WO2011158655A1 - Use with utilization of soluble klotho-, fgf23- and fgfr-complex formation mechanism in calcified tissue - Google Patents

Abstract

Provided are a method for screening an agent for controlling calcification, to which signal transduction relates, common to hard tissue and/or blood vessel; a calcification controlling agent; a method for examining calcification of a bone, calcification of a blood vessel and the pathological advance of a disease accompanying the same; and an examination kit therefor. The method for screening an agent for controlling calcification of hard tissue and/or blood vessel according to the first aspect comprises a step for, in a specimen in the absence and presence of a test compound, contacting soluble Klotho with FGFR and FGF23, and a step for selecting a compound causing a change in the specific signal transduction. The calcification controlling agent according to the second aspect comprises a compound capable of inhibiting or promoting the formation of a soluble Klotho-containing complex. The method for examining calcification according to the third aspect comprises a step for measuring the concentration of soluble Klotho, etc. The kit for examining calcification according to the fourth aspect contains a ligand having binding affinity to soluble Klotho, etc.

Description

Use of solubilized Klotho, FGF23 and FGFR complex formation mechanism in calcified tissue

The present invention relates to a screening method for a calcification regulating agent, a calcification regulating agent, and bone mineralization, vascular calcification, and pathological progression associated therewith, associated with signal transduction common to calcification of hard tissue and / or blood vessels. And a test kit for the same.

In recent years, it has been revealed that bone diseases such as osteoporosis correlate with circulatory diseases such as vascular calcification (bone vascular correlation). Furthermore, it has been reported that bone mass loss is associated with a high risk of death. Examples of the preventive / therapeutic agents for diseases involving bone or blood vessel calcification include calcium, vitamin D preparation, estrogen, ipriflavone, calcitonin, bisphosphonate, vitamin K2 preparation, phosphate adsorbent or statin. Including. However, more preferably, establishment of a novel mechanism-oriented therapeutic method, therapeutic agent, and prophylactic method that directly affects bone or blood vessel calcification is desired.

Fibroblast growth factor 23 (FGF23) is a protein identified as a causative factor of hereditary and neoplastic rickets / osteomalacia (see Non-Patent Document 1 to Non-Patent Document 3). Furthermore, in recent years it has been recognized as a physiological hormone that regulates phosphorus and vitamin D metabolism. Biological responses to the FGF family, including FGF23, take place through the binding of specific cell surface receptors to tyrosine kinase type receptors (FGFR, in particular FGFR1). However, the affinity of FGF23 for FGFR is low.

Under normal conditions, FGF23 is secreted mainly by osteoblasts and bone cells. After secretion, expression of type II sodium-phosphorous cotransporter (NaPiII) and expression of 25 hydroxyvitamin D ₃ -1α hydroxylase are suppressed targeting renal tubular epithelium. Therefore, the production of excess FGF23 suppresses reabsorption of phosphorus, and as the blood 1α, 25 dihydroxyvitamin D ₃ (1,25D) concentration decreases, absorption of phosphate from the intestinal tract is suppressed. Medium phosphorus concentration decreases. Furthermore, it has been clarified that FGF23 plays a central role in phosphorus homeostasis since Fgf23-deficient mice exhibit high phosphate and hypervitamin Demia.

In addition, regarding the relationship between FGF23 and bone, tooth or blood vessel calcification, it has been confirmed in vitro that FGF23 regulates the physiological mineralization of hard tissues including bone and the mineralization associated with pathological conditions such as blood vessels. ing. Specifically, FGF23 has been shown to suppress physiological calcification of bones and blood vessels (see Patent Document 1, Non-Patent Documents 1 and 2).

On the other hand, Klotho, which encodes a type I membrane protein with a molecular weight of about 130,000, has been identified as a responsible gene for mice that cause various human aging phenomena by mutation of a single gene. Klotho is expressed mainly in the main cells of renal tubules, brain choroid and parathyroid glands.

Klotho is involved in phosphorus reabsorption and vitamin D metabolism in the kidney and parathyroid hormone production in the parathyroid gland. Moreover, coupled with ^Na + ^/ K + -ATPase in a cell, in response to a decrease in calcium concentration outside the cell recruit ^Na + ^/ K + -ATPase to the cell membrane. On the other hand, the extracellular domain of membrane-type Klotho is cleaved and solubilized Klotho (hereinafter sometimes abbreviated as KLe) released from the extracellular domain in the blood circulates in the blood and is a secretor involved in development. It has been reported that it binds to the protein Wnt and inhibits the Wnt signal, or regulates the sugar chain modification of TRPV5 to regulate the function as a calcium channel.

Regarding the relationship between FGF23 and membrane type Klotho, as represented by Patent Document 2 and Non-Patent Document 3, it has been reported that both work together. That is, in CHO cells and the like in which both are forcibly expressed, FGF23, membrane-type Klotho and FGFR form a complex, and it is confirmed that phosphorylation of ERK, which is a specific signal of FGF23, is increased, or expression of Egr-1 is increased. Yes. In this cell, the same result can be obtained even if KLe is forcibly expressed instead of membrane-type Klotho. Furthermore, when CHO cells forcibly expressing KLe are transplanted into nude mice, FGF23 is overexpressed. Similarly, it is also described that serum 1,25D and phosphate concentrations decrease.

Patent Document 3 describes an invention related to a fusion protein (a fusion polypeptide) comprising a polypeptide containing such KLe and FGF (specifically, FGF23) (active fragment). Including a polypeptide consisting of: In detail, it describes about the invention of the method of treating or preventing the various disease state and metabolic disease state accompanying an aging phenomenon by using the said fusion protein.

JP 2008-17790 A JP 2006-240990 A US Patent Application Publication No. 2009/0192087

As described above, in Patent Document 2 and Non-Patent Document 3, it is described that membrane type Klotho or KLe converts FGFR to FGF23-specific receptor. It is explained that FGF23 is secreted by osteoblasts and bone cells and acts in the kidney and parathyroid gland through the bloodstream because the membrane type Klotho is localized in these tissues. However, at least in the kidney, membrane-type Klotho is localized in the distal tubular epithelium, so that membrane-type Klotho, FGFR, and FGF23 form a complex in the proximal tubule, which is the site of action of FGF23. It is hard to think. This problem is still not solved. On the other hand, membrane type Klotho is expressed only slightly in the proximal tubule, and it is involved in intracellular transport and degradation of NaPiIIa independently of FGF23 by its own β-glucuronidase activity, and reabsorbs phosphorus. (Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule. Hu MC, Shi M, Zhang J, Pastor J, NakataniazzT, Lanske Bazz Baum MG, Kuro-O M, Moe OW. FASEB J. 2010 May 21. http://www.fasebj.org/cgi/rapidpdf/fj.10-154765v2.pdf [Epub ahead of print]).

Thus, FGF23 is not necessarily involved in the action of Klotho in the kidney. In addition, in Patent Document 2 and Patent Document 3, there is a description that the two of FGF23 and KLe form a complex, but there is evidence that the two form a complex in a living body and other than muscle cells. Absent. Further, for example, when considering the case where such a complex is used in a living body, it binds nonspecifically to FGFR widely distributed in the living body, and the characteristics of FGF23 described above cannot be explained. In fact, there are recent papers that the two do not form a complex (Goetz R, Nakada Y, Hu MC, Kurosu H, Wang L, Nakatani T, Shi M, Eliseenkova AV, Razzaque MS, Moe OW, Kuro -o M, Mohammadi M. Proc Natl Acad Sci U S A. 2010 Jan 5; 107 (1): 407-412).

