JP2008017790A - Calcification-controlling agent and method for screening the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To elucidate blood vessel and bone calcification-controlling agent of FCF23 and to provide a prophylactic or therapeutic agent for controlling calcification of blood vessel, bone or tooth and to provide a method for screening a substance mutually interacting with an FGF23 receptor and an FGF23-specific signal molecule. <P>SOLUTION: The calcification controlling agent targets at a fibroblast growth factor 23 (FGF23), an FGF 23-specific receptor, a specific signal molecule or its specific signaling mechanism. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a prophylactic / therapeutic agent for osteoarthritis, ectopic ossification, arteriosclerosis or osteoporosis, rickets, osteomalacia, an alveolar bone resorption inhibitor associated with periodontal disease, tooth (dentin) , Cementitious). Furthermore, it is related with the screening method of these preventive / therapeutic agents.

  Vascular calcification has much in common with bone calcification, and vascular calcification is strongly involved in pathologies such as diabetes, chronic renal failure, and onset of arteriosclerosis. In addition, ectopic calcification includes inflammation, soft tissue ossification associated with tumors, and the like. Furthermore, as a disease in which ossification is a problem, osteoarthritis that causes pain due to ossification of a part of cartilage is known. Statins and bisphosphonates are candidates for the treatment of these diseases. The former is considered to show an effect by inhibiting phosphatization by inhibiting alkaline phosphatase activity, which is entrusted to the production of inorganic phosphate, among various activities. The latter inhibits calcification deposition due to the high affinity of calcium and phosphorus. However, there is no effective prophylactic / therapeutic agent that directly targets calcification of the vascular circulatory system, which seems to be particularly important clinically.

  In smooth muscle cells of calcified lesions, the expression of transcription factor Runx2, which is essential for osteoblast differentiation, is induced, and various osteoblasts such as osteoblast markers and sodium-dependent phosphate transporter (NPT) are common. Protein will be made. However, many molecular mechanisms of bone and blood vessel calcification are still unknown. A negative correlation is created between osteoporosis and vascular calcification in the changes in calcium / phosphorus metabolic hormones associated with aging and menopause. In recent years, attention has been paid to the relationship between blood vessels and bones, and so far, as a direct or indirect positive / negative factor for bone mineralization, type III NPT Pit-1 and its regulators (stanniocalcin) (Patent Literature) 1) is known.

  Fibroblast growth factor 23 (FGF23) belongs to the FGF family of 22 structural analogs with 25-36% homology (Non-Patent Document 1, Non-Patent Document 2). FGF23 was discovered as a responsible factor for hereditary / neoplastic hypophosphatemic rickets / osteomalacia (Non-patent Document 3, Non-patent Document 4, Non-patent Document 5). FGF23 is an important factor that inhibits absorption of Pi in renal tubules and intestinal tracts through suppression of expression of Npt2a and Npt2b belonging to type II NPT (Non-patent Document 6, Non-patent Document 7, Non-patent Document 8). . In these hypophosphatemic rickets and osteomalacia, blood FGF23 rises, reabsorption of Pi in the kidney and activation of vitamin D are suppressed, and hypophosphatemia is induced to induce bone tissue Causes calcification disorders. However, even in vitamin D-resistant X-linked hypophosphatemic rickets, the blood level of FGF23 is high (Non-patent Document 9), so it is said that the function of FGF23 is actually well understood. hard. On the other hand, it is speculated that not only these diseases but also FGF23 has an important role in physiological phosphate metabolism, but there are cases where the blood Pi level and the FGF23 level do not correlate (Non-patent Document 10). Its physiological significance or actual condition such as effects on bones are hardly clarified.

  The biological response to FGF is via binding of specific cell surface receptors to tyrosine kinase type receptors (FGFR). FGFR consists of six families and exerts various biological actions from the expression level in tissues, binding to ligands, and diversity of signal transduction pathways. FGF23 has been confirmed to bind to FGFR1 to 3 c-splice forms and FGFR4 among these 6 FGFRs (Non-patent Document 11).

  Furthermore, the binding of FGF23 and FGFRs involves the senescence-inhibiting gene product Klotho, which activates p42 / 44MAPK (ERK1 / 2) of mitogen-activated protein kinase (MAPK) (Non-patent Document 12). It has not yet been clarified how these FGF23 signal transduction mechanisms are actually related to the phosphate metabolism regulating action of FGF23.

