Expression and physiological role of CCN4⁄ Wnt-induced secreted protein 1 mRNA splicing variants in chondrocytes Takeshi Yanagita 1,2 , Satoshi Kubota 1 , Harumi Kawaki 1 , Kazumi Kawata 1 , Seiji Kondo 1 , Teruko Takano-Yamamoto 3 , Shinji Tanaka 4 and Masaharu Takigawa 1 1 Department of Biochemistry & Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan 2 Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan 3 Division of Orthodontics and Dentofacial Orthopedics, Tohoku University Graduate School of Dentistry, Miyagi, Japan 4 Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Japan The Wnt1-induced secreted proteins (WISPs) were identified together in a human study reported in 1998 and then later classified as members of the CCN family, which also includes cysteine-rich 61 (Cyr61 ⁄ CCN1), connective tissue growth factor (CTGF ⁄ CCN2) and nephroblastoma overexpressed (Nov ⁄ CCN3) proteins as its classical members [1–5]. In addi- tion, prior to the discovery of the human ortholog, murine CCN4 ⁄ WISP1 was identified as a novel gene with tumor-suppressor properties and that was initially Keywords cartilage; CCN family; chondrocyte differentiation; splicing variant; Wnt-induced secreted protein 1 (WISP1) Correspondence M. Takigawa, Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8525, Japan Fax: +81 86 235 6649 Tel: +81 86 235 6645 E-mail: takigawa@md.okayama-u.ac.jp Database The nucleotide sequence data described have been submitted to the GenBank data- base under the accession numbers EF025921, EF025922 and EF025923 (Received 20 November 2006, revised 15 January 2007, accepted 19 January 2007) doi:10.1111/j.1742-4658.2007.05709.x CCN4 ⁄ Wnt-induced secreted protein 1 (WISP1) is one of the CCN (CTGF ⁄ Cyr61 ⁄ Nov) family proteins. CCN members have typical structures composed of four conserved cysteine-rich modules and their variants lack- ing certain modules, generated by alternative splicing or gene mutations, have been described in various pathological conditions. Several previous reports described a CCN4 ⁄ WISP1 variant (WISP1v) lacking the second module in a few malignancies, but no information concerning the produc- tion of WISP1 variants in normal tissue is currently available. The expres- sion of CCN4 ⁄ WISP1 mRNA and its variants were analyzed in a human chondrosarcoma-derived chondrocytic cell line, HCS-2 ⁄ 8, and primary rab- bit growth cartilage (RGC) chondrocytes. First, we found WISP1v and a novel variant of WISP1 (WISP1vx) to be expressed in HCS-2 ⁄ 8, as well as full-length WISP1 mRNA. This new variant was lacking the coding regions for the second and third modules and a small part of the first module. To monitor the expression of CCN4 ⁄ WISP1 mRNA along chondrocyte differ- entiation, RGC cells were cultured and sampled until they were mineral- ized. As a result, we identified a WISP1v ortholog in normal RGC cells. Interestingly, the WISP1v mRNA level increased dramatically along with terminal differentiation. Furthermore, overexpression of WISP1v provoked expression of an alkaline phosphatase gene that is a marker of terminal dif- ferentiation in HCS-2 ⁄ 8 cells. These findings indicate that WISP1v thus plays a critical role in chondrocyte differentiation toward endochondral ossification, whereas HCS-2 ⁄ 8-specific WISP1vx may be associated with the transformed phenotypes of chondrosarcomas. Abbreviations ALP, alkaline phosphatase; CT, C-terminal cysteine knot; HUVEC, human umbilical vein endothelial cell; IGFBP, insulin-like growth factor binding protein-like; RGC, rabbit growth cartilage; TSP1, thrombospondin type 1; VWC, von Willebrand factor type C; WISP, Wnt-induced secreted protein. FEBS Journal 274 (2007) 1655–1665 ª 2007 The Authors Journal compilation ª 2007 FEBS 1655 described as ‘Expression in low-metastatic cells type 1 (Elm1)’ gene [6]. Currently, all six members tend to be referred to as CCN1–6, based on the unified nomenclature [1,7]. Members have been described as being involved in a variety of cell biological processes, such as apoptosis, mitosis, cell adhesion, the production of extracellular matrix and angiogenesis [2,8–12]. It should be noted that most have unfavorable roles in human fibrotic dis- orders and several types of malignancy [13]. As seen in the other CCN family members, CCN4 ⁄ WISP1 posses- ses a typical structure composed of four conserved cys- teine-rich modular domains encoded by separate exons [14], which share sequence homologies with the insu- lin-like growth factor binding protein (IGFBP: first), von