On the other hand, as described above, it has been confirmed that FGF23 is involved in the regulation of calcification of hard tissues and / or blood vessels (Patent Document 1). However, there are still many unclear points regarding the detailed mechanism of action. In Klotho mutant mice (kl / kl mice), abnormalities are observed in bones and teeth, but it has been confirmed that Klotho is not expressed in these tissues. Therefore, in order to establish a novel therapeutic method, therapeutic agent and preventive method that have an effect directly on calcification of hard tissue and / or blood vessels, the action mechanism of FGF23 in hard tissue and / or blood vessels is more specifically described. Elucidation and discovery of compounds that can participate in and regulate calcification suppression by FGF23 are desired.

The present invention has been made in view of the above circumstances, and a screening method for a calcification regulator, a calcification regulator, and bone calcification, which are associated with signals common to hard tissue and / or blood vessel calcification. An object of the present invention is to provide a method and a test kit for testing the degree, vascular calcification, and the progression of pathological conditions associated therewith.

In order to achieve the above-mentioned object, the present inventors have conducted intensive research and have found the following findings.
(1) KLe forms a functional complex with FGF23 and FGFR in Obc cells (see Examples), and is used for specific signal transduction in Obc cells (including suppression of bone mineralization by FGF23 associated with signal transduction). It is essential.
(2) The action of (1) is specific to bone and not seen in the kidney.
(3) The effect of (1) is also seen in calcification of vascular smooth muscle.

That is, the screening method for a calcification regulating agent for hard tissue and / or blood vessels according to the first aspect of the present invention comprises at least solubilized Klotho, FGFR, and FGF23 in a specimen in the absence and presence of a test compound. A step of contacting
Selecting a compound that changes FGF23-specific signaling in the sample in the presence of the test compound as compared to FGF23-specific signaling in the sample in the absence of the test compound;
It is characterized by including.

Preferably, the change in specific signaling is characterized by FGFR phosphorylation, ERK phosphorylation, or Egr-1 expression level as an index.

Or measuring the concentration of solubilized Klotho in the sample in the absence and presence of the test compound;
Selecting a compound that increases or decreases the concentration of the solubilized Klotho in the sample in the presence of the test compound as compared to the concentration of the solubilized Klotho in the sample in the absence of the test compound;
It is characterized by including.

Or measuring the complex formation of solubilized Klotho, FGFR and FGF23 in the specimen in the absence and presence of the test compound;
Selecting a compound that increases or decreases the complex formation in the sample in the presence of the test compound compared to the complex formation in the sample in the absence of the test compound;
It is characterized by including.

More preferably, the screening for the calcification regulating agent is performed in vivo.

The hard tissue and / or blood vessel calcification regulating agent according to the second aspect of the present invention is characterized by containing a compound that suppresses or promotes complex formation of solubilized Klotho, FGFR, and FGF23.

Preferably, the compound is a solubilized Klotho.

Alternatively, preferably, the compound is a ligand having binding affinity for solubilized Klotho, FGFR, or a complex of the solubilized Klotho, FGFR, and FGF23.

More preferably, the ligand is an antibody.

The method for testing calcification of hard tissue and / or blood vessels according to the third aspect of the present invention includes a concentration of solubilized Klotho, formation of a complex of solubilized Klotho, FGFR, and FGF23 in the specimen, or formation of the complex. And a step of measuring FGF-specific signal transduction associated with.

Preferably, the specimen is animal blood, plasma, serum, bone marrow, bone tissue or cell, or blood vessel tissue or cell.

More preferably, the animal is a human.

The calcification test kit for hard tissue and / or blood vessels according to the fourth aspect of the present invention contains a ligand having binding affinity for solubilized Klotho or a complex of solubilized Klotho, FGFR and FGF23. It is characterized by.

According to the screening method for a calcification regulating agent according to the first aspect of the present invention, a new calcification regulating agent that promotes or suppresses calcification in hard tissues and / or blood vessels can be screened. According to the calcification regulating agent according to the second aspect of the present invention, calcification in hard tissue and / or blood vessels can be suppressed or promoted. According to the calcification inspection method and the calcification inspection kit according to the third and fourth aspects of the present invention, calcification in a hard tissue and / or blood vessel can be inspected.

It is a figure which shows the mode of the result of the immunoprecipitation which concerns on Example 1, and immunoblotting. FIG. 6 shows data of real-time RT-PCR results at the Egr-1 mRNA level according to Example 2. It is a figure which shows the mode of the result of the Western blotting of ERK1 / 2 which concerns on Example 2, and phosphorylated ERK1 / 2. It is a figure which shows the data of the result of the mineralization parameter | index by rKLe addition by ALP / von Kossa dyeing | staining which concerns on Example 3. FIG. It is a figure which shows the mode of the result of the western blotting of rKLe which concerns on Example 3. FIG. It is a figure which shows the data of the result of the mineralization parameter | index by rKLe and rFGF23 addition by ALP / von Kossa dyeing | staining which concerns on Example 3. FIG. FIG. 6 shows data of real-time RT-PCR results at the Egr-1 mRNA level according to Example 4. It is a figure which shows the mode of the result of the Western blotting of ERK1 / 2 which concerns on Example 4, and phosphorylated ERK1 / 2. It is a figure which shows the mode in the fluorescence microscope of the plastic | slice piece of the skull doubly labeled with the calcein concerning Example 5. FIG. It is a figure which shows the data of the result of a label space | interval in the mode in the fluorescence microscope of FIG. It is a figure which shows the mode in the microscope of the paraffin section of the skull which carried out H & E dyeing concerning Example 5. FIG. It is a figure which shows the data of the number of osteoblasts in the mode in the microscope of FIG. It is a figure which shows the data of the number of bone cavities in the mode in the microscope of FIG. It is a figure which shows the data of the bone thickness in the mode in the microscope of FIG. FIG. 10 is a view showing a state of a plastic section of a skull stained with toluidine blue / von Kossa according to Example 5 under a microscope. It is a figure which shows the data of the thickness of the osteoid in the mode in the microscope of FIG. It is a figure which shows the result of the electronic probe microanalyzer which concerns on Example 5. FIG. It is a figure which shows the data of the result of the calcium content measurement which concerns on Example 6. FIG. It is a figure which shows the mode in the stereomicroscope of the aorta of the kl / kl mouse which concerns on Example 7. FIG. It is a figure which shows the mode of the result of the Western blotting of ERK1 / 2 which concerns on Example 8, and phosphorylated ERK1 / 2. It is a figure which shows the mode of the alizarin red / toluidine blue dyeing | staining of the paraffin section of the kl / kl mouse | mouth thoracic aorta concerning Example 9. FIG. It is a figure which shows the data of the alizarin red dyeing | staining range result of the kl / kl mouse | mouth based on Example 9, and a wild type mouse | mouth. It is a figure which shows the mode of the immuno-staining of the paraffin section of the kl / kl mouse | mouth thoracic aorta concerning Example 10. FIG.

Hereinafter, embodiments of the present invention will be described in detail. Note that “FGF23”, “FGFR1”, and “Klotho” of humans (Homoapisapiens) are registered under the accession numbers AAG09997, AAH15035, and BAA23382 in GenBank, respectively.

