal of ESC [44–46], but we did not see induction of YAP after FGF2 treatment (data not shown). The results indicate that there is a unique activation pathway for TAZ and YAP in response to certain extracellular signals, even though both are well-known Hippo signal effectors. At this point, it is notable that TAZ, not YAP, is stabilized by Wnt signal [47]. In embryonic stem cells, it was shown that FGF2 is a key signaling factor for stem cell self-renewal; FGF2 promotes self-renewal of hESCs by modulating the expression of TGFβ ligands and inhibition of FGF2 induced the neuroectodermal fate determinant PAX6 in hESC [48,49]. Also Fgf2 inhibits formation of early neural cells by epiblast intermediates [50]. However detailed mechanism was not clearly addressed. Recently, Varelas et al. show that TAZ is required for self-renewal of human embryonic stem cells through TGFβ signaling, and loss of TAZ leads to differentiation into a neuroectoderm lineage [9]. Thus, though further study is required, our results suggest that TAZ might regulate FGF2mediated embryonic stem cell self-renewal and is a common mediator for FGF2 signaling. In summary, our study demonstrates that FGF2 increases TAZ expression, and the induced TAZ stimulates osteogenic gene transcription

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Fig. 6. ERK inhibition suppresses FGF2 induced osteogenic differentiation. (A) U0126, a MEK inhibitor, inhibited FGF2-mediated osteogenic differentiation. C3H10T1/2 cells were incubated in osteogenic differentiation media, and 20 ng/ml FGF2 was added into the osteogenic differentiation medium with or without 10 μM of U0126. After 6 days of differentiation, the cells were stained for alkaline phosphatase activity. For quantifying alkaline phosphatase activity, enzymatic assay was assessed. (B) For quantifying alkaline phosphatase activity in (A), enzymatic assay was assessed. (C) Total RNA in (A) was prepared, and osteopontin (OPN), Runx2, and Dlx5 expression levels were analyzed by qRT-PCR analysis. Their relative expression was calculated after normalization to the GAPDH level. ** for p b 0.01, t-test.

through the activation of Runx2. Thus, we suggest that TAZ is a novel mediator in FGF2 induced osteogenic differentiation.

Acknowledgments This work was supported by the grants of the National Research Foundation (2011-0022926 and 2009-0001197) and the Korea Healthcare Technology R&D project, Ministry for Health & Welfare Republic of Korea (A120349).

Conflict of interest The authors declare that they have no conflicts of interest.

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Fig. 7. TAZ overexpression recovers osteogenic gene transcription which was suppressed by U0126. (A) The expression of TAZ was analyzed by immunoblot analysis. Con and TAZ indicate vector control cells and TAZ overexpressed cells, respectively. (B) Total RNAs were harvested from the cells in (A) and OPN, Dlx5, Runx2, TAZ and Col1A2 expression levels were analyzed by qRT-PCR. Their relative expression was determined after normalization to the GAPDH level. ** for p b 0.01, t-test.

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