Willebrand factor type C repeat (VWC: second), thrombospondin type 1 repeat (TSP1: third), and the C-terminal cysteine knot (CT: fourth), respectively. The multimodular architecture of WISP1, along with the production of its truncated isoforms in tumors, rai- ses interesting questions regarding the participation of each individual module in the various biological func- tions of this factor [15]. The involvement of CCN4 ⁄ WISP1 in malignant neo- plasms has been relatively well investigated [16]. In ear- lier studies, full-length WISP1 was also shown to restrain tumor growth and metastasis, as represented by its initial name, Elm1 [6]. Recently, a novel variant of WISP1 (WISP1v), which lack the VWC module, was reported in a few in human gastrointestinal cancer tissues, thus sug- gesting the involvement of the variant in malignant phe- notypes of certain cancers [17–20]. However, because of its initial discovery in mouse melanoma, no investigation was performed to clarify the distribution of WISP1 and its physiological roles in normal tissues. In this study, we analyzed the expression of full- length WISP1 mRNA and its splicing variants in nor- mal and malignant-transformed chondrocytes. For the first time, we describe the expression of WISP1v in nor- mal growth plate chondrocytes, which was regulated along with the terminal differentiation of those cells. Furthermore, we also identified a novel WISP1 mRNA variant in malignant-transformed human chondrocytes. The roles of these variants in chondrocyte biology have been analyzed and are now discussed. Results CCN4 ⁄ WISP1 mRNA splicing variants in human chondrocytic HCS-2/8 cells To characterize mRNAs from ccn4 ⁄ wisp1, RT-PCR was performed using total RNAs from HCS-2 ⁄ 8, human umbilical vein endothelial cells (HUVEC), HeLa, MDA231, SaOS2 and HEK293 cells with speci- fic primers that recognize the IGFBP (sense) and CT (antisense) coding exons. These primers were designed to amplify the splicing variant lacking the VWC mod- ule area (WISP1v), as well as full-length WISP1 (Fig. 1A). As a result, no ccn4 ⁄ wisp1 transcripts were Fig. 1. Analysis of CCN4 ⁄ WISP1 mRNA in several cell lines as evaluated by RT-PCR. (A) The sense and antisense primers were designed to recognize the sequences in IGFBP module-encoding exons and CT module-encoding exons, respectively. The length of the PCR products from the full-length CCN4 ⁄ WISP1 amplified by these primers was expected to be 606 bp. (B) The results of the RT-PCR analysis. CCN4 ⁄ WISP1 and WISP1v-cloned vectors were also amplified as controls. According to the DNA size markers analyzed together, the size of the PCR product I was  600 bp, whereas those of II and III were  400 and 200 bp, respectively. Experiments were repeated at least three times and representative results are shown. Equal sample load was confirmed by the RT-PCR analysis of gapdh mRNA. CCN4 splicing variants in chondrocytes T. Yanagita et al. 1656 FEBS Journal 274 (2007) 1655–1665 ª 2007 The Authors Journal compilation ª 2007 FEBS detected with kidney-derived HEK293, HeLa cervical cancer and MDA231 breast cancer cells. Vascular endothelial cells (HUVEC) and A371 melanoma cells expressed only full-length mRNA (Fig. 1B), whereas osteoblastic SaOS2 expressed both CCN4 ⁄ WISP1 and WISP1v. Interestingly, we obtained three distinct amplicons from the chondrocytic HCS-2 ⁄ 8 mRNA. Subsequent nucleotide sequence analysis revealed that the longest and intermediate bands were full-length CCN4 ⁄ WISP1 and WISP1v, respectively. However, the shortest band was a novel variant of WISP1 (WISP1vx) not previously described (Fig. 2A). In addi- tion, the presence of WISP1v in human osteogenic cell lines was also revealed for the first time. Structure and predicted translation product of the novel CCN4/WISP1 mRNA splicing variant, WISP1vx According to sequence analysis data, this novel vari- ant, WISP1vx, lacks VWC and TSP module-coding ex- ons and part of the IGFBP module-coding exon. Because the alternative splicing site in IGFBP is located slightly upstream of the authentic site, the IG- FBP-coding exon in WISP1vx was 23 bp shorter than the full-length exon. However, owing to the frame- shift, the IGFBP⁄ CT fusion coding frame is not trans- lated properly after the alternative splice site (Fig. 2A). The deduced protein from WISP1vx is a single IGFBP module, in which eight C-terminal authentic amino acid residues are removed, and an extra 14 residues are added in their place. Importantly, the nucleotide sequences of exon– intron