(Method for screening hard tissue and / or vascular calcification regulator)
Embodiment 1 of the present invention relates to a screening method for a calcification regulating agent for hard tissue and / or blood vessels. First, the step of contacting solubilized Klotho (KLe) with FGF23 and FGFR in a sample in the absence and presence of the test compound is compared with specific signal transduction in the sample in the absence of the test compound. And a method comprising a step of selecting a compound that changes the specific signal transduction in the specimen in the presence of the test compound. Preferably, the change in specific signaling can be indicated by FGFR phosphorylation, ERK phosphorylation, or Egr-1 expression level (see Examples).

In addition, the sample in the presence of the test compound is compared with the step of measuring the concentration of KLe in the sample in the absence and presence of the test compound and the concentration of KLe in the sample in the absence of the test compound. And a step of selecting a compound that increases or decreases the concentration of KLe. In addition, the formation of a complex of KLe, FGF23, and FGFR (hereinafter referred to as “complex containing KLe” or “complex”) may be measured and compared in a specimen. In this case, it is possible to compare the degree, presence, concentration, etc. of the formation of a complex containing KLe directly. These screenings are more preferably performed in vivo.

In the present invention, “solubilized Klotho (KLe)” means Klotho blood in mammals such as humans, mice, rats, hamsters, guinea pigs, monkeys, cows, pigs, horses, rabbits, sheep, goats, cats or dogs. Refers to the portion of the extracellular domain that circulates in Of these, KLe of human and non-human mammals (eg, rat, mouse, monkey or rabbit) having high homology with the amino acid sequences of human and human KLe are preferable. “High homology” means having a homology of 60% or more, preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more. Most preferably, it refers to human KLe. For example, a protein having the amino acid sequence set forth in SEQ ID NO: 1, an amino acid sequence in which one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 1 have been deleted, added, inserted or substituted, and FGF23 and Examples thereof include a protein having a function of forming a complex with FGFR and suppressing calcification, or a peptide fragment having the function. Thus, in the present invention, “solubilized Klotho (KLe)” is used as a meaning containing all of them.

In the present invention, “regulation” may include “promotion” or “activation” for positively controlling calcification, and “suppression” or “inhibition” for negative control. As an example of “positive” control, it is possible to show the inhibition of calcification due to the formation of KLe or a complex containing KLe and the activation of specific signal transduction associated therewith. In addition, as an example of controlling to “negative”, it is possible to show the formation of KLe or a complex containing KLe and the promotion of calcification by suppressing the specific signal transmission associated therewith. The increase or decrease in the formation of a complex containing KLe can include, for example, promotion or inhibition of binding of KLe to FGFR in hard tissue and / or blood vessels.

In the present invention, “specimen” means all target samples that can be used by those skilled in the art to measure KLe or a complex containing KLe and the accompanying FGF-specific signal transduction. For example, a biological sample in vivo or in vitro. In particular, the most preferable specimen suitable for the screening method according to the first embodiment refers to a non-human animal that is a target in vivo. Examples thereof include mice, rats, hamsters, guinea pigs, monkeys, cows, pigs, horses, rabbits, sheep, goats, cats and dogs.

In the present invention, “hard tissue” means living hard tissue, for example, bone tissue forming the skeleton of the skull and trunk, alveolar bone and cementum constituting periodontal tissue, or dentin (which comprises these) Including cells etc.). Most preferred is mammalian bone tissue. More preferably, it is a bone tissue of a non-human mammal (for example, rat, mouse, monkey or rabbit) having high gene homology with human.

In the present invention, “test compound” (“compound”) may be any substance capable of evaluating the formation of a complex containing KLe or KLe and the accompanying increase or decrease in specific signal transduction. For example, a low molecular compound, a nucleic acid, or a polypeptide can be used. The low molecular weight compound, nucleic acid, polypeptide or the like may be extracted and purified from a natural product, or may be artificially synthesized. Moreover, it is not restricted to what was refine | purified, For example, even an unpurified cell extract etc. can be used as a test compound. Moreover, not only a novel substance but a well-known substance or its improvement may be sufficient. For example, an existing therapeutic or preventive drug or derivative thereof can be evaluated by the screening method of the first embodiment.

In the present invention, “FGF23” (fibroblast growth factor 23) means not only human FGF23 protein but also FGF23 of a non-human mammal having high homology with the amino acid sequence of human FGF23 such as rat, mouse, monkey or rabbit. It also contains proteins or peptide fragments containing these active regions. The meaning of “high homology” is the same as described above.

In the present invention, “FGFR” also includes FGFR of a non-human mammal having high homology with the amino acid sequence of human FGFR. FGFR1, 3 and 4 that have been confirmed to bind to FGF23 (Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, Rosenblatt KP, Baum MG, Schiavi S, Hu MC, Moe OW, Kuro-o M. J Biol Chem. 2006 Mar 10; 281 (10): 6120-6123.), Most preferably FGFR1 (Non-patent Document 3 (Urakawa I., et al., Nature 444, 770-). 774, 2006)).

For specific methods of measuring and comparing KLe or KLe-containing complex formation in the specimen and the accompanying FGF23-specific signal transduction, any method for measuring and comparing them known to those skilled in the art can be used. can do.

In detail, for example, first, a test compound is administered or injected into a non-human mammal as described above. Next, the formation of KLe or a complex containing KLe in blood or the like of the mammal is measured. Further, FGF23 and KLe are directly brought into contact with a hard tissue or a part of hard tissue expressing FGFR, and complex formation in the hard tissue and the accompanying phosphorylation of FGFR, phosphorylation of ERK, or Egr-1 Expression levels can also be measured. In addition, an experimental system in which KLe, FGF23, or FGFR is forcibly expressed in an osteoblast, bone cell, or vascular smooth muscle cell as a specimen may be constructed so that measurement and screening can be performed more easily. Such cell configuration and DNA introduction are easy for those skilled in the art, and examples thereof include methods using DNA introduction plasmids or viral vectors, electroporation, lipofection, or microinjection. . Thus, the screening method according to the first embodiment can be performed in vivo or in vitro.

Regarding the measurement of complex formation including KLe and KLe in blood, hard tissue or cells and the accompanying FGF23-specific signal transduction, for example, intermolecular analysis, Western blotting, immunoprecipitation, ELISA (Enzyme-LinkedLinkImmunoSorbent Assay), radioimmunoassay (RIA), immunohistochemical staining, immunofluorescence, flow cytometry, and other known protein detection and quantification methods can be used by those skilled in the art for easy measurement.

Measured formation of KLe or a complex containing KLe in the presence of a test compound or the accompanying FGFR phosphorylation, ERK phosphorylation or Egr-1 expression increased or decreased as compared to the absence of the test compound If so, the test compound is likely to be a calcification regulator that suppresses or promotes calcification of the hard tissue and / or blood vessels, respectively.

The thus screened hard tissue and / or vascular calcification regulating agent that suppresses calcification is osteoarthritis, ectopic calcification, arteriosclerosis or as a disease / condition in which calcification is promoted. It can be used as a means for preventing or treating vascular calcification in renal failure. In the case of a calcification regulating agent that promotes calcification, it can be used as a prophylactic / therapeutic agent for osteoporosis, rickets or osteomalacia as a disease / condition in which calcification is inhibited or suppressed. Furthermore, use in fields such as other hard tissue diseases or regenerative medicine is also conceivable.

(Hard tissue and / or vascular calcification regulator)
Although the screening method of the mineralization regulator using the complex containing KLe or KLe and its downstream FGF23 specific signaling was described in detail, Kle itself or a compound related to the complex formation containing KLe is effective. As a component, there is a possibility that it can be used to control calcification. Therefore, Embodiment 2 describes a hard tissue and / or blood vessel calcification regulator.

