CCN2/CTGF Binds to Fibroblast Growth Factor Receptor 2 And

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FEBS Letters 586 (2012) 4270–4275

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CCN2/CTGF binds to ﬁbroblast growth factor receptor 2 and modulates its signaling ⇑ Eriko Aoyama a, Satoshi Kubota b, Masaharu Takigawa a,b,

a Biodental Research Center, Dental School, Okayama University, Okayama, Japan b Department of Biochemistry and Molecular Dentistry, Okayama University, Graduate School of Medicine, Dentistry, and Pharmaceutical Science, Okayama, Japan

article info abstract

Article history: CCN2 plays a critical role in the development of mesenchymal tissues such as cartilage and bone, and Received 26 July 2012 the binding of CCN2 to various cytokines and receptors regulates their signaling. By screening a pro- Revised 28 September 2012 tein array, we found that CCN2 could bind to fibroblast growth factor receptors (FGFRs) 2 and 3, with Accepted 23 October 2012 a higher affinity toward FGFR2. We ascertained that FGFR2 bound to CCN2 and that the binding of Available online 6 November 2012 FGFR2 to FGF2 and FGF4 was enhanced by CCN2. CCN2 and FGF2 had a collaborative effect on the Edited by Zhijie Chang phosphorylation of ERK and the differentiation of osteoblastic cells. The present results indicate the biological significance of the binding of CCN2 to FGFR2 in bone metabolism.

Keywords: CCN2 Structured summary of protein interactions: FGFR2 FGFR2 binds to CCN2 by protein array (View interaction) FGF2 FGFR1OP binds to CCN2 by protein array (View interaction) Osteoblast FGFR3 binds to CCN2 by protein array (View interaction) Differentiation Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction CCN2 as a probe and found fibroblast growth factor (FGF) receptor 2 (FGFR2) to be among the candidates as partners of CCN2. CCN2 is a member of the CCN family of proteins [1,2], and FGFR2 is a member of the FGF receptor family and is expressed CCN2-null mice show remarkable abnormalities in skeletal devel- in condensed mesenchyme and later in sites of endochondral and opment, as represented by bent costal cartilage and long bones intramembranous ossification [8]. In vitro assays show that FGFR2 with impaired endochondral ossification [3]. Also, in vitro studies regulates certain steps of skeletal development [9–11]. In humans, have shown that CCN2 is a stimulator of both the proliferation mutations of FGFR2 cause endochondral diseases such as Apert and and differentiation of chondrocytes, osteoblasts, vascular endothe- Crouzon syndromes [12–14]. FGFR2-deficient mice die at 10–11 lial cells, and mesenchymal stem cells [1,2]. These findings indicate days of gestation because of failure in the formation of their pla- that CCN2 plays a critical role in the metabolism of cartilage and in centa and limb buds [15]. These mice, deficient in FGFR2 specifi- bone formation. cally in the condensed mesenchyme, indicate that FGFR2 is Nevertheless, the molecular behavior determining the effect of essential for the normal proliferation of osteoblasts and osteogenic CCN2 has not been totally elucidated. CCN2 is known as a binding gene expression during postnatal bone development [16]. partner of many types of cytokines and extracellular matrix pro- Based on this background, we subsequently confirmed the binding teins, such as bone morphogenetic protein (BMP) 2, fibronectin, of CCN2 to FGFR2 and assessed its effects on FGFR2 signaling in and aggrecan [4–6]. Also, CCN2 was reported to be a modulator of osteoblastic cell line MC3T3-E1. Finally, we found that CCN2 enhanced signaling by BMP2, BMP4, and TGF-b [4,7]. Therefore, we assumed the signaling effected by FGF2-induced activation of extracellular reg- that proteins binding to CCN2 define the various effects of CCN2 ulated kinase (ERK), which action resulted in the collaborative in certain circumstances. Searching for new factors binding to enhancement of the differentiation of the osteoblastic cells. CCN2, we performed a protein-array experiment with recombinant 2. Materials and methods

2.1. Screening of CCN2-binding proteins ⇑ Corresponding author. Address: Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Phar- Proteins binding to CCN2 were screened with a ProtoArrayÒ maceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan. Fax: Human Protein Microarray ver. 4 (Invitrogen, Carlsbad, CA, USA). +81 86 235 6649. E-mail address: [email protected] (M. Takigawa). Experiments were professionally performed by Filgen Incorporated

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.10.038 E. Aoyama et al. / FEBS Letters 586 (2012) 4270–4275 4271