  Even in normal times, FGF23 expression has been confirmed in various tissues including vascular endothelial cells, and among them, the expression level in bone tissues is approximately 10 times or more that in other tissues (Non-patent Document 13). For example, osteoblasts are the main production cells of FGF23 (Non-patent Document 14). Therefore, blood FGF23 levels are expected to be strongly influenced by the regulation of expression in bone tissue, particularly osteoblasts. It has been reported that blood FGF23 level and FGF23 production in cultured osteoblasts are positively controlled by active vitamin D (Non-patent Document 15).

However, the detailed mechanism of FGF23 and its biological response on blood vessels and bone mineralization remains unknown.
JP 2005-281189 A Martin, G.M. R. Genes & Dev. 12: 1571-1586 (1998) Ornitz, D.M. M.M. and P.M. J. et al. Marie. Genes & Dev. 16: 1446-1465 (2002) ADHR Consortium. Nat. Genet. 26: 345-348. (2000) Jonsson, K. B. , Et al. N. Engl. J. et al. Med. 348: 1656-1663 (2003) White, K.K. et al. Kidney Int. 60: 2079-2086 (2001) Yamashita, T .; et al. J Biol. Chem. 277: 28265-28270 (2002). Yan, X. et al. Genes Cells 10: 489-502 (2005) Miyamoto, K. et al. et al. Ther. Apher. Dial. 9: 331-335 (2005) Yamazaki, Y. et al. et al. J. et al. Clin. Endocrinol. Metab. 87: 4957-4960 (2002) Weber, T .; J. et al. et al. J. et al. Bone Miner. Res. 18: 1227-1234 (2003) Yu, K .; et al. Development 130: 3063-3074 (2003) Kurosu, H .; et al. J. et al. Biol. Chem. 281: 6120-6123 (2006) Mirams, M.M. et al. Bone 35: 1192-1199 (2004) Mirams, M.M. et al. Bone 35: 1192-1199 (2004) Kolek, O .; I. et al. Am. Physiol. Gastrointest. Liver Physiol. 289: G1036-1042 (2005)

  Then, the present inventors elucidate the blood vessel and bone calcification regulation mechanism of FGF23, and provide a preventive / therapeutic agent that regulates calcification of blood vessels, bones, or teeth.

  Another object of the present invention is to provide a screening method for substances that interact with FGF23 receptor and FGF23-specific signal molecules.

In order to achieve the above object, the present inventors have conducted intensive research, and as a result, have found the following results.
(1) In a cultured bone formation model using fetal rat calvaria-derived (RC) cells, overexpression of human FGF23 using an adenovirus expression vector significantly reduced the number of bone nodules.
(2) Overexpression of human FGF23 in RC cells promoted FGFR and MAPK phosphorylation in the cells.
(3) Overexpression of human FGF23 in RC cells suppressed the NPT activity of the cells.
(4) Overexpression of human FGF23 in RC cells affected the expression of osteoblast differentiation marker and phosphate metabolism regulator mRNA.
(5) A specific inhibitor of FGFR tyrosine residue phosphorylation SU5402 inhibited the effect of overexpression of human FGF23 in RC cells.
(6) In a calcification model of rat aorta-derived smooth muscle cells by Pi loading, FGF23
Overexpression suppressed calcification.
(7) The high expression part of FGF23 was confirmed not only in osteoblasts but also in dental hard tissues such as cement blasts and odontoblasts.

  Based on these results, the present inventor has shown that FGF23 is a local factor that regulates calcification associated with pathological conditions such as physiological calcification of hard tissue or vascular calcification, and makes FGF23 positive or negative. It has been found that the calcification can be suppressed or promoted by controlling, and the present invention has been completed.

  From the above invention, it was revealed that FGF23 regulates calcification. This increase in the expression level of FGF23 or activation of its information transmission system is important in developing preventive / therapeutic agents for osteoarthritis, ectopic calcification, and arteriosclerosis in order to suppress calcification. Give means. On the other hand, since the decrease in the expression level of FGF23 or the inhibition of the information transmission system promotes calcification, it prevents or treats osteoporosis, rickets, osteomalacia, and an alveolar bone resorption inhibitor associated with periodontal disease, tooth It provides an important means in developing regenerative therapeutic agents (dentin, cementum).

  The present invention provides a regulator of calcification of FGF23. Examples of “modulation” may include “promotion” or “activation” that is positively controlled, “inhibition” or “suppression” that is negatively controlled. As an example of “positive” control, it is possible to show suppression of calcification by increasing the expression level of FGF23 or activating the FGF23-specific information transmission mechanism. Moreover, as an example of controlling to “negative”, an example of calcification promotion by decreasing the expression of FGF23, inhibiting the binding of FGF23 to a specific receptor, or inhibiting its signal transduction system can be shown.