Specifically, the regulator that suppresses calcification of hard tissue and / or blood vessels contains, as an active ingredient, a compound that promotes complex formation containing KLe, such as KLe. Further, the regulator that promotes calcification of hard tissue contains KLe, a complex containing KLe, or a ligand having binding affinity for FGFR. Preferably, the ligand is an antibody. Furthermore, it may be considered that a more effective calcification regulator can be obtained by simultaneously containing other inhibitors / promoters of specific signal transduction associated with the formation of a complex containing KLe as active ingredients. For example, inhibitors of ERK signals such as U0126 are readily available and may contain other modulators of specific signaling associated with complex formation involving such KLe.

KLe can be easily obtained, manufactured or purified. As shown in the examples, KLe can be easily purchased and obtained at, for example, R & D Systems. In addition, for example, a protein / peptide having the amino acid sequence shown in SEQ ID NO: 1 can be produced using a known protein / peptide synthesis method (eg, including a solid phase synthesis method, a liquid phase synthesis method, etc.). In addition, after the reaction, proteins and peptides can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like.

In the present invention, the “antibody” may be a monoclonal antibody or a polyclonal antibody, and is not particularly limited as long as it specifically binds to a complex containing KLe, FGFR or KLe. This also includes an antibody that promotes the formation of a complex of KLe, or an antibody that promotes the formation of a complex containing KLe. That is, any antibody may be used, either an agonist antibody or an antagonist antibody. Among the “antibodies”, the “neutralizing antibody” of KLe means an antibody that inhibits the function of specific signal transduction by KLe, such as an antibody that inhibits complex formation with FGFR and FGF23. . In addition, although shown in an Example, an anti- Klotho monoclonal antibody is mentioned, for example. Similarly, the “neutralizing antibody” of FGFR means an antibody that inhibits the function of specific signal transduction by FGFR. Similarly, a “neutralizing antibody” of a complex containing KLe means an antibody or the like that inhibits the function of specific signal transduction caused by the complex containing KLe.

Methods for producing antibodies are already well known to those skilled in the art, and the present antibodies can also be produced according to these conventional methods (Current protocol, molecular molecular biology, edit. Ausubel et al., 1983, Publish. John Wiley and Sons. Section 11.12-11.13, Antibodies; A Laboratory Manual, Lane, H, D. et al., Cold Spring Harber Laboratory Press Publishing New York 1989).

According to the regulator for suppressing calcification of hard tissue and / or blood vessels according to the second embodiment, in osteoarthritis, ectopic calcification, or arteriosclerosis, which are diseases / conditions in which calcification is promoted It can be used as a means for preventing the progression of vascular calcification. The modulator may be administered directly to the patient, or it may constitute a compound that also contains these other components and be administered to the patient. As the administration method, known methods such as oral, intravenous, intraarterial or subcutaneous injection can be used.

Also, according to the regulator that promotes calcification of hard tissue, it can be used as a means for prevention / treatment of osteoporosis, rickets or osteomalacia. The administration method and the like are the same as those of the regulator for suppressing calcification described above, but it is also conceivable to administer directly to a specific site (for example, a site where bone is decreased or excessively formed) by injection or the like.

(Method for examining calcification of hard tissue and / or blood vessels)
As described above, since KLe is an essential factor for suppressing calcification by FGF23-specific signal transduction, the suppression of calcification of hard tissue is caused by hard tissue, blood vessel or blood KLe (and FGF23 (Patent Document 1, Non-Patent Document). 1 and 2))) It is considered to be strongly influenced by the concentration.

Therefore, as a third embodiment, a method for examining calcification of hard tissue and / or blood vessels, which includes a step of measuring the concentration of KLe in a specimen can be mentioned. The degree of calcification can be estimated by measuring not only the concentration of KLe but also the formation of a complex containing KLe or the accompanying FGF23-specific signal transduction.

Since KLe circulates in the blood as described above, the measurement step is blood, plasma, serum, bone marrow, bone tissue or cell, or Kle concentration in blood vessel tissue or cell, formation of a complex containing KLe Alternatively, it is more convenient and preferable to measure the accompanying FGF23-specific signal transduction. As a measurement method, any method known to those skilled in the art can be used. For example, the concentration of KLe or the formation of a complex containing KLe may be measured. For example, when measuring the concentration of KLe in blood, methods such as immunoprecipitation, Western blotting, ELISA, RIA, immunohistochemical staining, immunofluorescence, or flow cytometry can be used. Measurement of FGF23-specific signal transduction associated with complex formation can be performed by measuring FGFR phosphorylation, ERK phosphorylation, or Egr-1 expression level, as described above.

The measured concentration of KLe in blood or the like can be evaluated by, for example, comparing the degree of calcification and the progression of the pathological condition accompanying it, and screening for active substances, by comparing with a normal value that has been previously measured and averaged. It is considered possible. In addition, by using human blood, plasma, serum, bone marrow, or the like as a specimen in the calcification test method according to the fourth embodiment, vascular calcification in osteoarthritis, ectopic calcification, or arteriosclerosis is achieved. There is a high possibility that the disease state is grasped and monitored, or the disease state is grasped and monitored for bone mineralization failure in osteoporosis, rickets or osteomalacia.

(Hard tissue and / or blood vessel calcification test kit)
As described in Embodiment 3, it is considered that the measurement of the concentration of KLe in blood and the formation of a complex containing KLe can serve as a measure of the degree of calcification of hard tissues and / or blood vessels. Therefore, as a fourth embodiment, hard tissue and / or blood vessel containing a ligand having binding affinity for KLe or a complex containing KLe for use in the calcification testing method described in the third embodiment. Mention may be made of a calcification test kit.

The ligand means a substance that specifically binds to a measurement target (KLe or a complex containing KLe). For example, an antibody of KLe or a complex as described above (part of which can be a Klotho antibody) and the antibody Refers to the secondary antibody against. In addition, for example, various reagents, enzymes, buffers, reactor materials, and / or instructions for comparing and evaluating the degree of calcification failure may be included. The method of using these is the same as the method described in the third embodiment.

Unless defined otherwise, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. 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.

Hereinafter, the present invention will be described in more detail using examples, but the examples are not intended to limit the present invention. In addition, about the reagent used in the Example, the thing of Sigma-Aldrich was used unless otherwise indicated. Klotho mutant mice (hereinafter referred to as kl / kl mice) were obtained from Clea Japan, and C57BL / 6J mice and Wistar rats were obtained from Japan Charles River.

Example 1
In Example 1, an example relating to formation of a complex of KLe with FGF23 and FGFR1 in osteoblasts / bone cells will be described in detail.

First, the mature osteoblast / bone cell (Obc cell) and the method for growing and culturing the Obc cell used in Examples 1 to 3 will be described. Cells were isolated from calvaria derived from embryonic day 21 fetal rats (see Yoshiko Y, et al., Endocrinology 144, 4134-4143, 2003), and the calvarial cells were separated from 10% FBS (fetal bovine serum, Fetal Bovine Serum ) (HyClone) and α-MEM (Modified Essential Medium) medium containing 50 μg / ml ascorbic acid. The culture period was 10 days, and the cells were cultured in a usual manner at 37 ° C. in a wet 5% CO ₂ gas phase. After the calvarial cells formed osteoid-like nodules (nodules), cells were selectively removed from the nodules by collagenase (type I) treatment. Thereafter, the removed cells were cultured under the above-described conditions and used as Obc cells. In order to confirm the characteristics of mature osteoblasts and bone cells, the expression levels of Ocn (osteocalcin), Dmp-1 (dentin matrix protein-1) and Sost were determined using real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction). Measured by the method (not shown).