boundaries of WISP1vx strictly conserve the general rule of mRNA splicing sites in higher eukaryo- tes (Fig. 2B). These findings indicate that WISP1vx is not an artifact of PCR amplification, but a splicing variant uniquely present in HCS-2 ⁄ 8 chondrocytic cells. Detection of CCN4 ⁄ WISP1 proteins in HCS-2/8 cells In order to examine whether CCN4 ⁄ WISP1 and WISP1v mRNAs yielded corresponding proteins in HCS-2 ⁄ 8 cells, we analyzed the HCS-2 ⁄ 8 cell lysate using western blotting with an anti-(CCN4 ⁄ WISP1) serum targeting the CT module. First, mammalian expression vectors for FLAG epitope-tagged CCN4 ⁄ WISP1, WISP1v and WISP1vx proteins were con- structed, and the proteins were overexpressed by DNA transfection of these expression vectors (Fig. 3A) into HCS-2 ⁄ 8 cells. The overexpressed CCN4 ⁄ WISP1, WISP1v and WISP1vx served as pos- itive controls for western blotting. Because the FLAG epitope itself is a small octapeptide, the exogenesis CCN4 ⁄ WISP1 and its variants are indistinguishable from endogenous ones on SDS⁄ PAGE. As shown in Fig. 3B, analysis of transfectants with the anti- (CCN4 ⁄ WISP1) clearly showed strong enhancement of the CCN4 ⁄ WISP1 and WISP1v signals that were also present in HCS-2 ⁄ 8 cells without the overexpres- sion. As expected, no WISP1vx signal was detected, because it lacked the CT module in which the epitope of H-55 antibody was located. In contrast, when we used anti-FLAG IgG, we detected CCN4 ⁄ WISP1, WISP1v and WISP1vx only in overexpressed cells at the expected electromobility. This indicates that A B Fig. 2. Exon–intron boundaries and the putative translation products of the novel variant (WISP1vx). (A) Nucleotide sequences of the amplicons I, II and III from HCS-2 ⁄ 8 cDNA were analyzed and the structures of the corresponding mRNAs and deduced translational products are illustrated. Owing to the alternative splicing, a frame- shift mutation was introduced, thus resulting in a premature termin- ation of the translation (III), as detailed in Fig. 2B. (B) Genomic, mRNA and the deduced amino acid sequences are displayed at upper, middle and lower, respectively. Alternative and authentic spli- cing sites are indicated with an emphasis of consensus sequences. Owing to the frame-shift mutation, a stop codon appears at the fifteenth codon downstream of the alternatively splice site. SD and SA denote splicing donors and acceptors, respectively. T. Yanagita et al. CCN4 splicing variants in chondrocytes FEBS Journal 274 (2007) 1655–1665 ª 2007 The Authors Journal compilation ª 2007 FEBS 1657 CCN4 ⁄ WISP1 and WISP1v proteins were actually translated from CCN4 ⁄ WISP1 and WISP1v mRNAs in HCS-2 ⁄ 8 cells. A rabbit ortholog of the reported CCN4/WISP1- splicing variant (WISP1v) that is associated with human malignancies HCS-2 ⁄ 8 was derived from a human chondrosarcoma and it is one of the cell lines known to retain the characters of chondrocytes, as represented by the expression of type II and type X collagen genes. To assess whether WISP1v and WISP1vx are associated with malignant phenotypes, or with normal chondro- cytic phenotypes, we investigated whether CCN4 ⁄ WISP1 mRNA and its variants are also expressed in normal primary chondrocytes. Rabbit growth carti- lage (RGC) cells were isolated, and chondrocytic differentiation was induced in vitro. Thereafter, RT- PCR was performed to analyze the ccn4 ⁄ wisp1 mRNA expression pattern in RGC cells. Interestingly, WISP1v, as well full-length CCN4 ⁄ WISP1 mRNA, was distinctly detected in normal RGC, whereas WISP1vx was not. As summarized in Fig. 4, nucleo- tide sequence analysis indicated that the splicing sites used to generate rabbit WISP1v are at the same posi- tion as those observed in the human gene, indicating that it is exactly the same ortholog as that of human WISP1v. Regulated expression of WISP1v along with the chondrocytic differentiation of RGC cells In order to assess whether WISP1v is associated with the development of growth cartilage, primary growth cartilage cells collected from rabbits were cultured until they became confluent. Afterwards, a long-term culture for the induction of terminal differentiation was car- ried out for over one month. Fluctuation in type II collagen as a marker gene of chondrocyte differenti- ation showed that RGC cells were properly differenti- ated in vitro (Fig. 5C). Alizarin red staining of RGC cells revealed increased mineralization by the cells from 2 weeks after reaching confluency (Fig. 5A). Interest- ingly, the expression level of