(Nagoya, Japan) by using an oligohistidine-tagged recombinant hu- 2.7. Quantitative real-time PCR analysis man CCN2 (BioVendor Laboratory Medicine, Brno, Czech Republic) as a probe. Total RNA was isolated by using an RNeasy Mini Kit (Qiagen, Hilden, Germany) and was reverse-transcribed to cDNA by using 2.2. Solid-phase binding assay a Takara RNA PCR kit (AMV), Version 3.0 (Takara Shuzo, Tokyo, Japan). Ampliﬁcation reactions were performed with a SYBRÒ For the examination of the direct interaction between CCN2 and Green Real-time PCR Master Mix (Toyobo; Tokyo, Japan) by using FGFR-Fc, a solid-phase binding assay was carried out, as described StepOne™ Software v2.1 (Applied Biosystems, Foster City, CA, previously [5,6]. Brieﬂy, wells of ELISA plates previously coated USA). The nucleotide sequences of the primers used were as 0 0 with 1 lg/ml of rCCN2 (BioVendor Laboratory Medicine) or bovine follow: 5 -GCCAAAAGGGTCATCATCTC-3 (forward) and reverse 0 0 0 serum albumin (BSA) were blocked with blocking buffer. Diluted 5 -GTCT TCTGGGTGGCAGTGAT-3 (reverse) for Gapdh;5-CTCACA- 0 0 recombinant human FGFR2a (IIIc)/Fc (FGFR2-Fc) or recombinant GATGCCAAGCCCA-3 (forward) and reverse 5 -CCAAGGTAGCGCCG- 0 human FGFR3a (IIIc)/Fc (FGFR3-Fc; R&D Systems, Minneapolis, GAG TCT-3 (reverse) for Osteocalcin, as reported earlier [17,23]. MN) was then added to the wells, and incubation was conducted for 2 h at 37 °C. Thereafter, the wells were washed and incubated 2.8. Statistical analysis with horseradish peroxidase (HRP)-conjugated anti-human Fc antibody (Sigma Aldrich, St Louis, MO); and the bound HRP was Statistical analyses were performed by using Student’s t-test, if ⁄ ⁄⁄ then monitored by using 3,3,5,5-tetramethylbenzidine (TMB) per- necessary. p < 0.05, and p < 0.01, as compared with the values for oxidase substrate (Sigma–Aldrich). The effect of CCN2 on the bind- control wells. ing of FGFR2-Fc to FGF2 or FGF4 was assayed on ELISA plates coated with 1 lg/ml of FGF2 or FGF4 (R&D Systems). FGFR2-Fc 3. Results was incubated with CCN2 for 30 min before being added to the wells, and detection of signals was performed as described above. 3.1. FGFR2 and 3 as candidate CCN2-binding proteins