In this Example 1, Obc cell cultures cultured under conditions of serum deficiency of 0.1% FBS were used. Furthermore, the cells contain both recombinant KLe (rKLe) (≦ 500 ng / ml (R & D ＆ Systems)) (described in SEQ ID NO: 2) and recombinant FGF23 (rFGF23) (≦ 500 ng / ml (R & D Systems)). The cells were cultured for 15 minutes under any of the following conditions: conditions containing only rKLe, conditions containing only rFGF23, or conditions containing none.

Each cell thus cultured was solubilized with 1% Igepal CA 630 and 50 mM Tris-HCl (pH 8.0). Immunoprecipitation (IP) with anti-Klotho monoclonal antibody (αKlotho Mab) (Calbiochem) or anti-FGFR1 monoclonal antibody (αFGFR1 Mab) (Abcam), and immunoblotting with αFGFR1 Mab or anti-FGF23 polyclonal antibody (αFGF23) (R & D Systems) ( IB) was performed. For immunoprecipitation, solubilized cell extract was incubated with protein G coupled to magnetic microbeads (μMACS protein G microbeads, Miltenyi Biotec). At that time, either αKlotho Mab or αFGFR1 Mab was added to obtain an immune complex (MACS (registered trademark) Separator User Manual, Miltenyi Biotec). Thereafter, this sample was subjected to SDS-PAGE (Poly-Acrylamide Gel Electrophoresis), immunoblotting (IB) with αFGFR1 Mab or αFGF23 (Santa Cruz) was performed, and a signal was detected by chemiluminescence.

FIG. 1 is a view showing the results of immunoprecipitation and immunoblotting according to Example 1. As shown in FIG. 1, FGFR1 and FGF23 formed a complex with Klotho and FGFR1 only in the presence of both rKLe and rFGF23, respectively. From these results, it was revealed that KLe and FGF23 and FGFR1 form a complex in osteoblasts / bone cells.

(Example 2)
In Example 2, an example relating to the correlation between FGF23-specific signal transduction and KLe in Obc cells will be described in detail.

Egr-1 is a gene whose expression is promoted by FGF23-specific signal transduction (see Non-Patent Document 3). Therefore, the present inventors measured the mRNA level of Egr-1 in Obc cells by real-time RT-PCR.

The cells used were cultured for 30 minutes under the same culture conditions as described in Example 1. First, total RNA of cells was prepared using TRIzol reagent (Invitrogen). Real-time RT-PCR was performed using the prepared RNA (Y. Yoshiko, GA Candeliere, N. Maeda, JE Aubin, Mol Cell Biol 274, 465-4474, 2007, and H. Wang, Y. Yoshiko, R Yamamoto, T.Minamizaki, K.Kozai, K.Tanne, JE Aubin, N.Maeda, J Bone Miner Res 23, 939-948, 2008). Ribosomal protein L32 was used as an internal standard. The Egr-1 forward primer is the one described in SEQ ID NO: 3, the reverse primer is the one described in SEQ ID NO: 4, the L32 forward primer is the one described in SEQ ID NO: 5, and the reverse primer is the sequence SEQ ID NO: 4. The thing of No. 6 was used.

FIG. 2 is a diagram showing data of the results of real-time RT-PCR at the Egr-1 mRNA level according to Example 2. In addition, there is a significant difference compared to the solvent alone (−). In FIG. 2, ** (significance level): p <0.01 and n = 4.

On the other hand, phosphorylation of ERK1 / 2 is also an index of FGF23-specific signal transduction (see Non-Patent Document 3). Therefore, the present inventors performed Western blotting in order to examine ERK1 / 2 activity.

Cells were treated for 5 to 30 minutes under the same conditions as in the culture described in Example 1, and then a cell extract (100 μmM KCl, 1 mM EDTA, 0%) to which a phosphorylation inhibitor (Phosphatase Inhibitor Cocktail (Nacalai Tesque)) was added. Solubilize cells with 5% Nonidet P-40, 1 mM phenylmethylsulfonylfluoride, Complete Protease Inhibitor Cocktail (Roche Diagnostics), 50 mM Tris-HCl (pH 7.5), and perform SDS-PAGE, immunoblotting and chemiluminescence detection It was. As the primary antibody, ERK1 / 2 and phosphorylated ERK1 / 2 (p-ERK1 / 2) were used.

FIG. 3 shows the results of Western blotting of ERK1 / 2 and phosphorylated ERK1 / 2 according to Example 2. As shown in FIG. 3, phosphorylation of ERK1 / 2 was detected for those treated for 10 minutes under conditions including both rFGF23 and rKLe. Thus, in both the results of FIG. 2 and FIG. 3, increase of mRNA level of Egr-1 and activation of ERK1 / 2 were confirmed only in the presence of rFGF23 and rKLe.

From these results, it was considered that a complex of KLe, FGF23, and FGFR promotes FGF23-specific signal transduction in Obc cells.

(Example 3)
In Example 3, an example relating to the correlation between calcification failure due to FGF23-specific signaling in Obc cells and KLe will be described in detail.

In order to confirm the correlation between KLe and calcification suppression by FGF23-specific signaling in Obc cells, the present inventors detect the activity of alkaline phosphatase (ALP), which is an osteoblast marker enzyme, and calcification. Kossa staining was performed. In addition, since the present inventors have confirmed that 1α, 25 dihydroxyvitamin D ₃ (1,25D) strongly promotes the expression of FGF23, not only rFGF23 but also 10 nM 1,25D was added. The cultured ones were also examined. FBS was confirmed to contain KLe (not shown, confirmed by Western blotting), so that 5% of FBS was removed from FBS by μMACS protein G microbeads and αKlotho Mab. To the culture medium. rKLe was used for recovery experiments. The culture was terminated 36 hours after the addition of the reagent, washed with PBS, fixed with 10% neutral buffered formalin, and then stained. In the group to which rFGF23 was added, the culture supernatant (conditioned media) was collected, and the level of the added rKLe was confirmed by Western blotting. In order to induce calcification, 3 mM β-glycerophosphoric acid was added. Thereafter, the number of ALP-positive calcified nodules was counted under a stereomicroscope. The numerical value was expressed with the negative control (solvent only) as 100.

FIG. 4 is a diagram showing data of the result of the calcification index by addition of rKLe in ALP / von Kossa staining according to Example 3. In addition, there is a significant difference compared to the solvent alone (−). In FIG. 4, * (significance level): p <0.05, ** (significance level): p <0.01, and n = 4. As shown in the calcification index (Mineralized foci) in FIG. 4, in both 1,25D and rFGF23, calcification is not suppressed, but rather promoted, as compared with the control when rKLe is not added. However, it has returned to the state of calcification suppression again by adding rKLe. FIG. 5 is a diagram showing the result of Western blotting of rKLe according to Example 3. As shown in FIG. 5, the rKLe concentration of the culture supernatant was consistent with the concentration dependency of the calcification inhibiting action shown in FIG. These results in FIGS. 4 and 5 suggest that the degree of calcification depends on the rKLe concentration.