WISP1v increased gradu- ally along with the mineralization of chondrocytes (Fig. 5D,E), thus suggesting that WISP1v plays a spe- cific role therein. In contrast, full-length CCN4 ⁄ WISP1 was observed to be expressed almost constantly throughout all stages of chondrocyte differentiation (Fig. 5D), thus implying a more basic biological role for full-length CCN4 ⁄ WISP1 mRNA in growth plate chondrocytes. Distribution of CCN4/WISP1 proteins in growth cartilage in vivo In general, the distribution of a protein in a certain tis- sue is closely associated with its function in vivo. Fig. 3. Production of the WISP1v proteins in HCS-2 ⁄ 8 cells and their overexpression. (A) Molecular constructs for the overexpression of WISP1, WISP1v and WISP1vx. A mammalian expression vector, pFLAG-CMV was employed as the parental vector. The resultant constructs are illustrated. These plasmids were designed to express WISP1, WISP1v and WISP1vx with the FLAG epitopes at the N-termini. (B) West- ern blotting analysis probed by anti-(human CCN4 ⁄ WISP1) serum (left), or anti-FLAG serum (right) with the lysate of HCS-2 ⁄ 8 cells, together with those overexpressing WISP1, WISP1v, WISP1vx. Apparent molecular masses of specific signals in SDS ⁄ PAGE are shown. Positions of molecular mass markers are shown in the middle. Specific signals are marked by arrows. Experiments were repeated twice, yielding com- parable results. CCN4 splicing variants in chondrocytes T. Yanagita et al. 1658 FEBS Journal 274 (2007) 1655–1665 ª 2007 The Authors Journal compilation ª 2007 FEBS Fig. 4. The expression of WISP1v orthologue in normal RGC cells and its structure. (A) The detection of WISP1v as well as CCN4 in RGC cells. Total RNA from RGC cells (right) that had been cultured 3 weeks at confluence was analyzed. The expected sizes of the amplicons were 550 bp from rabbit CCN4 ⁄ WISP1 and 289 bp from rabbit WISP1v, respectively, which were consistent with the electromobility seen here (left). A representative result of four independent series of experiments with similar results is displayed. (B) Detailed structure of the exon–intron boundary of rabbit WISP1v in comparison with human WISPv. Fig. 5. Regulated expression of WISP1v along with chondrocytic differentiation. RGC cells were differentiated in vitro, and the expression of a marker gene and WISP1v was analyzed along with the time course. (A) Alizarin red staining of RGC cells representing mineral deposition. (B) Agarose gel electrophoresis analysis confirming the quality of the total RNA at each stage. (C) Expression of the type II collagen gene as evaluated by quantitative real-time PCR. (D) RT-PCR analysis with the primers recognizing WISP1v. (E) A quantitative real-time PCR analysis performed with rabbit-specific WISP1v primers. The sampling time points are represented as the numbers of weeks after the cell reached confluence. Representative results of four independent series of experiments are displayed with error bars (SD of real-time PCR evaluation). T. Yanagita et al. CCN4 splicing variants in chondrocytes FEBS Journal 274 (2007) 1655–1665 ª 2007 The Authors Journal compilation ª 2007 FEBS 1659 Therefore following mRNA analysis, we examined the distribution of CCN4 ⁄ WISP1 and WISP1v proteins in developing mouse growth cartilage by immunohisto- chemistry (Fig. 6). Because no anti-(CCN4 ⁄ WISP1) serum was able to detect the loss of VWC, which is the structural determinant of WISP1v that distingui- shes it from full-length WISP1 mRNA, we used an antibody that recognized a common module between the full-length CCN4 ⁄ WISP1 mRNA and WISP1v proteins. As a result, positive signals representing full- length CCN4 ⁄ WISP1 mRNA and WISP1v were observed, and were particularly strong in the hyper- trophic zone of the developing tibial sections of the mouse embryo. Although these signals also include those from the full-length CCN4 ⁄ WISP1 mRNA, these in vivo findings are consistent with the total outcome of the constitutive expression of the full-length and the differentiation stage-dependent expression of WISP1v in vitro. Effect of overexpression of CCN4/WISP1 and its variants on the phenotypes of human chondrocytic HCS-2 ⁄ 8 cells Finally, the biological function of WISP1v and WISP1vx was evaluated using overexpression experi- ments. FLAG epitope-tagged WISP1v and WISP1vx were overexpressed by DNA transfection of mamma- lian expression vectors into HCS-2 ⁄ 8 cells. First, over- expression of WISP1v and WISP1vx was confirmed by western blotting with anti-FLAG