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The Japanese Society of Developmental Biologists

Develop. Growth Differ. (2012) 54, 474–480

doi: 10.1111/j.1440-169X.2012.01333.x

Original Article

OBIF, an osteoblast induction factor, plays an essential role in bone formation in association with osteoblastogenesis Koji Mizuhashi, 1,2 Takashi Kanamoto, 3 Masako Ito, 4 Takeshi Moriishi, 5 Yuki Muranishi, 1 Yoshihiro Omori, 1,6,7 Koji Terada, 1 Toshihisa Komori 5 and Takahisa Furukawa 1,6 * 1

Department of Developmental Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka, 565-0874; 2Graduate School of Medicine, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto, 606-8501; 3Department of Orthopedic Surgery, Graduate School of Medicine, Osaka University, Osaka 565-0871; 4Department of Radiology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki, 852-8588; 5Department of Cell Biology, Unit of Basic Medical Sciences, Nagasaki University, Graduate School of Biomedical Science, 1-7-1 Sakamoto, Nagasaki, 852-8588; 6Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Osaka 565-0874; and 7 Precursory Research for Embryonic Science and Technology, Furuedai, Suita, Osaka, 565-0874, Japan

In vertebrate bone formation, the functional mechanisms of transcription factors in osteoblastic differentiation have been relatively well elucidated; however, the exact roles of cell-extrinsic molecules are less clear. We previously identified human and mouse Obif, an osteoblast induction factor, also known as Tmem119, which encodes a single transmembrane protein. OBIF is predominantly expressed in osteoblasts in mouse. While exogenous Obif expression stimulated osteoblastic differentiation, knockdown of Obif inhibits the osteoblastic differentiation of pre-osteoblastic MC3T3-E1 cells. In order to investigate an in vivo role of OBIF in bone formation, we generated Obif-deficient mice by targeted gene disruption. Analyses of micro-computed tomography (mCT) revealed that Obif) ⁄ ) mice exhibit significantly reduced cortical thickness in the mid-shaft of the femur at postnatal day 14 (P14). Furthermore, progressive bone hypoplasia is observed after 8 weeks. The expression levels of osteoblast marker genes, Collagen 1a1, Osteopontin, Runx2, and Osterix, in the calvaria were decreased in Obif) ⁄ ) mice at P4. These data indicate that Obif plays an essential role in bone formation through regulating osteoblastogenesis. Key words: bone formation, micro computed tomography, Obif, osteoblast, transmembrane protein.

Introduction The vertebrate skeleton consists of endochondral and membranous bones. Endochondral bones, which constitute the majority of bones, develop from cartilaginous templates (Mackie et al. 2011). Chondrocytes and osteoblasts are derived from common precursors. Both the activities of intrinsic cues and signals from extrinsic cues play pivotal roles in the cell fate decision of these cell types (Karsenty & Wagner 2002; Zelzer & Olsen 2003; Hartmann 2006). Mice with a targeted mutation *Author to whom all correspondence should be addressed. Email: [email protected] Received 9 January 2012; revised 25 January 2012; accepted 25 January 2012. ª 2012 The Authors Development, Growth & Differentiation ª 2012 Japanese Society of Developmental Biologists

of Runx2 or Osterix, transcriptional regulators in osteoblast progenitor, show the absence of mature osteoblasts, indicating that these regulators are prerequisites for osteoblastogenesis (Ducy et al. 1997; Komori et al. 1997; Otto et al. 1997). A recent study showed that the inhibitory effect of Sox9 on osteoblastic and chondrocyte maturation via the repression of Runx2 function is an essential mechanism for osteochondroprogenitor cell fate determination (Zhou et al. 2006). Osterix, which contains three zinc finger motifs, belongs to the specificity protein ⁄ Kru¨ppel-like factor (SP) family of transcription factors. Osterix-null mice showed a complete lack of osteoblasts, demonstrating that Osterix is essential for osteoblast differentiation functioning downstream of Runx2 (Nakashima et al. 2002). An important role for Wnt signaling in bone formation was revealed. The Wnt molecule activates the canonical pathway by interacting with receptors of the Frizzled family and co-receptors of