Furthermore, the correlation between calcification suppression by rKLe and rFGF23 and FGF23-specific signal was confirmed. Obc cells obtained by culturing under the condition of serum deficiency of 0.1% FBS in the same manner as described in Example 1 were used for 2 hours with 10 μM of ME01 / 2 inhibitor U0126 or solvent alone for 2 hours. Pretreatment was performed. After washing, only rKLe and rFGF23 or a solvent were added, and 36 hours later, ALP / von Kossa staining was performed as described above. FGFR neutralizing antibody (αFGFR) 2 μg / ml (CHEMICON) or its solvent alone (negative control) was added when rKLe and rFGF23 were added. Further, as described above, β-glycerophosphate was added simultaneously with rKLe and rFGF23.

FIG. 6 is a diagram showing data on the result of calcification index by adding rKLe and rFGF23 in ALP / von Kossa staining according to Example 3. In addition, there is a significant difference compared to the solvent alone (−). In FIG. 6, ** (significance level): p <0.01 and n = 4. As shown in FIG. 6, the suppression of calcification by rKLe and rFGF23 is largely recovered by the FGFR neutralizing antibody or U0126, so that signal transduction based on the formation of a complex containing KLe is strongly involved in the suppression of calcification. It became clear to do. Therefore, they are effective as therapeutic agents for suppressing calcification based on the formation of a complex containing KLe. Further, it is suggested that KLe is effective as a regulator that suppresses calcification, and that a neutralizing antibody of a complex containing KLe and KLe is effective as a regulator that promotes calcification.

From the results of Example 3 and Example 1 and Example 2, KLe forms a functional complex with FGF23 and FGFR in Obc cells, and FGF23-specific signaling in Obc cells (of bone by FGF23) (Including calcification failure). In this example, Wnt and TRPV5 that react with KLe may be affected, but Wnt was similarly stained with ALP / von Kossa and confirmed that it was not affected (not shown). TRPV5 is not present in the bone and therefore has no effect.

Example 4
In Example 4, an example relating to the correlation between FGF23-specific signal transduction and calcification failure and KLe in bone and kidney will be described in detail.

RKLe was administered to Klotho mutant mice lacking Klotho (kl / kl mice) in order to confirm whether the action of KLe shown in the above-mentioned Examples was also observed in the living body. In addition, the present inventors have confirmed that the concentrations of phosphate, calcium, 1,25D and FGF23 are significantly higher in kl / kl mouse serum than in WT mouse serum. (Not shown, 1 and 25D were measured by RIA kit (TFB), and the concentration of FGF23 was measured by FGF23 ELISA kit (Kainos). Phosphoric acid and calcium were Phospha C-Test Wako and Calcium C-Test Wako, respectively Thus, although the FGF23 concentration in the blood of kl / kl mice is extremely high (the cause is unknown), it is estimated that FGF23 does not act because Klotho is deficient. The

For administration, 10 μg of male kl / kl mouse calvaria on the 10th day after birth was mixed with 0.3% atelocollagen (Koken) and injected at intervals of 3 days, and samples were collected on the 22nd day after birth. Calcein 10 mg / kg / time was injected intraperitoneally, divided into 2 times 2 days before and 6 days before the sample collection. First, total RNA was extracted from the parietal bone and kidney, and Egr-1 mRNA levels were measured by real-time RT-PCR. For RT-PCR samples, real-time RT-PCR primers and other detailed methods are the same as in Example 2 described above.

FIG. 7 is a diagram showing data on the results of real-time RT-PCR at the Egr-1 mRNA level according to Example 4. As for the parietal bone (Bone), wild type mice (WT) that were not administered rKLe were also examined. There is a significant difference compared to the bone data of kl / kl mice not administered with rKLe. In FIG. 7, * (significance level): p <0.05 and n = 9. As shown in FIG. 7, in the bones of kl / kl mice, administration of rKLe increases Egr-1 mRNA levels. However, in the kidney (Kidney) of the same mouse, the mRNA level of Egr-1 is hardly changed by administration of rKLe.

Further, in order to examine ERK1 / 2 activity as in Example 2, Western blotting was also performed. The mice used for the samples were kl / kl mice administered in the same manner as described above, and samples of parietal bone and kidney cells of the mice were used. The Western blotting method and other conditions are the same as in Example 2 described above.

FIG. 8 shows the results of Western blotting of ERK1 / 2 and phosphorylated ERK1 / 2 according to Example 4. In addition, a figure shows the representative example of the individual of 9 examples implemented for each group. As shown in FIG. 8, promotion of ERK1 / 2 phosphorylation was detected in the bones of mice administered with rKLe. However, in the kidney, activation of ERK1 / 2 was not detected in the group administered with rKLe. That is, the results shown in FIGS. 7 and 8 suggest that FGF-specific signal transduction by administration of rKLe to kl / kl mice is specific to bone and not found in the kidney. That is, it can be said that KLe does not show a cooperative action with FGF23 in the kidney.

(Example 5)
In this Example 5, a specific example in vivo relating to the correlation between calcification inhibition by FGF23-specific signal transduction in Kbc cells and KLe will be described in detail.

In order to more specifically confirm the function of inhibiting mineralization in the FGF23-specific signal transduction of KLe in bone as described in Examples 1 to 4, the present inventors have conducted histomorphometric measurements of this function. Analysis was carried out. That is, the parietal bone of kl / kl mice administered with rKLe was fixed with 4% paraformaldehyde, and plastic sections or paraffin sections were prepared according to a conventional method, and histologically evaluated. The type of mouse and the administration method of rKLe are the same as those described in Example 4.

First, fluorescence detection with a double label of calcein will be described. FIG. 9 is a diagram showing a state of a plastic section of the parietal bone double-labeled with calcein according to Example 5 under a fluorescence microscope. The figure shows a representative example of the 9 individuals in each group implemented. FIG. 10 is a diagram showing data of the result of the label interval in the state of the fluorescence microscope of FIG. In addition, FIG.10, FIG.12, FIG.13, FIG.14 and FIG. 16 which show the data in this Example 5 have a significant difference compared with the WT mouse | mouth and kl / kl mouse | mouth which administered the solvent. In these figures, * (significant level): p <0.05 compared to WT mice, and # (significant level): p <0.05 compared to kl / kl mice. As shown in FIGS. 9 and 10, in the parietal bone of kl / kl mice administered with rKLe, the label interval is significantly smaller than in kl / kl mice not administered with rKLe. That is, calcification was suppressed in the parietal bone of kl / kl mice administered with rKLe.

Next, H & E (Hematoxilin-Eosin) staining will be described. FIG. 11 is a view showing a state of a paraffin section of the skull subjected to H & E staining according to Example 5 under a microscope. In addition, a figure shows the representative example of the individual of 9 examples implemented for each group. In the figure, the portion “]” indicates an osteoid. FIG. 12 is a diagram showing data on the number of osteoblasts in the state of the microscope of FIG. FIG. 13 is a diagram showing data of the number of bone cavities in the state of the microscope of FIG. FIG. 14 is a diagram showing bone thickness data in the state of the microscope of FIG. As shown in FIG. 12, FIG. 13 and FIG. 14, unlike the above-mentioned labeling with calcein, the kl / kl mice did not show any significant changes regardless of whether or not rKLe was administered.

Furthermore, the results of von Kossa staining of plastic sections and the results obtained with an electron probe microanalyzer will be described. In von Kossa staining, counterstaining was performed with toluidine blue.

FIG. 15 is a view showing a state of a plastic section of a skull stained with toluidine blue / von Kossa according to Example 5 under a microscope. FIG. 16 is a diagram showing data on the thickness of the osteoid in the state of the microscope of FIG. As shown in FIGS. 15 and 16, the parietal bone of kl / kl mice administered rKLe is markedly more osteoid (noncalcified bone) than that of kl / kl mice not administered rKLe. Had increased. In addition, the figure shows a representative example among 9 individuals in each group.