First, we used a protein array to screen for proteins binding to 2.3. SPR (surface plasmon resonance) measurements CCN2. Fig. 1 shows the CCN2 binding profile of the FGF-associated molecules included in the protein array. Among the top 3 candi- For the calculation of dissociation constant (Kd) values, SPR dates, we focused on FGFR2 and 3, because their mutants are measurements were performed, as described previously [4,6]. known to result in genetic disorders of bone formation, in which Briefly, FGFR2-Fc was diluted to concentrations of 1.6, 8, 40, 200, CCN2 plays a critical role. Thus, these 2 molecules were forwarded and 1000 nM with HBS-EP (GE Healthcare UK, Ltd, Little Chalfout, for subsequent investigation. UK, Little Chalfout, UK); and each was reacted with rCCN2 immobilized on CM5 sensor chips (GE Healthcare) according to 3.2. Specific binding of CCN2 to FGFR2 and FGFR3 the standard amine coupling procedure recommended by the manufacturer. Binding and dissociation of these molecules were To confirm the binding of CCN2 to FGFR2 and 3, we initially em- monitored with a Biacore X (GE Healthcare) and the data were fit- ployed a solid-phase binding assay, which results are displayed in ted using the BIAevaluation software version 4.1 (GE Healthcare) Fig. 2(A) and (B). Though FGFR2-Fc and FGFR3-Fc increasingly bound with the single cycle kinetics support package (GE Healthcare). to CCN2 depending upon the doses, the bound FGFR2-Fc is more The sensorgrams were corrected by subtracting the signal of the than twice as much as FGFR3-Fc in each dose. Both FGFR2 and 3 reference cell and were forwarded for Kd computation. bound to CCN2 hardly bound to wells coated with BSA, which protein was used as a negative control for CCN2 protein. Similarly, 2.4. Cell culture human IgG used as a negative control for FGFR fused to human IgG Fc domain did not bind to CCN2 at all (data not shown). These results Cells of the mouse osteoblastic cell line MC3T3-E1 were mean that the binding of FGFR2 and 3 to CCN2 was specific and the cultured in a humidified incubator (5% CO2 in air) at 37 °C and binding of FGFR2 appeared to be stronger than that of FGFR3. On the maintained in 10% FCS/DMEM supplemented with 50 lg/ml strep- basis of these results, we decided to further investigate the mode tomycin and 100 units/ml penicillin. and functional significance of the binding of FGFR2 to CCN2. First we analyzed the affinity of CCN2 to FGFR2 by performing 2.5. Immunoprecipitation and western blotting SPR analysis (Fig. 2(C)). SPR assay indicated the Kd value for the binding of CCN2 to FGFR2 to be 7.71 nM. According to previous The cell lysate mixed with oligohistidine-tagged rCCN2 was studies, the Kd values for FGF1, 2 and 4, which are known as li- incubated for 2 h at 4 °C and reacted with Protein G Sepharose 4 gands of FGFR2, were reported to be 0.791 nM, 0.490 nM, and Fast Flow beads (GE Healthcare) having adsorbed anti-FGFR2 (San- 0.412 nM, respectively [17]. These data taken together indicate ta Cruz Biotechnology, Santa Cruz, CA) or control goat IgG for 1 h at that the binding affinity of CCN2 for FGFR2 was not equivalent, 4 °C. The precipitated protein was assayed by western blotting but at least comparable, to that of known FGFR2 ligands. analysis using rabbit anti-6-His Antibody Affinity Purified (Bethyl Next, the binding of FGFR2 in vivo to CCN2 was ascertained by Laboratories, Inc., Montgomery, TX, USA). co-immunoprecipitation of FGFR2 on MC3T3-E1 cells with rCCN2 (Fig. 2(D)). Much more CCN2 protein was detected in the immuno- 2.6. Evaluation of ERK protein phosphorylation precipitant with anti-FGFR2 Ab than with control Ab. These data represent CCN2 bound to FGFR2 expressed on MC3T3-E1. Cells lysates were collected in RIPA buffer after stimulation of the cells with FGF2, rCCN2 or FGFR2-Fc. Western blotting was car- 3.3. Effect of CCN2–FGFR2 interaction on the binding of FGFs to FGFR2 ried out with Anti-ACTIVEÒ MAPK pAb, Rabbit pTEpY (Promega Corporation, Madison, Wl) or p44/42 MAPK antibody (Cell Signal- The major function of FGFR2 is to transmit the signals from its ing Technology, Danvers, MA). The density of each band was quan- authentic ligands. Therefore, we verified whether or not this bind- tified by Multi Gauge Ver3.0 (FUJIFILM, Japan). ing of CCN2 to FGFR2 could influence the binding of FGFR2 to its 4272 E. Aoyama et al. / FEBS Letters 586 (2012) 4270–4275

Fig. 1. Protein array analysis suggesting strong interaction of CCN2 with FGFR2 and FGFR3. The ProtoArrayÒ Human protein microarray was probed with His-tagged CCN2, and FGF and associated factors were extracted from the results and ranked by signal intensities in descending order.