IgG (Fig. 3B). Thereafter, we collected total RNA and comparatively analyzed the expression levels of the marker genes of chondrocyte differentiation and mineralization by real- time quantitative RT-PCR. No significant difference was observed in the mRNA level of type II collagen and aggrecan core protein genes; however, the alkaline phosphatase (ALP) mRNA level increased remarkably with WISP1v overexpression (Fig. 7). Discussion In this study, we obtained some clues to clarify the physiological roles of WISP1v in normal cells. In par- Fig. 6. Immunohistochemical staining of CCN4 ⁄ WISP1 and WISP1v in the developing tibia of a mouse embryo. The tibia at embryonic day of 17 was probed using anti-(CCN4 ⁄ WISP1) serum recognizing the CT module. Dark brown signals indicate the presence of CCN4 ⁄ WISP1 and ⁄ or WISP1v protein (right). A negative control without primary antibody is also shown (left). Cartilaginous ECM was counterstained by methyl green. Fig. 7. Effects of the overexpression of WISP1v and WISP1vx in HCS-2 ⁄ 8 on the differentiation marker genes of growth plate chondrocytes. Quantitative real-time PCR analysis was performed with WISP1v and WISP1vx overexpressing HCS-2 ⁄ 8 cells. The relative values against the expression of gapdh were computed and displayed with error bars (SD). Data are derived from three independent evaluations. Statistical analysis was performed by Tukey–Kramer test. *P<0.05, significantly different as indicated by the bracket. CCN4 splicing variants in chondrocytes T. Yanagita et al. 1660 FEBS Journal 274 (2007) 1655–1665 ª 2007 The Authors Journal compilation ª 2007 FEBS ticular, expression of WISP1v among normal chondro- cytes and osteoblastic cells was confirmed, which has heretofore not been elucidated. Expression of WISP1v increased during the course of chondrocytic terminal differentiation, thus suggesting a role for WISP1v therein. This hypothesis was supported by the fact that overexpression of WISP1v enhanced the gene expres- sion of one of the mineralization markers in chondro- cytic cells. Moreover, the presence of a novel splicing variant not described previously was discovered in HCS-2 ⁄ 8 cells. These findings are summarized in Fig. 8. To date, WISP1v has been investigated only from the point of view of tumorigenesis and malignant phe- notypes of tumors [16]. Indeed, expression of WISP1v has been reported only in malignant tumor cells [7,17]. Scirrhous carcinoma of the stomach is known to be associated with WISP1v and it is characterized by rapid growth with a vast fibrous stroma, high invasive- ness and a substantially poor prognosis [20]. Because little is known about the molecular pathogenesis of the disease, a pathological role for WISP1v should be investigated further. It should be noted that most CCN family members are associated with certain types of malignancy. Regarding physiological functions, some CCN fam- ily members play an important role in skeletal growth, as typically represented by CCN2 ⁄ CTGF [1,4,5]. CCN2 ⁄ CTGF was found to be highly expressed in hypertrophic chondrocytes, as shown by in situ hybrid- ization of both whole-mount neonates and their longi- tudinal sections [21]. A series of in vitro evaluations revealed that CCN2 ⁄ CTGF plays an important role in cartilage formation and endochondral ossification. RGC and rabbit articular cartilage cells transfected with recombinant adenoviruses generating mRNA for CCN2 ⁄ CTGF, thus result in increased proteoglycan synthesis [22,23]. Moreover, rCCN2 ⁄ CTGF effectively increased both the ALPase activity and the matrix cal- cification of RGC cells [24,25]. In the developing skel- etal system, CCN1 ⁄ Cyr61 is expressed in the cartilage of a number of skeletal elements. Regarding these ele- ments, CCN1 ⁄ Cyr61 is first expressed in condensing mesencymes, with expression persisting throughout the stages of chondrogenesis [26]. Strong expression of CCN1 ⁄ Cyr61 in the developing cardiovascular and skeletal systems suggests that it might play an import- ant role in angiogenesis and⁄ or chondrogenesis [27– 31]. In contrast, no report has described the role of CCN4 ⁄ WISP1 in cartilage development and endochon- dral ossification. Here, we showed for the first time the expression and possible function of the CCN4 ⁄ WISP1 variant at a late stage of endochondral ossification. In addition to the effect on ALP gene expression, we also evaluated the effect of WISPv overexpression on type X collagen gene expression, but no significant effect was observed (data not