The electronic probe microanalyzer used JXA-8200 (JEOL) according to the analyzer's instruction manual (irradiation current value 20 nA, acceleration voltage 15 kV, measurement time 0.05 sec / 1 pixel).

FIG. 17 shows the results of the electronic probe microanalyzer according to Example 5. The figure shows a representative example of the 9 individuals in each group implemented. In each figure, element mapping of magnesium (Mg), calcium (Ca), and phosphorus (P) is shown from the left. As shown in FIG. 17, in the skull of kl / kl mice administered with rKLe, Mg, Ca, and P are decreased compared to the case of kl / kl mice not administered with rKLe. That is, the results of the electron probe microanalyzer also showed that administration of rKLe decreased bone mineralization.

In this Example 5, it is suggested that FGF23 in Obc cells acts cooperatively with rKLe in vivo. This includes the results of the above-mentioned Examples, and the level of circulating KLe is directly related to bone mineralization via FGF23 signaling in osteoblasts and bone cells (the higher the circulating level of KLe, the higher the level). , Bone mineralization is suppressed). That is, it suggests that bone mineralization and formation can be controlled by adjusting the amount of KLe circulating in the body or the binding between FGF23 and KLe.

(Example 6)
The present inventors considered that the function of KLe as described above is also involved in the calcification of blood vessels from the bone-vessel correlation. Therefore, in this sixth embodiment, an embodiment relating to the measurement of calcium content in aortic smooth muscle cells will be described in detail.

Rat aortic smooth muscle cells were cultured in 15% FBS DMEM (Dulbecco's Moodified Eagle's Medium) in the incubator in the same manner as described in Example 1. After the cells became confluent, the culture was continued under the condition of 0.1% FBS serum deficiency and loaded with 2 mM sodium hydrogen phosphate. Two days after the start of culture under the condition of serum deficiency, rKLe and rFGF23 were added individually or simultaneously, and cultured for another week. The medium was replaced with fresh medium every 2-3 days. After completion of the culture, the cells were washed with PBS and treated with 0.6N hydrochloric acid overnight. The amount of calcium in the supernatant was measured by the aforementioned calcium C-test Wako.

FIG. 18 is a diagram showing data on the results of calcium content measurement according to Example 6. In addition, there is a significant difference compared to the solvent alone (−). In FIG. 18, ** (significance level): p <0.01 and n = 4. As shown in FIG. 18, calcification of vascular smooth muscle due to phosphate load is suppressed by the simultaneous addition of rKLe and rFGF23, suggesting that a complex containing KLe is involved in the suppression of vascular calcification.

(Example 7)
In this Example 7, an example relating to calcification of the aorta in vivo in order to confirm whether or not the involvement of the complex for inhibiting vascular calcification shown in Example 6 is also observed in the living body is detailed. Explained.

In the experiment of kl / kl mice shown in Example 4 and Example 5, aorta was collected from kl / kl mice administered with rKLe and kl / kl mice administered with solvent (PBS-Atelocollagen) in the same manner. did. The collected aorta was fixed with 4% paraformaldehyde, washed with water, and permeated with 0.5% potassium hydroxide. Thereafter, the solution was stained in a solution in which 20 μg / ml alizarin red was dissolved.

FIG. 19 is a diagram showing a state of an aorta of a kl / kl mouse according to Example 7 with a stereomicroscope. The figure shows a representative example of the 9 individuals in each group implemented. The left is a diagram of the aorta of a kl / kl mouse administered only with a solvent, and the right is a diagram of the aorta of a kl / kl mouse administered with rKLe. Each figure shows a representative example of nine examples. As shown in FIG. 19, the calcified nest stained with alizarin red is clearly reduced in the mice administered with rKLe compared to the mice administered with solvent alone. That is, it is considered that a complex containing KLe suppresses vascular calcification even in in vivo.

(Example 8)
In the eighth embodiment, a further embodiment for confirming the dependency between the rKLe concentration and the degree of calcification, which is simply shown in the third embodiment, will be described in detail.

Into 4-week-old male C57BL / 6J mice, 1,25D was administered subcutaneously twice a day at 6 μg / kg / 1 day to induce the expression of FGF23. 24 hours after the second administration, 2 or 10 μg of rKLe was intravenously injected once. After 2 hours, the parietal bone and kidney were collected, and cell extract (similar to that used in Example 2) was acceptable. Solubilized. This extract was subjected to SDS-PAGE in the same manner as described in Example 1, and phosphorylation of ERK1 / 2 was confirmed by Western blotting in the same manner as described in Example 2.

FIG. 20 shows the results of Western blotting of ERK1 / 2 and phosphorylated ERK1 / 2 according to Example 8. The figure shows a representative example of 5 individuals in each group implemented. As shown in FIG. 20, enhanced phosphorylation of ERK1 / 2 in the parietal bone was observed depending on the dose of rKLe. That is, in consideration of the result of the dependence between the rKLe concentration and the degree of calcification briefly shown in Example 3, the rKLe concentration and lime in cells, tissues, etc. of mouse blood, plasma, serum, bone marrow, bone and blood vessels This means that the dependence on the degree of conversion has been strongly suggested. When this result is used, there is a high possibility that the degree of calcification of hard tissue and / or blood vessels can be examined by measuring the concentration of KLe in the blood of animals such as humans. In such determination of the degree of examination and calcification, for example, it is conceivable to compare with the concentration of KLe in the blood at normal time in the animal. Such phosphorylation promotion was not observed in the kidney (not shown).

Example 9
In this Example 9, an example relating to alizarin red / toluidine blue staining of calcification of the aortic blood vessel wall in vivo will be described in detail.

First, rKLe was administered directly into the male kl / kl mouse at the age of 4 weeks (day 28) using an Alzette (registered trademark) osmotic pump (flow rate of 150 ng / h). After administration of rKLe, sampling of the thoracic aorta of kl / kl mice was performed at 6 weeks of age (day 42). The mouse thoracic aorta was placed in 4% paraformaldehyde / PBS for 16 hours at 4 ° C. and then lavaged. Thereafter, in a conventional manner, a 12 μM paraffin section of the thoracic aorta of the mouse was prepared, and the paraffin section was stained with alizarin red / toluidine blue. The detailed method of the alizarin red / toluidine blue staining is the same as in Example 5 and Example 7 described above. On the other hand, paraffin sections of the thoracic aorta of kl / kl mice administered with solvent were prepared and stained in the same manner.

FIG. 21 is a view showing a state of alizarin red / toluidine blue staining of a paraffin section of a kl / kl mouse thoracic aorta according to Example 9. The figure shows a representative example of the kl / kl mouse thoracic aorta performed. -RKLe is a paraffin section of the thoracic aorta of a kl / kl mouse administered with solvent, and + rKLe is a paraffin section of the thoracic aorta of a kl / kl mouse administered with rKLe. As shown in FIG. 21, in the paraffin section of the aorta of kl / kl mice administered with rKLe, the calcified nest stained with alizarin red is reduced, and the range of only toluidine blue used for counterstaining is shown. It was increasing.