  That is, an activator of increased expression of FGF23 or an FGF23-specific information transmission mechanism can be used for suppression of calcification, and as a disease / condition in which calcification is promoted, osteoarthritis, ectopic calcification, arteries It can be used for prevention and treatment of sclerosis.

  In addition, FGF23 or an inhibitor of FGF23-specific information transmission mechanism can be used to promote calcification, and as a disease / condition in which calcification is inhibited or suppressed, it is used as a prophylactic / therapeutic agent for osteoporosis, rickets and osteomalacia. Available. Furthermore, since FGF23 has been expressed at a high level in tooth tissues (cement blasts and odontoblasts), it can be used in fields such as dental region diseases such as periodontal disease and regenerative medicine.

  FGF23 can be mentioned as a specific example of the calcification regulator according to the present invention. FGF23 consists of the amino acid sequence set forth in SEQ ID NO: 2, but the present invention is not limited to this, and has a function capable of regulating a specific signal transmitter of FGF23. In the amino acid sequence represented by SEQ ID NO: 2, Any FGF23 mutant having a sequence similar to the amino acid sequence in which one or more amino acids are substituted, deleted, or added may be used.

  Among the proteins of the present invention, those present in nature can be prepared as natural proteins through operations such as extraction and purification. For example, the protein having the amino acid sequence of SEQ ID NO: 2 can be prepared from human cells, body fluids (blood, urine, etc.) and the like. The cells here are not particularly limited as long as they produce or express the protein of the present invention, and for example, spleen cells, immune system cells and the like can be used.

  The present invention also provides DNA encoding FGF23 or a variant thereof. Examples of the DNA encoding human FGF23 include the DNA described in SEQ ID NO: 1. Further, the sequence encoding the FGF23 variant can be prepared, for example, by modifying the DNA encoding human FGF23 described in SEQ ID NO: 1.

  The FGF23 cDNA can be prepared by methods known to those skilled in the art. The DNA of the present invention can be prepared from a suitable genomic DNA library or cDNA library. In this case, a probe composed of a DNA fragment encoding a part of the protein of the present invention is prepared, and the DNA of the present invention is screened by using a known hybridization method. The DNA of the present invention can be prepared by a PCR method using an appropriate primer using an appropriate genomic DNA library or cDNA library as a template. As the primer, any primer designed to specifically amplify a desired DNA fragment based on the DNA sequence encoding the protein of the present invention can be used. The genomic DNA library or cDNA library used for preparing the DNA of the present invention may be any as long as it contains the DNA of the present invention. For example, a commercially available DNA library can be used. . Alternatively, a cDNA library prepared by a well-known method using mRNA extracted from appropriate cells as a template can also be used.

  Although FGF23 cDNA can be used as it is, it can also be used by carrying it on a vector.

  Yet another aspect of the present invention relates to a vector carrying the DNA of the present invention. In other words, the DNA of the present invention can be used by inserting it into an appropriate vector. Any vector can be used as long as it can insert the DNA of the present invention, but it is suitable depending on the purpose of use (cloning, protein expression) and in consideration of the type of host cell. It is preferable to select and use a vector. Examples of vectors include M13 vectors, pUC vectors, pT7 vectors, etc., when Escherichia coli is used as a host. The DNA of the present invention can be inserted into a vector by a known method using a restriction enzyme and DNA ligase (Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York).

  Yet another aspect of the present invention relates to a transformant carrying the DNA of the present invention. That is, the present invention relates to a transformant obtained by transforming a host cell with the DNA of the present invention. For example, the DNA of the present invention can be obtained by the calcium phosphate method, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, PL et al., Proc. Natl. Acad. Sci. US 84, 7413-7417 (1984)), microinjection (Graessmann, M. & Graessmann, A., Proc. Natl. Acad. Sci.U.S.A 73,366-370 (1976)) can be introduced into a host cell and transformed. In addition, the transformant of the present invention can be obtained by transforming a host cell with the vector of the present invention. Various host cells can be used depending on the purpose, and for example, CHO cells, COS cells, HeLa cells, insect cells, yeasts and the like can be used. Further, not only eukaryotic cells but also prokaryotic cells such as Escherichia coli can be used. By culturing the transformant of the present invention under appropriate conditions, it is possible to produce a large amount of the expression product (protein) of the DNA of the present invention.