OBIF regulates osteoblastogenesis

the LRP5 ⁄ 6 family (Logan & Nusse 2004). Thus, in osteoblastic differentiation, the functional mechanisms of transcription factors are relatively well understood; however, the possible roles of extrinsic molecules in osteoblastic differentiation are less clear. In our previous report, we identified a gene encoding a single transmembrane protein, Obif, which is predominantly expressed in osteoblasts during mouse development. We performed functional analyses on this gene in vitro (Kanamoto et al. 2009). We found that osteoblast differentiation is stimulated when Obif is overexpressed and inhibited when Obif is knocked down in preosteoblastic MC3T3-E1 cells. In order to investigate the biological function of Obif in bone formation, we generated Obif mutant mice by targeted gene disruption. Micro computed tomography (CT) analysis revealed that Obif) ⁄ ) mice showed a significant decrease of cortical thickness in the mid-shaft of the femur at P14. In addition, progressive bone hypoplasia was observed after 8 weeks. The expression levels of osteoblast marker genes, including Collagen 1a1, Osteopontin, Runx2 and Osterix, were significantly reduced in the calvaria of Obif) ⁄ ) mice at P4. These data suggest that Obif plays an essential role in bone formation in association with reduced osteoblastogenesis.

Materials and methods

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Quantitative real-time PCR Quantitative real-time polymerase chain reaction (QPCR) was performed using SYBR Green ER Q-PCR Super Mix (Invitrogen) and Thermal Cycler Dice Real Time System single MRQ TP870 (Takara) according to the manufacturer’s instructions. Quantification was performed by Thermal Cycler Dice Real Time System software Version 2.0 (Takara). The sequences of primers for amplification of Collagen 1a1, Osteopontin, Osteocalcin, Runx2, Osterix, RANKL, and OPG were described previously (Maruyama et al. 2007). Histological analysis For histological analyses of femurs using decalcified sections, mice were euthanized at 8 weeks. To prepare sections for Sirius red staining and immunohistochemistry, we fixed mice with 4% paraformaldehyde ⁄ phosphate-buffered saline (PBS) buffer, and decalcified femurs in 10% ethylenediaminetetraacetic acid (EDTA) (pH 7.4) and embedded in paraffin. Sections (4 lm thick) were stained with Sirius red, and subjected to immunohistochemical analysis using an anti-rabbit Collagen I antibody (LSL). Immunohistochemistry was performed according to the manufacturer’s protocol. The specimens were observed under a laser confocal microscope (LSM710; Carl Zeiss).

Generation of Obif mutant mouse We obtained Obif genomic DNA clones from a screen of the 129S6 (129 ⁄ SvEv Taconic) mouse genomic DNA library (Stratagene). We subcloned an 8.3 kb SacII-EcoRI fragment and a 5.4 kb NcoI-HindIII fragment from Obif genomic clones into a modified pPNT vector (Deng et al. 1996; Muranishi et al. 2011), and transfected the linearized targeting construct into the 129S6 embryonic stem cell line. The culture, electroporation and selection of 129S6 were carried out as previously described (Sato et al. 2008). Embryonic stem cells that were heterozygous for the targeted gene disruption were microinjected into C57BL ⁄ 6 blastocysts to obtain chimeric mice. All procedures were approved by the Institutional Safety Committee on Recombinant DNA Experiments and the Animal Research Committee of Osaka Bioscience Institute. We confirmed the deletion in the genomic DNA of Obif-deficient mice by Southern blot.