FIG. 22 is a diagram showing data on the results of alizarin red staining range of kl / kl mice and wild-type mice according to Example 9. That is, it is a figure which shows the calcification range (Calcification area). In FIG. 22, the value of the kl / kl mouse (kl / kl (+)) administered with rKLe when the kl / kl mouse (kl / kl (−)) administered with the solvent is taken as 100, and the same The figure shows the value of a wild-type mouse (WT (−)) administered with a solvent prepared and stained for paraffin sections. The kl / kl mouse (+) is significantly different from the kl / kl mouse (−), and in FIG. 22, * (significance level): p <0.05. As shown in FIG. 22, when rKLe was administered to kl / kl mice, the vascular calcification range of kl / kl mice was suppressed.

(Example 10)
In Example 10, an example relating to immunostaining of calcification of the aortic blood vessel wall in vivo will be described in detail.

This Example 10 is performed on kl / kl mice administered with a solvent and kl / kl mice administered with rKLe, and is the same as Example 9 described above until the preparation of paraffin sections in the experimental method. Thereafter, blocking was performed using Protein® Block (DAKO) for 1 hour at room temperature, followed by washing. Anti-Runx2, Anti-phosphorylated phosphoERK, Anti-FGF23 and Anti-Klotho (both are SantauzCruz, dilution factor × 50) were used as primary antibodies and placed at 4 ° C. for 16 hours. Next, a Cy2 / 3-labeled secondary antibody (Jackson Immunoresearch Lab., Dilution factor × 400) was used for 1 hour at room temperature. Then, each was observed using the fluorescence microscope.

FIG. 23 is a view showing a state of immunostaining of a paraffin section of a kl / kl mouse thoracic aorta according to Example 10. The figure shows a representative example of the kl / kl mouse thoracic aorta performed. -RKLe is a paraffin section of the thoracic aorta of a kl / kl mouse administered with solvent, and + rKLe is a paraffin section of the thoracic aorta of a kl / kl mouse administered with rKLe. In each figure of FIG. 23, the portion L delimited by a line indicates the lumen of the blood vessel, the arrow M indicates the portion of the media of the blood vessel, and the arrow A indicates the portion of the outer membrane of the blood vessel. The primary antibody used is shown in the upper right of each figure.

As shown in FIG. 23, when a primary antibody of -rKLe Klotho, FGF23, or p-ERK was used, the aortic wall did not show any positive reaction with any antibody. On the other hand, Runx2 antibody was able to confirm Runx2-positive osteoblast-like cells in a partially calcified region of the thoracic aorta of kl / kl mice. However, by comparing -rKLe Runx2 and + rKLe Runx2, it was also confirmed that the progress of calcification was suppressed by administration of KLe. Further, from the figure of Klotho and FGF23 of + rKLe, it was confirmed that accumulation of endogenous FGF23 and administered KLe was observed outside the medial wall of the vascular wall of the aorta of kl / kl mice. Furthermore, the figure of p-ERK clearly indicates that FGF23-specific signal transduction is activated in this region because the accumulation site of KLe and FGF23 coincides with the phosphorylation site of ERK. Yes.

The rKLe used in each example is also verified for boiling and deactivated, and it is added that the result was the same as when only the solvent was used in each example (not shown).

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.

The contents of the papers, published patent gazettes, patent gazettes, etc. specified in this specification are incorporated by reference in their entirety.

This application is based on Japanese Patent Application No. 2010-136637 filed on June 15, 2010. In this specification, the specification, claims, and entire drawings of Japanese Patent Application No. 2010-136737 are incorporated by reference.

INDUSTRIAL APPLICABILITY According to the present invention, a calcification regulating (suppression or promotion) agent capable of suppressing or promoting hard tissue (for example, bone) and / or vascular calcification by FGF23, a screening method thereof, and hard tissue by FGF23 A calcification inspection method and a calcification inspection kit capable of inspecting calcification in blood vessels and / or blood vessels are provided. The mineralization regulator is an important means for preventing and treating osteoporosis, rickets, osteomalacia, osteoarthritis, ectopic calcification, or arteriosclerosis. A screening method for a calcification regulating agent is an important means for developing new preventive / therapeutic agents for these medical conditions. Providing a calcification test method / test kit is likely to prevent these symptoms associated with calcification inhibition.

In particular, among the above-mentioned diseases, many therapeutic agents for bone diseases have been developed, but for the therapeutic agents related to vascular calcification, the mechanism of signal transduction (due to a complex of KLe, FGF23, and FGFR) It has not been clearly elucidated and has not been developed much. Therefore, for the development of a therapeutic agent for arteriosclerosis caused by vascular calcification, it is considered that the use of the present invention leads to the development of a new and useful drug.

Claims (15)

1. Contacting at least solubilized Klotho, FGFR, and FGF23 in a sample in the absence and presence of the test compound;
   Selecting a compound that changes FGF23-specific signaling in the sample in the presence of the test compound as compared to FGF23-specific signaling in the sample in the absence of the test compound;
   A screening method for a calcification regulating agent for hard tissue and / or blood vessels, comprising:
2. 2. The regulation of hard tissue and / or blood vessel calcification according to claim 1, wherein the change in specific signaling is indicated by phosphorylation of FGFR, phosphorylation of ERK or expression level of Egr-1. Agent screening method.
3. Measuring the concentration of solubilized Klotho in the sample in the absence and presence of the test compound;
   Selecting a compound that increases or decreases the concentration of the solubilized Klotho in the sample in the presence of the test compound as compared to the concentration of the solubilized Klotho in the sample in the absence of the test compound;
   A screening method for a calcification regulating agent for hard tissue and / or blood vessels, comprising:
4. Measuring the complex formation of solubilized Klotho, FGFR and FGF23 in the specimen in the absence and presence of the test compound;
   Selecting a compound that increases or decreases the complex formation in the sample in the presence of the test compound compared to the complex formation in the sample in the absence of the test compound;
   A screening method for a calcification regulating agent for hard tissue and / or blood vessels, comprising:
5. The screening method for a hard tissue and / or blood vessel calcification regulator according to claim 1, wherein the calcification regulator is screened in vivo.
6. The screening method for a hard tissue and / or blood vessel calcification regulator according to claim 3, wherein the calcification regulator is screened in vivo.
7. The screening method for a calcification regulating agent for hard tissue and / or blood vessels according to claim 4, wherein the screening for the calcification regulating agent is performed in vivo.
8. A hard tissue and / or vascular calcification regulator comprising a compound that suppresses or promotes complex formation of solubilized Klotho, FGFR, and FGF23.
9. The hard tissue and / or vascular calcification regulator according to claim 8, wherein the compound is solubilized Klotho.
10. 10. The hard tissue and / or blood vessel according to claim 9, wherein the compound is a ligand having binding affinity for solubilized Klotho, FGFR, or a complex of the solubilized Klotho, FGFR, and FGF23. Calcification regulator.
11. 11. The hard tissue and / or blood vessel calcification regulator according to claim 10, wherein the ligand is an antibody.
12. Measuring the concentration of solubilized Klotho, the formation of a complex of solubilized Klotho, FGFR and FGF23, or FGF-specific signal transduction associated with the complex formation in a specimen, and / or Or blood vessel calcification testing method.
13. 13. The hard tissue and / or blood vessel calcification test according to claim 12, wherein the specimen is animal blood, plasma, serum, bone marrow, bone tissue or cells, or blood vessel tissue or cells. Method.
14. The method for inspecting calcification of hard tissue and / or blood vessels according to claim 13, wherein the animal is a human.
15. A kit for examining calcification of hard tissue and / or blood vessels, comprising a ligand having binding affinity in a complex of solubilized Klotho or solubilized Klotho, FGFR and FGF23.

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