  Examples of the method of applying the protein of the present invention include direct administration to a patient. Moreover, the composition which uses the protein of this invention as an active ingredient can be comprised, and this can also be administered to a patient. As an administration method, a known method such as oral or intravenous, intraarterial, or subcutaneous injection can be used. Moreover, it can also administer directly to a specific site | part (for example, site | part in which the bone has decreased or formed excessively) by injection etc. In addition, the DNA of the present invention can be introduced into cells to express the protein of the present invention in vivo. The method for introducing DNA is not particularly limited, and can be performed by a known method such as a method using a DNA introduction plasmid or a viral vector, electroporation, lipofection, microinjection, or the like.

  The present invention also includes functional nucleic acids such as antisense nucleotides, double-stranded RNAs, ribozymes, and the like that target any site, including transcribed regions and promoter regions of the FGF23 gene, and lime containing such functional nucleic acids as active ingredients. Provide oxidization regulators. Such a functional nucleic acid may be either siRNA, double-stranded RNA, or siRNA or double-stranded RNA containing a modified RNA strand in at least one strand. The functional nucleic acids described above can be administered to a patient by intravenous injection, subcutaneous injection, oral delivery, liposome delivery or intranasal delivery and accumulate in the patient's target systemic, organ, tissue or cell type.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Examples "Example 1" FGF23 suppresses osteoblast differentiation and substrate calcification in rat fetal calvarial (RC) cell cultures. Experiments were performed by the following method.
1) Construction of adenovirus vector having human FGF gene Full-length human FGF23 cDNA was synthesized from total RNA of human heart using RT-PCR, and EcoR1 at the cloning site of pTRE-shuttler2 vector (BD Bioscience, PaloAlto, CA) and Inserted between BamH1 sites. Tet-responsible expression cassette was isolated by I-Ceu1 and PI-Sce1 and introduced into Adeno-X viral DNA (BD Bioscience). Recombinant Adeno-X FGF23 DNA (Ad-FGF23) was prepared by digesting with PacI and transfecting linear recombinant Ad-FGF23 DNA into HEK 293 cells according to a conventional method. An adenovirus vector (Ad-Tet-off) for control of expression and a control adenovirus vector (Ad-βgalactosidase; AD-βgal) were prepared according to the attached materials. The adenovirus particles produced in the culture supernatant were purified using BD-Adeno-X virus purification kit (BD Bioscience), and the concentrated adenovirus particles were calculated for plaque forming units (pfu) using HEK cells.
2) Isolation of RC cells from fetal rat calvaria-derived (RC) cell culture and adenovirus infected fetal rats (Yoshiko Y, et al. Endocrinology. 144: 4134-4143. (2003)), 0.3X10 ⁴ / cm 70% confluence with 5% fetal serum (FCS), 50 μg / ml ascorbic acid, α-modified essential medium (α-MEM) (Gibco BRL-LifeTechnologies, Gaithersburg, MD) with antibiotics at a cell density of ² Cultured until complete. At this point (d4), Ad-FGF23 or Ad-βgal was infected with Ad-Tet-off at a titer of 20 pfu / cell. Overexpression of FGF23 was expected 2 days after differentiation was initiated. Four hours after infection, fresh medium was added and replaced with fresh medium the next day. On the other hand, RC cells at the mature stage (d14) were similarly infected under the condition of 20 pfu / cell. In order to induce calcification of nodules, 50 mM β-glycerophosphate (βGP) was added 2 days before the end of the culture (d15). In culture, the medium was changed at 37 ° C. in the presence of 5% CO 2 generally every 2 to 3 days.
3) Western Blot Ad-FGF23 or Ad-βgal infected cells were removed after 48 hours with cell lysate (100 mM KCl, 1 mM EDTA, 0.5% Nonident P-40, 1 mM phenylmethylsulfonfluoride, complete protease inhibitors, biosensors, biosensors ).
Similarly, the culture supernatant of the infected RC cells was collected, and a part thereof was subjected to enzyme-linked immunoassay (ELISA, described later). The remainder was subjected to acetone precipitation at −20 ° C., and the precipitate was dissolved in the cell lysate.