Micro CT analysis Quantitative mCT analysis was conducted as described previously (Maruyama et al. 2007). Briefly, mCT was performed with a mCT system (lCT-40; ScancoMedical). Data were analyzed three-dimensionally to calculate femoral morphometric parameters, including bone volume fraction, trabecular connective density, trabecular number, trabecular thickness, trabecular separation and cortical bone thickness. Trabecular bone parameters were measured at the distal femoral metaphysis. CT scans were performed at the distal metaphysis to calculate trabecular parameters, and at mid-shaft to calculate cortical thickness, in P14, 8-, 24-week-old mice. The cranio-caudal scan lengths were 1.2 mm in 8-week-old and 2.4 mm in 24-week-old mice in the distal metaphysis, and the scan length was 0.24 mm at the mid-shaft.

Results

X-ray photography X-ray photography was performed using Laboratory CT (LaTheta, Aloka). Mice were treated with isoflurane (2% v ⁄ v) anesthesia, and analyzed by X-ray photography according to the manufacturer’s protocol.

Generation of Obif-deficient mouse To determine a possible role of Obif in bone formation, we generated Obif-deficient mouse by targeted gene

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disruption. Mouse Obif mRNA contains a single long open reading frame (ORF) encoding 280 amino acids, including a putative signal sequence (amino acid residues 1–18) and a single transmembrane domain (amino acid residues 92–112), suggesting that OBIF is a type IA transmembrane protein (Kanamoto et al. 2009). OBIF protein contains a glutamic acid rich region in its C-terminus but no other known domain or motif. Since the entire Obif ORF is encoded on exon 2, we deleted the entire exon 2 to generate a targeted allele of Obif gene by homologous recombination in ES cells (Fig. 1A). We confirmed the deletion of exon 2 in the genome of the Obif mutant mouse by Southern blot analysis (Fig. 1B). Obif homozygous mutant (Obif) ⁄ )) mice were born at the expected Mendelian frequency and were indistinguishable in appearance from control littermates before weaning. However, at 8 weeks, the significant stunted growth of Obif) ⁄ ) mice became manifest (Fig. 1C). The body length of 8 weeks Obif) ⁄ ) mice is significantly shorter than that of littermate control mice (16.4 ± 0.1 cm in control, 13.4 ± 0.1 cm in Obif) ⁄ ) mice) (Fig. 1D). We also observed a significantly decreased body weight of Obif) ⁄ ) mice at 8 weeks

(19.3 ± 0.6 g in control, 13.4 ± 0.3 g in Obif) ⁄ )) (Fig. 1E). To determine the gross skeletal features of Obif) ⁄ ) mice, we performed X-ray photographic analysis. We did not observe skeletal composition differences between littermate controls and Obif) ⁄ ) mice (Fig. 1F). Obif) ⁄ ) mice exhibit severe bone hypoplasia We then performed mCT analysis on Obif) ⁄ ) mice. We analyzed femoral sections of littermate control mice and Obif) ⁄ ) mice at P14 (lactation period) and 8 weeks (adolescent period). We found that cortical thickness was already significantly lower in Obif) ⁄ ) mice than that in control mice at P14 (Fig. 2A,B). Furthermore, this feature was observed in 8-week-old Obif) ⁄ ) mice as well (Fig. 2C,D). In addition, bone volume fraction (BV ⁄ TV), connective density, and trabecular number (Tb.N) in Obif) ⁄ ) mice were significantly lower than in control mice. In contrast, trabecular separation (Tb. Sp) was significantly higher in 8-week-old Obif) ⁄ ) mice. However, we observed no obvious difference of trabecular thickness (Tb.Th) between control and Obif) ⁄ ) mice (Fig. 2D). Notably, cortical thickness in femurs of Obif) ⁄ ) mice showed