representative ligands, FGF2 and FGF4, which are also essential for protein. These results suggest the requirement of FGF signaling normal bone formation (Fig. 3(A) and (B)). As a result, we found that for the promotion of osteoblastic differentiation by CCN2. the binding of FGFR2 to FGF4 was less strong than that to FGF2. However, in the case of both FGF2 and FGF4, the presence of CCN2 dose-dependently enhanced the binding of FGFR2 to these 4. Discussion FGFs. This report demonstrates for the first time the novel interac- 3.4. Functional significance of the CCN2-FGFR2 interaction tion between human CCN2 and FGFR2. We confirmed that the binding of FGFR2 to CCN2 was comparably efficient as that to Finally, we examined the effect of CCN2 on the intracellular sig- its known ligands by performing the solid-phase binding assay naling after FGFR2 in an osteoblastic cell line, MC3T3-E1. Fig. 4 and SPR analysis. Interestingly, the presence of CCN2 enhanced shows that CCN2 enhanced the activation of ERK triggered by the binding of FGFR2 to its authentic ligands, FGF2 and FGF4, FGF2. The activation of ERK induced by FGF4 (10 ng/ml) was in a CCN2 dose-dependent manner. Moreover, intracellular FGF weaker than that by FGF2, but was also increased by CCN2 (data signaling was enhanced in the presence of CCN2. A previous re- not shown). The activation of ERK was suppressed by the addition port showed that CCN2 could also bind to FGF2 [22]. These find- of a fusion protein of the extracellular domain of FGFR2 and immu- ings together indicate that CCN2 may enhance the cross-linking noglobulin FC region, indicating that FGF2 and CCN2 induced this of FGFR2 and these FGFs via forming tri-molecular complexes. phosphorylation of ERK essentially via signaling from FGFR2. Furthermore, according to another report, CCN2 binds to heparin According to previous reports, FGFR2 signaling increases the [23], which is known as a cofactor for the binding of FGFs to their expression of osteoblastic markers in MC3T3-E1 cells [8,10,11]; receptors. Therefore, the fully functional FGFR2 complex that however, the effect of FGF2 on the differentiation of osteoblasts accepts FGF signals at best efficiency on the cell surface may still remains controversial [18–21]. To gain new insight into this is- be composed of CCN2, proteoglycan, and FGFR2. Indeed, we sue, we evaluated the combinational effect of FGF2 and CCN2 on assessed the possibility that CCN2 acted as a co-receptor like the expression of osteocalcin, a typical osteoblastic marker. CCN2 heparin in vitro, but CCN2 did not increase or decrease the dimer- was added with or without FGF2 to subconfluent cultures of ization of FGFR2 in the presence or absence of heparin (data not MC3T3-E1 cells. After 7 days, the expression of osteocalcin was as- shown). sayed as described in Section 2. As shown in Fig. 5, CCN2 and FGF2 According to previous studies, the signaling via FGFR2 enhances cooperatively enhanced the expression of the osteocalcin gene. Of osteoblastic differentiation of pre-osteoblast cell lines or bone note, the enhancement was suppressed in presence of FGFR2-Fc marrow stromal cells [9,11]. However, although FGF2 is a potent E. Aoyama et al. / FEBS Letters 586 (2012) 4270–4275 4273

Fig. 2. Binding of CCN2 to FGFR2 conﬁrmed by results of solid-phase binding assay and SPR analysis. Solid-phase binding assay showed that FGFR2-Fc (A) and FGFR3-Fc (B) bound to CCN2 in a dose dependent manner. FGFR-Fc protein was incubated on ELISA plates pre-coated with CCN2 or BSA (a negative control for CCN2 protein). Bound FGFR2- Fc or FGFR3-Fc was detected with anti-Fc antibody-HRP and HRP substrate (TMB). The amount of FGFR-Fc binding to wells was represented as the absorbance (450 nm) in each well corrected by subtracting that of blank wells. Data are representative of 2 independent experiments. The data are mean values ±S.D. of 3 wells. (C) The indicated dilutions of FGFR2-Fc were injected onto a CCN2 -immobilized Biacore sensor chip. The Kd value was calculated by using BIAevaluation software, as described in Section 2. (D) Recombinant CCN2 bound to FGFR2 expressed on MC3T3-E1 cells. MC3T3-E1 cells suspended in with 10% FCS/aMEM were plated at the density of 10 105/dish per 6-cm dish and cultured in the medium for a week. The media was changed at day 3. The cell lysate (740 lg protein) mixed with oligohistidine-tagged rCCN2 (2.5 lg) was incubated for 2 h and precipitated with 50 ll protein G Sepharose gel having adsorbed anti-FGFR2 Ab or control goat IgG. The proteins in the gel and rCCN2 (2 lg/lane) were subjected to western blotting analysis using anti-His Ab. Positions of molecular weight standards are indicated at the right. Arrowhead indicates the CCN2 protein.

ligand of FGFR2, its role in the differentiation of osteoblasts is not in the reports using other cell lines [20,24]. We also tried a few perfectly clear. In this study we demonstrated that CCN2 and FGF2 doses up to 2–5 times higher than the optimal ones, but no stron- cooperatively enhanced the osteoblastic differentiation of MC3T3- ger effects were observed (data not shown). Debiais et al. described E1. However CCN2 or FGF2 alone did not necessarily enhance the that the positive or negative effects of FGF2 on the mineralization osteoblastic differentiation under the experimental conditions em- and expression of osteocalcin depend on the stage of cell differen- ployed, though both of them were used at optimal doses described tiation [20]. At the early stage, FGF2 decreases this phenotype; 4274 E. Aoyama et al. / FEBS Letters 586 (2012) 4270–4275