The sample was electrophoresed by SDS-PAGE, transferred to a nitrocellulose membrane, reacted with an anti-FGF23 antibody (× 500, Santa Cruz Biotechnology, Santa Cruz, Calif.) At 4 ° C. overnight, and then a peroxidase-labeled secondary antibody ( × 2,000, Santa Cruz) at room temperature for 1 hour. Subsequently, chemiluminescence was detected using an ECL Western blotting detection reagent (Lumi-Light ^PLUS Western Blotting Substrate, Roche Diagnostics).
4) ELISA
FGF23 secreted into the culture solution was quantified by a Kinos ELISA kit (Tokyo).
5) Identification of calcified nodules RC cells were fixed with 10% neutral buffered formalin after culturing , and nodules were identified by alkaline phosphatase (ALP) staining and calcification was identified by von Kossa staining.
6) Cell proliferation The cell proliferation rate was measured by 3 (4,5-dim4thyl-thiazoyl-2yl) 2.5-diphenyltetrazulimide thiol blue colorimetric method (MTT assay).

  The cultured bone formation model using RC cells and the experimental protocol are shown in FIG. 1A. Undifferentiated RC cells proliferate and reach confluence in 6 days. At the same time as confluence, the cells differentiate into preosteoblasts with the formation of nodules consisting of an organic matrix corresponding to osteoids. On the 14th day of culture, the cells mature as osteoblasts. When β-glycerophosphate (βGP) is added at this time, osteoid-like nodules are mineralized within 2 days. The adenovirus-human FGF23 vector (Ad-hFGF23) construct is shown in FIG. 1B. Two days after infection, the expression of FGF23 in Ad-hFGF23 and Ad-βgal-infected cells and the level of FGF23 in the culture supernatant were compared by Western blotting and ELISA. As a result, significant expression and increased secretion were confirmed in Ad-hFGF23-infected cells. (FIG. 2A, B). In addition, in the culture supernatant of Ad-hFGF23-infected cells, an intact molecular size (about 32 kDa) and two bands (about 26 kDa and about 16 kDa) that seem to be a degradation product although weak were recognized (FIG. 2C).

  FIG. 3A shows an example of a stained image after completion of culture (d17) by infecting Ad-hFGF23 or Ad-βgal with d4 and d14 matured RC cells. From the measured values of the obtained stained images, it was revealed that overexpression of FGF23 in RC cells suppressed both nodule formation and calcification (FIGS. 3A and 3B).

  When RC cells infected with Ad-hFGF23 or Ad-βgal were subcultured at a low density after 2 days and the cell growth curves were compared over time using the MTT assay, there was no significant difference between the two. (FIG. 3C).

  Example 2 FGF23 suppresses bone formation in parietal bone organ culture.

The experiment was carried out by the following method.
1) Rat parietal bone organ culture The parietal bone of the fetal rat on day 21 was used for organ culture. The parietal bone was cut in half along the sagittal suture and further divided in half. A piece of parietal bone was digested with collagenase and the total number of cells was counted. After the parietal bone fragment was infected with Ad-FGF23 or Ad-βgal at 20 pfu / cell for 4 hours, it was placed on a cell culture insert (pore size 0.4-μm; Millipore, Bedford, NJ). The medium used was BGjb medium supplemented with 20% FCS. To confirm the amount of bone formation, calcein (1 μg / ml) was added the next day. The medium was changed every day, and after further culturing for 3 days, it was fixed with 4% PFA at 4 ° C. for 2 hours, and it was made into a 7 μm frozen section and analyzed histologically.
2) Immunostaining anti FGF23 antibody (× 100, Santa Cruz) was reacted overnight at 4 ° C., after washing peroxidase-labeled secondary antibody and ABC reagent (Vector Laboratories, Burlingame, CA) was reacted analogously to the instructions of the . Sections were counterstained with hematoxylin. Negative subjects used a specific blocking peptide (Santa Cruz) according to the formulation (not shown).

  When Ad-hFGF23 was infected, a stronger FGF23 signal was confirmed in the cell group on the bone surface compared to Ad-βgal (FIGS. 4A and B). The expansion of the osteoid layer identified by toluidine blue staining was confirmed by the infection with Ad-hFGF23 (FIGS. 4C and D). In addition, the fluorescence area of accumulated calcein decreased (FIGS. 4E and F). Their quantitative values are shown in FIG. 4G.

[Example 3] Co-expression of FGF23 and FGFR in RC cells The experiment was performed by the following method.
1) Semiquantitative RT-PCR and real-time quantitative RT-PCR analysis Total RNA of RC cells was collected over time using TRIzol (Invitrogen, Carlabad, CA). cDNA was synthesized at 50 ° C. from 2 μg of total RNA using RiverTra Ace (Toyobo, Tokyo). PCR (Qiagen, Hilden, Germany) was performed under conditions optimal for the target gene. Ribosomal protein L32 was used as an internal control. Real-time quantitative PCR was carried out using 1 × SYBR Green Realtime PCR Master Mix (Toyobo) and LightCycler (Roche Diagnostics). Osteopontin (OPN), ALP, bone sialoprotein (BSP), osteocalcin (OCN), FGF23, FGFR1 (R1), FGFR2 (R2), FGFR3 (R3), two splice forms (b and c), FGFR4 (R4) (Yan, X., et al. Genes Cells. 10: 489-502. (2005)) and L32 primer sequences are shown in Table 1.