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Fig. 1. Generation of Obif mutant mice by targeted gene disruption. (A) Strategy for the targeted deletion of Obif gene. Exon 2 was replaced with a phosphoglycerine kinase (PGK)-neo cassette. The probe used for Southern blot analysis is shown (3¢ probe). (B) Southern blot analysis of genomic structure of Obif locus was performed to confirm the deletion of Obif exon2. EcoRI-digested genomic DNA was hybridized with 3¢ probe. (C) Obif) ⁄ ) mice show slightly stunted growth compared with control mice (Scale bar = 3 cm). (D, E) Obif) ⁄ ) mice are shorter in body length and smaller in body weight compared with those in littermate control mice at 8 weeks. (F) Total body radiographs of 8-week-old control littermate and Obif) ⁄ ) mice. Scale bar = 3 cm, n = 3, *P < 0.03. Student’s t-test. S, SacII; E, EcoRI; N, NcoI; H, HindIII; DT-A-pA, diphtheria toxin A fragment cassette with a polyadenylation signal. ª 2012 The Authors Development, Growth & Differentiation ª 2012 Japanese Society of Developmental Biologists

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Fig. 2. Micro computed tomography (CT) analysis on femurs of Obif) ⁄ ) mice. Two- and three-dimensional reconstruction of slices from micro CT analysis of trabecular bone architecture of distal femoral metaphysis were prepared using mice at P4 (A) and 8 weeks (C). Cortical thickness was already significantly lower in Obif) ⁄ ) mice (j) than that in littermate control mice at P14 (h) (B). Obif) ⁄ ) mice exhibited severe bone hypoplasia at 8 weeks (D) (control at 8 weeks [h]; Obif) ⁄ ) at 8 weeks [j]. Obif) ⁄ ) mice (j) exhibit no significant difference in trabecular bone and cortical bone formation compared with those of littermate control mice at 24 weeks (h) (E). Trabecular bone parameters were measured at distal femoral metaphysis, and cortical thickness was measured at femoral diaphysis. BV (bone volume) ⁄ TV (tissue volume), Trabecular bone volume fraction; Conn. Dens., Trabecular connectivity density; Tb.N, Trabecular number; Tb.Th, Trabecular thickness; Tb.Sp, Trabecular separation. (A, B) n = 3. (C, D) n = 5. (E) control mice, n = 6, Obif) ⁄ ) mice, n = 3. *P < 0.03. Student’s t-test.

approximately a 20% reduction compared with that of control mice at 8 weeks. These data suggest that Obif promotes bone formation and ⁄ or reduces bone reabsorption during the adolescent period in vivo. To determine whether Obif) ⁄ ) mice show abnormality even at the senescent stage, we analyzed the bone parameters of femurs in Obif) ⁄ ) mice at 24 weeks. We

observed no significant difference between Obif) ⁄ ) mice and control littermate mice in all bone parameters analyzed (Fig. 2E). BV ⁄ TV values in control littermate mice at 24 weeks were decreased compared with those of 8week-old mice (12.2 ± 2.7% at 24 weeks, 17.3 ± 1.7% at 8 weeks) (Fig. 2D,E); however, those in Obif) ⁄ ) mice were similar between 24-week-old and 8-week-old

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mice (11.0 ± 2.6% at 24 weeks, 12.6 ± 1.4% at 8 weeks) (Fig. 2D,E). These results suggest that bone development and maturation in Obif) ⁄ ) mice catch up with that in control mice by 24 weeks. Osteoblastogenesis is perturbed in Obif) ⁄ ) mouse In our previous report, we identified that Obif mRNA is initially detected in the limb bud during development, and then Obif is highly expressed in osteoblasts at the stage of endochondral ossification. In addition, mCT analyses revealed that Obif) ⁄ ) mice show severe bone hypoplasia in trabecular and cortical bone in femurs. These results suggest that Obif is essential for endochondral ossification in femurs at least. In contrast, membranous ossification is observed in calvaria. To examine if Obif is involved in membranous ossification, we analyzed gene expression levels of osteoblast markers in the calvariae of Obif) ⁄ ) mice at P4 using real-time PCR. Collagen 1a1 and Osteopontin are early markers of osteoblast development (Nakashima et al. 2002). Osteocalcin is a late marker of osteoblast development (Nakashima et al. 2002). We found that expression of Collagen 1a1 and Osteopontin was markedly decreased in Obif) ⁄ ) mice compared with littermate control mice, whereas, expression of Osteocalcin was unaltered between Obif) ⁄ ) mice and control mice (Fig. 3). Runx2 and Osterix are transcription factors essential for osteoblast cell fate determination and osteoblast development, respectively (Komori et al. 1997; Nakashima et al. 2002). We found significant decreases of both Runx2 and Osterix expression in Obif) ⁄ ) mice (Fig. 3). In contrast, the expression lev-