shown). Together with the fact that osteoblastic cells also expressed WISP1v, this variant is thought to play a role in mineralization rather than hypertrophy of chondrocytes. In comparison with CCN1 ⁄ Cyr61 and CCN2 ⁄ CTGF, the role of CCN4 ⁄ WISP1 in chondrocyte dif- ferentiation appears more specific to the mineralizing stage of endochondral ossification. This fundamental difference may be interpreted from an evolutionary point of view. Genome-wide prediction of CCN ortho- logs in several species indicated that CCN4 ⁄ WISP1 or- thologs were found only in animals with a calcified skeleton, whereas CCN1 ⁄ Cyr61 and CCN2 ⁄ CTGF could be identified in invertebrates such as Ciona intes- tinalis. Thus, CCN4 ⁄ WISP1 is thought to be required for the establishment of a calcified endoskeleton. This hypothesis is consistent with our findings on WISP1v. However, it may not apply exactly to full- length CCN4 ⁄ WISP1, because no clear change was observed according to the chondrocytic differentiation. Fig. 8. Formation and function of CCN4 ⁄ WISP1 variants. Structure, expres- sion and function of the CCN4 ⁄ WISP1 vari- ants are summarized. All of the findings were obtained in this study except for that of gastrointestinal cancers reported by Tan- aka et al. [17,20]. ++, enhanced expression evaluated by real-time RT-PCR; ±, modest expression evaluated by real-time RT-PCR; –, expression undetectable. T. Yanagita et al. CCN4 splicing variants in chondrocytes FEBS Journal 274 (2007) 1655–1665 ª 2007 The Authors Journal compilation ª 2007 FEBS 1661 In addition, no substantial induction of ALPase activ- ity was seen in the overexpression experiment (data not shown). We suspect that full-length CCN4⁄ WISP1 does not have such a close relationship with endochon- dral mineralization, but rather another basic cell biolo- gical role in chondrogenesis may exist. In fact, our preliminary data indicate the increasing expression of CCN4 ⁄ WISP1 end ⁄ or its variant along with chondro- genesis of mesenchymal stem cells (Kawaki et al., manuscript in preparation). The functional significance of WISP1vx remains to be investigated. Expression of WISP1vx was detected only in chondrosarcoma-derived HCS-2 ⁄ 8 cells. We are cur- rently collecting clinical samples of chondrosarcomas to verify the significant relationship between WISP1v expression and phenotypes of particular type of malig- nancy. Further functional evaluations are also required to elucidate the pathological roles of this variant. Experimental procedures Cell culture A human chondrosarcoma-derived chondrocytic cell line, HCS-2 ⁄ 8 [32], human cervical carcinoma-derived HeLa, human melanoma cell line A371, human osteoblastic SaOs2 and human embryonic kidney-derived HEK293 [33] cells were grown in DMEM supplemented with 10% fetal bovine serum. HUVECs derived from human umbilical cords were purchased from Biowhittaker (Walkersville, MD) and used between passages three and seven. These cells were cultured in EBM-2 complete medium (Clonetics, San Diego, CA). RGC cells were isolated from growth car- tilage in ribs of 5-week-old rabbits, as described previously [25]. The primary RGC cells were cultured in a-modifica- tion of minimum essential medium (Sigma, St Louis, MO) containing 10% fetal bovine serum. Cell cultures were maintained in a humidified 5% CO 2 atmosphere at 37 °C without any passage until analysis. Alizarin red staining RGC cells in six-well plates were washed with NaCl ⁄ P i and fixed using 4% paraformaldehyde ⁄ NaCl ⁄ P i . Following fix- ation cells were washed again in NaCl ⁄ P i and stained with a 1% alizarin red solution, as described previously [34]. Macroscopic images were captured after 15 extensive washes with NaCl ⁄ P i . DNA transfection HCS-2 ⁄ 8 cells were seeded onto six-well plates at 500 000 cells per well. Cells were transfected with 2 lg of plasmid DNA using 8 lL of FuGENE6 transfection reagent (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions. Cells were lysed in 50 lLof1· passive lysis buffer (Promega, Madison, WI), and collected for western blotting at 48 h after transfection. RNA was also extracted from cells for RT-PCR at 48 h after transfec- tion. RNA extraction and RT-PCR analysis Total RNA was extracted from the cells using the RNeasy kit, according to the manufacturer’s instructions (Qiagen, Valencia, CA). Total RNA (500 ng) was reverse-transcribed to cDNA by using an oligo(dT) with avian myeloblastosis virus reverse transcriptase (Takara, Tokyo, Japan). For semiquantitative PCR analysis, the