  Primer set used for RT-PCR

  In RC cells, R1c, R2IIIc, and R3c mRNA expression was confirmed, but no other subtype mRNA was confirmed (FIG. 5A). In quantitative real-time RT-PCR analysis, the continuous increase in the expression level of OPN, ALP, BSP, OCN is differentiated from undifferentiated osteogenic cells to premature osteoblasts through mature osteoblasts as expected by RC cells (FIG. 5B). R1c and RIIIc levels showed peak values at the pre-osteoblast stage, whereas R3c showed strong specificity at the mature stage of osteoblasts.

  Example 4 FGF23 promotes phosphorylation of FGFR.

  The experiment was carried out by the following method.

  The supernatant obtained by culturing RC cells infected with Ad-hFGF23 and Ad-βgal according to [Example 1] for 5 days was recovered (overexpression of FGF23 was confirmed by ELISA) and diluted 10-fold with α-MEM. . Mature (d14) RC cells were subcultured on glass slides and allowed to stand for 1 day, and the serum concentration was lowered to 0.1% and further cultured for 1 day. The above diluted culture supernatant was added thereto, and immediately after 15 minutes, it was fixed with cold acetone for 5 minutes. After blocking the cells, the primary antibody (anti-Runx2 antibody, anti-phosphorylated FGFR antibody, x100, Santa Cruz) was reacted overnight at 4 ° C. The secondary antibody used was labeled with fluorescein isothiocyanate (FITC) (Santa Cruz Biotechnology, Inc), Cy3 (Jackson ImmunoResearch).

  In Ad-hFGF23 and Ad-βgal-infected RC cells, reactivity to anti-phosphorylated FGFR antibodies (anti-pFGFRs) was clearly strongly positive for Ad-FGF23-infected cells, whereas Ad-βgal-infected cells were Almost no staining (FIG. 6A). Moreover, these cells were FGF23 and FGFR1-FGFR3 positive (FIG. 6B).

[Example 5] An experiment in which FGF23 suppresses calcification via FGFR of RC cells was performed by the following method. As a phosphorylated FGFR inhibitor, SU5402 (CALBIOCHEM, La Jolla, Calif.) Was used.

  Ad-hFGF23 and Ad-βgal were infected at the mature stage (d14) as in [Example 1]. The above inhibitor (10 μM) with medium change from the day after infection. After completion of the culture (d17), ALP / von Kossa staining was performed as described in [Example 1], and nodules and calcification were measured.

  An inhibitor of FGFR phosphorylation partially restored the suppression of nodule calcification by FGF23 (FIG. 7).

  Example 6 FGF23 regulates the expression of osteoblast differentiation marker and phosphate metabolism regulator mRNA.

  The experiment was carried out by the following method.

  According to [Example 1], 70% confluence (d4) and matured (d14) RC cells were infected with Ad-hFGF23 or Ad-βgal, and after completion of the culture, total RNA was recovered according to [Example 3] The sample was subjected to quantitative real-time RT-PCR. In addition to the osteoblast markers in Table 1 of [Example 3], as a phosphate metabolism regulator, matrix extracellular phosphoprotein (MEPE) (Gowen, L. C. et al. J Biol Chem. 278: 1998-2007. 2003)), secreted frozen-related protein (sFRP) -4 (Berndt T. et al., J Clin Invest 112: 785-794, (2003)), stanniocalcin (STC) 1 (Yoshiko, o. 144: 4134-4143 (2003)) and secreted proteins and Pit-1, Pit-2, phosphate-regula ing gene with homologies to endopeptidases on X chromosome (Phex) (Drezner, M.K Kidney Int.57:. 9-18 (2000)) were analyzed membrane protein. The respective primer sequences are shown in Table 2.