els of receptor activator of nuclear factor-jB (NF-jB) ligand (RANKL), a major determinant of osteoclastogenesis, and Osteoprogerin (OPG), an osteoblastsecreted decoy receptor that functions as a negative regulator, were unaffected between control and Obif) ⁄ ) mice (Fig. 3). These results suggest that Obif is important for both membranous ossification and endochondral ossification in association with osteoblastogenesis. To further investigate a phenotype of Obif) ⁄ ) mice in terms of osteoblastogenesis, we performed histological analysis using sections of femoral epiphysis. Sirius red specifically stains collagen fibrils in tissues. Sirius red staining of tissue sections from femurs of 8-week-old mice showed that collagen fibrils were significantly reduced in Obif) ⁄ ) mice (Fig. 4A). Collagen 1a1 and 1a2 are known to be the major collagens expressed in osteoblasts (Marks & R. 2002). Using an antibody that recognizes both Collagen 1a1 and ⁄ or 1a2, we confirmed that Collagen 1a1 and ⁄ or 1a2 are markedly decreased in Obif) ⁄ ) mice (Fig. 4B). These results show that osteoblastogenesis is perturbed in Obif) ⁄ ) mice.

Discussion In our previous report, we found an uncharacterized membrane protein, Obif, which is highly expressed in osteoblasts in developing mouse embryos. Obif transcripts increase during osteoblastic differentiation in several skeletal cell lines (Kanamoto et al. 2009). In preosteoblastic MC3T3-E1 cells, matrix mineralization was stimulated upon overexpression of Obif. In contrast, matrix mineralization was inhibited when Obif

Fig. 3. Quantitative expression analysis of osteoblast markers in Obif-null mice. Quantitative real-time polymerase chain reaction (PCR) analysis using RNA from calvariae at P4 in littermate control mice and Obif) ⁄ ) mice. Expression of Collagen 1a1 and Osteopontin was markedly decreased in Obif) ⁄ ) mice (j), compared with those in littermate control mice (h). n = 4, *P < 0.03; Student’s t-test. ª 2012 The Authors Development, Growth & Differentiation ª 2012 Japanese Society of Developmental Biologists

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Fig. 4. Histological expression analysis in Obif-null mice. (A) Sirius red staining, which specifically stains collagen fibrils, of femoral epiphysis in Obif) ⁄ ) mice and littermate control mice at 8 weeks. Collagen fibrils significantly decreased in Obif) ⁄ ) mice. Scale bar = 500 lm. (B) Immunohistochemical analysis of the femur using an anti-Collagen 1 (a marker for early osteoblast development) antibody showed markedly decreased femoral epiphysis in Obif) ⁄ ) mice at 8 weeks. Scale bar = 100 lm.