cDNAs were amplified with Blend-Taq Plus (Toyobo, Osaka, Japan) with a regular thermal cycler. The sets of synthetic primers used for amplification are described below, where the numbers in parentheses indicate the expected sizes of the PCR products: human glyceralde- hyde-3-phosphate dehyrogenase gene (gapdh), sense 5¢-GCC AAAAGGGTCATCATCTC-3¢ and antisense 5¢-GTCTTC TGGGTGGCAGTGAT-3¢ (215 bp); human ccn4 ⁄ wisp1, sense 5¢-CTCAGCAGCTTGGGGACAAC-3¢ and antisense 5¢-GATGCCTCTGGCTGGTACAC-3¢ (606 bp); rabbit ccn4 ⁄ wisp1, sense 5¢-ATCGGGGCCTCTACTGCGACTA CAG-3¢ and antisense 5¢-TGGCTGGTACACAGCCAGA CACTTC-3¢ (550 bp); human wisp1v, sense 5¢-GCAATAG GAGTGTGTGCACAGGTGG-3¢ and antisense 5¢-GAT GCCTCTGGCTGGTACAC-3¢ (345 bp). The amplification conditions were as follows: for human ccn4 ⁄ wisp1 and human wisp1v cDNA, 94 °C (5 min) for 1 cycle, followed by 94 °C (30 s), 65 °C (30 s), 72 °C (45 min) for 35 cycles, and a final incubation at 72 °C for 5 min; for rabbit ccn4 ⁄ wisp1 and rabbit wisp1v cDNAs, 94 °C (5 min) for 1 cycle, followed by 94 ° C (30 s), 60 °C (30 s), 72 ° C (30 s) for 35 cycles, and a final incubation at 72 °C for 5 min. PCR products were electrophoresed in a 1% agarose gel containing ethidium bromide and visualized under UV light. Photographs of the stained gels were taken and analyzed quantitatively with transilluminator fasiii (Toyobo). Antibodies and molecular clones An anti-(CCN4 ⁄ WISP1 H-55) serum recognizing the CT module (Santa Cruz Biotechnologies, Santa Cruz, CA) and an anti-(FLAG M2) mAb (Sigma) were utilized in western blotting or immunohistochemistry. For molecular cloning of wisp1-related cDNA fragments, PCR products were fractionated by agarose gel electrophor- esis and were extracted from the gels using the Gel Extrac- tion kit (Qiagen). Next, purified PCR products were inserted into pGEM T-easy vector according to the manu- facturer’s instructions (Promega). CCN4 splicing variants in chondrocytes T. Yanagita et al. 1662 FEBS Journal 274 (2007) 1655–1665 ª 2007 The Authors Journal compilation ª 2007 FEBS The original human CCN4 ⁄ WISP1 and WISP1v mam- malian expression vectors were given by Shinji Tanaka (Tokyo Medical and Dental University), in which human cDNAs were inserted in pCR3.1 (Invitrogen, San Diego, CA) [17]. Three FLAG epitope-tagged expression vectors were newly constructed. ccn4 ⁄ wisp1 and wisp1v cDNAs were excised from the pCR3.1-based plasmids by digestion with HindIII and XbaI, and then inserted between the corresponding restric- tion enzymatic sites of the Mammalian Amino-Terminal FLAG Ò vector (Sigma). The resultant expression plasmids contain the ccn4 ⁄ wisp1 and its variant cDNAs fused in- frame to the FLAG epitope tag. We designated these plas- mids CMV-FLAG–WISP1, CMV-FLAG–WISP1v and CMV-FLAG–WISP1vx, respectively, and utilized them in the overexpression experiments. DNA sequence analysis The nucleotide sequences of the cloned PCR products and newly constructed expression plasmids were determined by automated fluorescence DNA sequencing using the ABI Prism Dye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA), following the manufac- turer’s recommendations. The primer recognizing the bac- teriophage T7 promoter was employed in the sequencing reactions for the TA-cloned cDNAs. At least three inde- pendent clones were analyzed for each mRNA variant and identical results were obtained. To confirm the nucleotide sequence of the expression vectors, CMV- FLAG–WISP1, CMV-FLAG–WISP1v and CMV-FLAG– WISP1vx, ccn4 ⁄ wisp1-specific primers used in RT-PCR analysis were utilized. Products of the sequencing reac- tions were then purified using SIGMA SPIN columns (Sigma), and the samples were dissolved in 20 lL of Tem- plate Suppression Reagent and analyzed by ABI Prism 310 Automated DNA Sequencing System (PE Applied Biosystems). Quantitative real-time PCR Optimal primer concentrations were determined based on the protocols of Toyobo SYBR Green PCR Master Mix Manual (Toyobo). Reactions were performed in 20 lLofa reaction mixture (Toyobo) containing 2 lL cDNA, 5 lm each primer and 1· SYBR Green Master Mix. The primer sets used for the evaluation of ccn4 ⁄ wisp1 and its variants were the same as the regular RT-PCR. The nucleotide sequences of the primer sets for the quantitative evaluation of human alkaline phosphatase (ALP), type II collagen and aggrecan cDNAs were: human alp, sense 5¢-TGG AGCTTCAGAAGCTCAACACCA-3¢ and antisense 5¢-AT CTCGTTGTCTGAGTACCAGTCC-3¢ (443 bp); human type II collagen, sense 5¢-GAGGGCAATAGCAGGTTCA