  Primer set used for RT-PCR

  When Ad-hFGF23 was infected to d4, the mRNA levels of OCN, Phex, and MEPE were significantly reduced, and the levels of ALP, STC1, and Pit-1 mRNA were also decreased as compared to Ad-βgal. On the other hand, BSP and sFRP-4 mRNA levels increased and OPN remained unchanged (FIG. 8A). On the other hand, when Ad-hFGF23 was infected with d14, an increase in ALP, Pit-1, Pit-2, MEPE, STC1 mRNA levels, and a decrease in Phex and OCN mRNA levels were observed. However, OPN, BSP, and sFRP-4 mRNA levels were unchanged (FIG. 8B).

  "Example 7" FGF23 suppresses Pi-induced calcification of cultured vascular smooth muscle.

  The experiment was carried out by the following method.

Rat aorta-derived smooth muscle cells (within passage 15) were plated at 0.5 to 0.8 × 10 ⁴ / cm ² using 10% FCS-added D-MEM. When cells reached confluence, they were loaded with 1.5 mM sodium phosphate (pH 7.4) (calcification medium) and simultaneously infected with Ad-hFGF23 or Ad-βgal to 20 pfu / cell. The basic procedure of infection followed [Example 1]. The fresh calcification medium was replaced every 2-3 days.
On day 10 after infection, the cells were washed, washed with Tris buffer, treated with 0.6N hydrochloric acid, and calcification was measured with Calcium C Test-Wako (Wako Pure Chemical Industries, Tokyo).

  In Ad-hFGF23-infected smooth muscle, calcification due to phosphate load was significantly suppressed as compared to Ad-βgal-infected smooth muscle (FIG. 9).

  Example 8 FGF23 is also produced in dentin and cementum.

  Total RNA was collected from adult male (8 weeks old) and fetal (embryonic day 21) rat tissues according to [Example 3], and the expression of FGF23 was confirmed by real-time RT-PCR. In addition, FGF23 immunostaining was performed on the dental tissues of the rats by the ABC method (Vector Laboratories, Burlingame, Calif.). The lower jaw of an adult male rat was fixed with 4% paraformaldehyde at 4 ° C. overnight, then decalcified with a citric acid / formic acid solution or EDTA to obtain a paraffin specimen. Sections of 5 to 6 μm were prepared, and anti-FGF23 antibody (Santa Cruz) was reacted at 4 ° C. overnight. As a result of real-time PCR, high expression was confirmed in tooth tissues other than bone tissues (FIG. 10). In the immunostaining, odontoblasts and cementoblasts were FGF23 positive (FIG. 11). From this, it was shown that FGF23 is produced in cells (excluding enamel blasts) that are subjected to hard tissue matrix formation.

  The mechanism by which FGF23 clarified by the above invention regulates calcification, specifically, the increase in the expression of FGF23 or the activation of its signal transduction system suppresses calcification, so that osteoarthritis and ectopic lime are suppressed. It can be used as a preventive / therapeutic agent for keratosis and arteriosclerosis. On the other hand, the decrease in the expression of FGF23 or the inhibition of its information transmission system promotes calcification, so that it is a preventive / therapeutic agent for osteoporosis, rickets and osteomalacia, as well as dental diseases such as periodontal disease or regeneration in dentistry. It can also be used for medical treatment. Furthermore, it can be used as a method for screening for preventive / therapeutic agents for these diseases.