was knocked down using a shRNA against Obif. These results strongly suggest that Obif plays a significant functional role in osteoblastogenesis. To elucidate a role for Obif during developmental osteogenesis, we generated Obif-deficient mice by targeted gene disruption. Skeletal preparations show no obvious phenotype in Obif ) ⁄ ) mice at P0, however, real-time PCR analysis demonstrated that osteoblastic markers including Collagen 1a1, Osteopontin, Runx2, and Osterix, were significantly decreased in the femur of Obif ) ⁄ ) mice at P4. Micro CT analyses revealed that Obif) ⁄ ) mice exhibit bone hypoplasia in the femur at P14. Furthermore, bone hypoplasia is more severe at the adolescent stage. Notably, cortical thickness showed a 20% decrease in Obif) ⁄ ) mice compared with control mice. These results suggest that OBIF is mainly involved in postnatal bone formation. In contrast, we observed no obvious difference in RANKL and OPG expression between the Obif) ⁄ ) mice and control littermate mice, suggesting that Obif is not involved in osteoclast function. Osteoblastogenesis consists of multiple processes conducted by transcription factors and secreted growth factors. The centerpiece in the transcription control of osteoblast differentiation is the RUNX2-mediated pathway. Runx2-deificient mice develop cartilaginous skeleton without any osteoblasts, indicating that osteoblast differentiation is arrested as early as E12.5 (Komori et al. 1997). OSTERIX, a zinc finger-containing protein that is known to be downstream of RUNX2, is

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expressed in osteoblast progenitors. Homozygous Osterix mutant mice also lack mature osteoblasts (Nakashima et al. 2002). Although other transcription factors have been reported to be involved in osteoblastic differentiation, the precise roles of these factors and their relationship with RUNX2 are less understood (Acampora et al. 1999; Jochum et al. 2000; Sabatakos et al. 2000; Satokata et al. 2000; Bialek et al. 2004; Yang et al. 2004). A previous report suggested that Obif is one of the molecules downstream of Runx2 (Hecht et al. 2007). In addition, Obif promoters contain a number of putative RUNX2-binding sites in multiple species (Kanamoto et al. 2009). We found that expression level of Runx2 is downregulated in Obif) ⁄ ) mice. There may be reciprocal regulation between Runx2 and Obif. To determine whether OBIF can influence bone metabolism at the senescent stage, we analyzed the femurs of aged Obif) ⁄ ) mice. Our data showed that bone components of femurs are unaltered between Obif) ⁄ ) and control mice at 24 weeks. In control mice, trabecular bone marker, BV ⁄ TV, Tb.N and Tb.Th, were reduced at the senescence stage compared with that at the adolescent stage; however, Obif) ⁄ ) mice show a similar reduction of trabecular bone markers, suggesting that bone development in Obif) ⁄ ) catches up with that in control mice at the senescent stage. Obif seems to exert an essential function mainly in the adolescent period but not in the senescence period in osteogenesis. Our current results suggest that OBIF promotes bone formation. Obif) ⁄ ) mice showed decreased expression of osteoblastic marker genes, including Collagen 1a1, Runx2, and Osterix, and mCT analysis revealed that Obif) ⁄ ) mice exhibit severe bone hypoplasia. These results imply that OBIF protein may be useful for the treatment of osteoporosis in human. The majority of osteoporosis patients are currently prescribed anti-reabsorptive drugs, including bisphosphonate; however, the patients recover insufficient bone volume (Rodan & Martin 2000). On the other hand, parathyroid hormone is the only single drug currently available to the public that improves bone mass by osteoblastmediated bone formation (Canalis et al. 2007; Garrett 2007). Administration of OBIF in combination with antireabsorptive drug may enhance the therapeutic effect in osteoporotic patients.

Acknowledgments This work was supported by A-Step (Adaptable and seamless technology transfer program through targetdriven R&D), CREST, and PRESTO from Japan Science and Technology Agency, a grant for Grants-in-Aid for Scientific Research on Priority Areas, Grant-in-Aid for

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Scientific Research (B), Specially Designated Research Promotion and Scientific Research on Innovative Areas ‘‘Intracellular Logistics’’ from the Ministry of Education, Culture, Sports and Technology of Japan, The Takeda Science Foundation, The Uehara Memorial Foundation, Novartis Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research and The Naito Foundation, Senri Life Science Foundation, Kato Memorial Bioscience Foundation. We thank Dr Urade, Dr Aritake, and H. Suzuki for laboratory CT, and M. Kadowaki, A. Ishimaru, T. Tsujii, A. Tani, H. Abe, and S. Kennedy for technical assistance.

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