CGTA-3¢ and antisense 5¢-TGGGTGCAATGTCAATGA TGG-3¢ (133 bp); human aggrecan, sense 5¢-TCTTCA GTCCCGTTCTCCAC-3¢ and antisense 5¢-AACATCACT GAGGGCGAAGC-3¢ (93 bp), The amplification condi- tions were as follows: for human ccn4 ⁄ wisp1 and human alp cDNA, 95 °C (1 min) for 1 cycle, followed by 95 °C (1 s), 59 °C (1 s), 72 °C (30 s) for 50 cycles, and a melting process from 50 to 95 °C for 5 min, for rabbit ccn4 ⁄ wisp1 and rabbit ccn4 ⁄ wisp1v cDNAs, 94 °C (5 min) for 1 cycle, followed by 94 °C (5 s), 60 °C (5 s), 72 °C (30 s) for 45 cycles, and melting process from 50 to 95 °C for 5 min, human aggrecan,94°C (30min) for 1 cycle, followed by 94 °C (5 s), 60 °C (0 s), 72 °C (30 s) for 45 cycles, and melting process from 50 to 95 °C for 5 min. Similarly, rab- bit gapdh cDNA fragment (283 bp) was analyzed with sense (5¢-TCACCATCTTCCAGGAGCGA-3¢) and antisense (5¢-CACAATGCCGAAGTGGTCGT-3¢) primers under the following conditions: 94 °C (30 s) for 1 cycle, followed by 94 °C (5 s), 65 °C (10 s), 72 °C (15 s) for 45 cycles, and melting process from 50 °Cto95°C for 5 min. Rabbit type II collagen cDNA fragment (370 bp) was analyzed with sense (5¢-GACCCATGCAGTACATGGG-3¢) and antisense (5¢-AGCCGCCATTGATGGTCTCC-3¢) primers under the conditions of; 94 °C (30 s) for 1 cycle, followed by 94 °C (5 s), 65 °C (0 s), 72 °C (60 s) for 45 cycles, and melting process from 50 °Cto95°C for 5 min. Each ampli- fication reaction was performed and checked for absence of nonspecific PCR products by the melting curve analysis in a LightCycler TM system (Roche). Relative cDNA copy numbers were computed based on the data with a serial dilution of a representative sample for each target gene. Immunohistochemistry Paraffin-embedded sections of normal mouse tibia were deparaffinized with xylene, rehydrated and washed with NaCl ⁄ P i . After blocked in a blocking agent (Histofine: Nichirei, Tokyo, Japan), the samples were incubated over- night at 4 °C in an anti-(CCN4 ⁄ WISP1 H-55) serum (Santa Cruz Biotechnologies) at a concentration of 1 : 50. After washing several times in NaCl ⁄ P i , the sections were immersed in methanol, containing 0.3% H 2 O 2 for 30 min to block endogenous peroxidase activity. To reduce nonspe- cific binding, 10% goat serum (Vector Laboratories, Burlin- game, CA) in NaCl ⁄ P i (GS-NaCl ⁄ P i ) was applied to the specimens for 30 min. Sections were then incubated with a specific antibody against CCN4 ⁄ WISP1 (1 : 50) diluted in GS-NaCl ⁄ P i at 4 °C overnight. Specific signals were probed by a horseradish peroxy- dase-conjugated secondary antibody (Histofine: Nichirei), and visualized using 3,3-diaminobenzidine tetrachloride (Sigma). Finally, the samples were counterstained with methyl green solution (Wako Pure Chemical Industries, Osaka, Japan). For negative controls, we skipped the primary antibody reaction step. T. Yanagita et al. CCN4 splicing variants in chondrocytes FEBS Journal 274 (2007) 1655–1665 ª 2007 The Authors Journal compilation ª 2007 FEBS 1663 Western blotting The cell lysate was prepared in a SDS sample buffer con- taining 2.5% b-mercaptoethanol. Each sample (25 lL) was heated for 5 min at 95 °C and then was separated by 15% PAGE, then the separated proteins were transferred to a poly(vinylidene difluoride) membrane (Immobilon, Milli- pore, Bedford, MA). Following the established protocol for western blotting, FLAG-tagged proteins were detected using a mouse anti-(FLAG M2) mAb (Sigma). Horseradish peroxidase-conjugated goat anti-(mouse IgG) (Chemicon, Temecula, CA) was used as a secondary antibody. These antibodies were used at dilutions of 1 : 1000 and 1 : 5000, respectively. Similarly, CCN4 ⁄ WISP1 proteins were detec- ted, using anti-(CCN4 ⁄ WISP1 H-55) serum (Santa Cruz Biotechnologies). Horseradish peroxidase-conjugated anti- (rabbit IgG) (DAKO-Japan, Tokyo, Japan) was used as a secondary antibody. These antibodies were used at dilutions of 1 : 200 and 1 : 1000, respectively. Throughout the proce- dure, 3% BSA–NaCl ⁄ P i was used as a blocking solution. Signals were detected using ECL TM western blotting detec- tion regaments (Amersham Biosciences, Uppsala, Sweden). Autoluminograms were obtained with an ECL mini-camera (Amersham Biosciences). Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research (S) (to MT) and (C) (to SK) from Japanese Society for Promotion of Science (JSPS) and for Exploratory Research (to MT) from the Ministry of Education, Culture, Sports, Science, and Technol- ogy of Japan. 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