(A): Cultured bone formation model and experimental protocol using RC cells. (B): Structure of hFGF23 recombinant adenovirus vector. ITR, inverted terminal repeats (inverted terminal repeats). hCMV-1, a tetracycline responsive element-added cytomegalovirus promoter (minimum immediate early promoter of cytomegalovirus). Confirmation of overexpression of hFGF23 in RC cells. (A): Expression of hFGF23 gene and protein in cells. (B): Quantification of hFGF23 in the culture supernatant by ELISA. (C): Properties of hFGF23 protein in the culture supernatant. Effect of hFGF23 overexpression in a cultured bone formation model of RC cell culture. d4 (A) and d14 (B) RC cells were infected with the viral vector and sampled at d17. (A): ALP and von Kossa staining. (B): Number of ALP positive nodules and calcified nodules by staining of (A). Numbers are mean ± SD. The graph in (A) and the graph in the lower row of (B) are both significantly different from Ad-βgal ((1) P <0.05, (2) P <0.01). n = 4. (C): Growth curve by MTT assay of cells subcultured after infection of d4RC cells with viral vector. Numbers are mean ± SD. There is no significant difference between the two on the same day. n = 4. Effect of FGF23 overexpression in parietal bone organ culture. (A): FGF23 overexpression by immunostaining. (B): Fluorescent image of calcein. (C): Quantification of calcein fluorescence amount of (B). Numbers are mean ± SD. All are significantly different from Ad-βgal (P <0.05). n = 8. Co-expression of FGF23 and FGFRs in RC cells. (A): Expression of FGFR subtype mRNA in RC cells by RT-PCR. (B): Osteoblast differentiation marker associated with osteoblast differentiation by real-time quantitative RT-PCR, mRNA levels of FGF23 and FGFR. Numbers are mean ± SD. n = 3. Activation of FGFR by overexpression of hFGF23 in RC cells. (A): Fluorescent immunostaining with anti-FGFR phosphorylated antibodies (anti-pFGFRs). (B): Staining with anti-FGF23 antibody and anti-FGFR1-3 antibody. FGFR phosphorylation inhibitor against calcification suppression by overexpression of hFGF23 in RC cells. Numbers are mean ± SD. There is a significant difference in comparison with Ad-βgal (P <0.05). n = 4. Effect of FGF23 overexpression on osteoblast markers of RC cells and Pi metabolism-related factor mRNA levels. d4 (A) and d14 (B) RC cells were infected with the viral vector and sampled at d17. Numbers are mean ± SD. n = 3. Effect of FGF23 overexpression on Pi-induced calcification of rat aortic smooth muscle cells. Numbers are mean ± SD. There is a significant difference in comparison with Ad-βgal (P <0.05). n = 4. FGF23 mRNA expression level. Numbers are mean ± SD. All are significantly different from the fetus (* P <0.05, ** P <0.01). n = 3. Femur (femur), Calvaria (parietal bone), Incisor (incisor), Brain (brain), Thymus (thymus), Spleen (spleen), Kidney (kidney), Liver (liver), Heart (heart), S. et al. intestine. Localization of FGF23 in adult male rat teeth and periodontal tissues. Methyl green is counterstained. Panel a shows a mandibular sagittal cut including incisors (ir), molars (mo), and alveolar bone (ab). Pdl, de, and ce pu respectively indicate periodontal ligament, dentin, cementum, and dental pulp. Panels b to d are strong enlargements of panels a to B. Cylindrical odontoblasts (§), cementoblasts (Θ), and alveolar bone osteoblasts (*) show stronger staining than pdl and pu cells. Some of the cement cells (∞) embedded in the matrix as well as the alveolar bone cells (arrowheads) are positive. Panel e is a negative control. Corresponds to area C of panel a. The bar is 50 μm.

Claims (11)

  A calcification regulator that targets fibroblast growth factor 23 (FGF23), an FGF23-specific receptor, a specific signal molecule, or a specific signal transduction mechanism thereof.   A calcification inhibitor for osteoarthritis, ectopic calcification, and arteriosclerosis, comprising FGF23 as an active ingredient.   A DNA (Fgf23) comprising the base sequence represented by SEQ ID NO: 1 as an active ingredient according to claim 2 or a protein (FGF23) comprising an amino acid sequence represented by SEQ ID NO: 2.   4. The calcification inhibitor for osteoarthritis, ectopic calcification, and arteriosclerosis according to claim 2 and 3, wherein the DNA is carried on a vector.   The calcification inhibitor of osteoarthritis, ectopic calcification, or arteriosclerosis according to claim 2 or 3, wherein the protein is a recombinant.   6. The variant according to claim 2 to 5, wherein the active ingredient is a FGF23-specific signal molecule such as a mitogen-activated protein kinase containing at least JNK downstream of the FGF23-specific receptor or a compound that activates a specific signal transduction mechanism. Calcification inhibitor for osteoarthritis, ectopic calcification, and arteriosclerosis.   An osteoporosis, rickets, osteomalacia calcification promoter, alveolar bone resorption inhibitor associated with periodontal disease, and regenerative treatment of teeth (dentin, cementum) containing an inhibitor of FGF23 as an active ingredient.   The osteoporosis according to claim 7, wherein the inhibitor is a FGF23-specific antisense, a nucleic acid such as RNAi, a neutralizing antibody, a receptor antagonist, a specific signal molecule or a compound that inhibits a specific signal transduction mechanism, Calcification promoters for rickets and osteomalacia, alveolar bone resorption inhibitors associated with periodontal disease, and regenerative treatments for teeth (dentin and cementum).   The calcification regulating agent according to claim 1, relating to FGF23 targeting a diseased site such as bone, cartilage, blood vessel, muscle, or tooth.   A drug delivery system for the therapeutic agent according to claim 1 to 9.   A screening method for a calcification regulating agent that interacts with FGF23, FGF23 receptor or FGF23-specific signal molecule.

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