Fig. 3. CCN2 enhances the binding of FGFR2-Fc with FGF2. FGFR-Fc protein and CCN2 were incubated on ELISA plates pre-coated with FGF2 (A) or FGF4 (B). The bound FGFR- Fc was detected with anti-Fc antibody-HRP and TMB substrate. Optical absorbance in each well was measured and corrected by subtracting that of blank wells. Data are representative of 2 independent experiments and are the mean values ±S.D. of 3 wells. ⁄p < 0.05 and ⁄⁄p < 0.01, as compared with the value for the control wells incubated with FGFR2-Fc alone.

via FGFR2, which increased the expression of osteocalcin. Never- theless, in this experiment, FGF2 alone did not clearly influence the expression of this osteoblastic marker. This result appears con- tradictory to the study cited above, but 2 possibilities that may ac- count for this discrepancy can be presented. Firstly, MC3T3-E1 cells under our conditions might not have expressed enough FGFR2 to fully respond to FGF2. Secondly, in the experiments performed in the previous study, CCN2 could be already present at a sufficient amount to support the signaling via FGFR2. In fact, a recent report showed that CCN2 gene expression in osteoblasts reaches its peak after 14 days following the induction of differentiation in vitro [25,26]. Another study using a different experimental system also showed that CCN2 displays a cooperative effect with FGF2 [26], but the machinery at work was not elucidated. Based on the results obtained in this study, we propose the possible machinery yielding the effect of CCN2 via FGFR2 on osteoblastic differentiation. Our present study clearly showed that CCN2 enhanced the ERK- mediated signal emitted by the FGFR2/FGF2 complex via direct molecular interaction, which enhanced the differentiation of osteoblastic cells, and such effects were nullified by a FGFR2 signaling inhibitor. These findings collectively indicate the FGFR2-dependency of one aspect of CCN2 function, confirming the molecular action of CCN2 as a signal conductor for harmonized bone development and regeneration. In addition, it remains to be clarified whether this FGFR2/CCN2 interaction is critically involved in similar effects of CCN2 on cells other than osteoblasts.

Acknowledgments

Fig. 4. CCN2 promotes the activation of ERK by FGF2 in osteoblastic MC3T3-E1 Masaharu Takigawa would like to dedicate this article to the cells. MC3T3-E1 cells in a-modiﬁcation of MEM containing 10% FCS were plated at the density of 4 105/well in 6-well plates. After a 24-h incubation, the cells were memories of his father, Dr. Masami Takigawa, and Professor cultured in 0.5% FCS/aMEM for another 24 h and then stimulated with FGF2 (2 ng/ Emeritus Yoshiro Takeda, both of whom passed away while this re- ml) with or without CCN2 (100 ng/ml) and FGFR-Fc. The lysates were collected at search was being conducted. We thank Drs. Takako Hattori and 30 min after stimulation for western blotting analysis with the indicated antibodies. Takashi Nishida for their helpful suggestions and Ms. Eri Yashiro The blots are representative of 2 independent experiments. The lower panel shows for her secretarial assistances. This work was supported in part the ratio between the optical densities of phospho-ERK and total ERK. by grants from the programs Grants-in-Aid for Scientiﬁc Research (S)[to M.T.], (B)[to M.T.] and (C)[to S.K.] and for Exploratory Re- whereas at the later stage, the same factor increases it. Also, the search (to M.T.) from the Japan Society for the Promotion of Sci- expression of FGFR2 is up-regulated after the middle stage. Our ences; and by a research grant (to S.K.) from Terumo Life Science present data indicates that CCN2 supported the signaling of FGF2 Foundation. E. Aoyama et al. / FEBS Letters 586 (2012) 4270–4275 4275

Fig. 5. FGF2 and CCN2 enhance the expression of osteocalcin in MC3T3-E1 cells. (A) MC3T3-E1 cells in 10% FCS/aMEM were plated at the density of 3 105/well in 6-well plates. After 7 d, the sub-conﬂuent cells were stimulated with FGF2 (10 ng/ml) and/or CCN2 (100 ng/ml) for another 7 d. The mRNA levels were quantiﬁed by RT-real time PCR analysis, as described in Section 2. The data are the mean values ±S.D. of values for 6 wells from 2 independent experiments, each performed in triplicate. ⁄p < 0.05 as compared with the value for the control wells. (B) The enhancement of expression of osteocalcin by FGF2 and CCN2 was suppressed in the presence of FGFR2-Fc. The data are the mean ±S.D. values from 3 wells. ⁄p < 0.05 as compared with the value from the control wells.

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