JP4934812B2 - NFAT2 expression suppression method - Google Patents

Description

  The present invention relates to a method for suppressing the expression of NFAT2, which is a transcription factor in the osteoclast differentiation process. More specifically, a method of suppressing the expression of NFAT2 by suppressing the signal transduction pathway of L-serine and / or L-glycine, and further, prevention and / or treatment of NFAT2-related diseases by controlling the expression of NFAT2 The present invention relates to a drug and a screening method thereof.

The bone tissue undergoes bone remodeling (remodeling) by repeating bone formation by osteoblasts and bone resorption by osteoclasts. However, when “osteoporosis” or “rheumatoid arthritis” develops, bone resorption exceeds the amount of bone formation, resulting in a decrease in bone mass. On the other hand, “marble bone disease” is a pathological condition caused by bone resorption failure. When osteoclasts are abnormally differentiated and there are no osteoclasts in the bone tissue or there are abnormalities in the function of osteoclasts. It is thought that it develops when bone resorption cannot be performed.
Conventionally, osteoporosis treatment methods include direct administration of calcium agents, use of calcitonin and bisphosphonates, administration of hormone agents such as parathyroid hormone (PTH) and growth hormone, and administration of vitamin D3. Is not a definitive treatment. Calcium preparations need to be ingested in a very large amount, calcitonin has the disadvantage that the appearance of antibodies and oral administration are impossible, hormonal preparations have strong side effects, and vitamin D3 has low effects. Anti-RANKL antibodies against osteoclast differentiation factor (receptor activator of NFκB ligand; hereinafter abbreviated as RANKL) are also candidates, but in the case of proteins, there are problems such as antigenicity and inability to administer orally. is there. Therefore, development of a novel osteoporosis therapeutic agent has been demanded.

In addition, osteoporosis is caused by the loss of the balance between bone resorption and bone regeneration and increased formation of osteoclasts responsible for osteolysis. Although this mature osteoclast is a multinucleated cell, it is known that the NFAT2 (NFATc1) gene is essential in the process of cell fusion (see Patent Document 1). Therefore, if the expression of the NFAT2 gene can be suppressed, osteoclast maturation can be suppressed, and osteoporosis may be prevented or treated. Techniques for controlling osteoclast formation using such signal transduction include methods for promoting or suppressing Ca ²⁺ signal transduction (see Patent Document 1) and methods including a step for promoting or inhibiting IFN signal transduction (patent) Documents 2 and 3) are known. However, these methods need to be administered locally because they control Ca ²⁺ signaling, or have problems such as antigenicity and inability to be administered orally because they are administered proteins. Therefore, development of a novel screening method based on a novel mechanism has been demanded.
JP 2004-154132 A International Publication No. 2002/024228 Pamphlet International Publication No. 2002/024229 Pamphlet

  The present invention relates to a preventive or therapeutic agent for NFAT2-related diseases such as osteoporosis based on a novel mechanism, a method for preventing or treating NFAT2-related diseases such as osteoporosis, and a screening method for preventive or therapeutic agents for NFAT2-related diseases such as osteoporosis. The purpose is to provide.

In the osteoclast differentiation process, osteoclast progenitor cells express various genes responsible for the progression of differentiation in response to signals from RANKL. The inventor used the osteoclast differentiation induction system consisting of the mouse cell line RAW264 and the differentiation-inducing factor RANKL protein last year to combine the gene expression by microarray analysis and the phenomenon that was uniquely found to suppress differentiation by cell seeding density. The transcription factor NFAT2, whose expression is induced by RANKL, was found to be important for multinucleated cell formation. Normally, in osteoclast precursor cells, NFAT2 is phosphorylated in the cytoplasm and in an inactive state. However, when a signal from RANKL is received, Ca ²⁺ and Ca ²⁺ -CaM are bound and activated by calcineurin. NFAT2 is dephosphorylated. Dephosphorylated NFAT2 moves into the nucleus approximately 24 to 48 hours after stimulation with RANKL, and induces gene expression related to osteoclast formation and functional expression such as various enzyme groups responsible for cell fusion and osteolysis reaction . Conversely, osteoclast precursor cells treated with tacrolimus (hereinafter also referred to as FK506), which is an inhibitor of NFAT2, or cyclosporine do not differentiate into osteoclasts.

  Recently, using the same experimental system as described above, the inventor found that either amino acid L-serine or L-glycine in the medium components is essential for induction of NFAT2 by RANKL. To confirm this, L-serine analogues were used to confirm that D-serine and L-homoserine competitively inhibit the activity of L-serine and suppress the induction of NFAT2 and the formation of multinucleated cells. From this, it was suggested that this phenomenon has a mechanism that recognizes the specificity of the molecular structure. As described above, since disturbance of bone resorption due to relative activation of osteoclasts is a factor of bone diseases such as osteoporosis, the formation of osteoclasts by the amino acids L-serine and L-glycine Control of activity opens the way to new molecular targeted drugs for bone metabolic diseases.

The present inventor has found that the amino acid L-serine or L-glycine is essential for the expression of the NFAT2 gene, and that the NFAT2 gene is not expressed in a state where L-serine and L-glycine are deficient. As a result, the present inventors have found that osteoclasts do not fuse and osteoclasts do not mature. That is, the present invention relates to a method for controlling the expression of NFAT2 essential for osteoclast formation, a drug and a medicament for use in the control, and a screening method thereof, more specifically,
(1) A method for suppressing the expression of NFAT2, characterized by suppressing a signal transduction pathway of L-serine and / or L-glycine,
(2) The method according to (1), wherein the suppression of NFAT2 expression is due to suppression of NFAT2 gene expression,
(3) The method according to (1) or (2) above, wherein the signal transduction pathway of L-serine and / or L-glycine is suppressed by contacting with a substance that antagonizes L-serine.
(4) The method according to (3) above, wherein the substance that antagonizes L-serine is an L-serine analog.
(5) The method according to (4) above, wherein the L-serine analog is D-serine, L-homoserine, a salt thereof, or an ester thereof.
(6) A method for preventing and / or treating an NFAT2-related disease, which comprises suppressing the expression of NFAT2 by the method of any one of (1) to (5) above,
(7) A method for preventing and / or treating an NFAT2-related disease, comprising suppressing a signal transduction pathway of L-serine and / or L-glycine,
(8) The prevention and / or treatment method according to (6) or (7) above, wherein the NFAT2-related disease is osteoporosis,
(9) Inhibition of L-serine and / or L-glycine signal transduction pathway in vivo, comprising administration of an effective amount of D-serine, L-homoserine, a salt thereof or an ester thereof process,
(10) A preventive and / or therapeutic agent for NFAT2-related diseases, comprising a substance that antagonizes L-serine,
(11) The preventive and / or therapeutic agent according to (10) above, wherein the substance that antagonizes L-serine is D-serine, L-homoserine, a salt thereof, or an ester thereof.
(12) The preventive and / or therapeutic agent according to (10) or (11), wherein the NFAT2-related disease is osteoporosis,
(13) A method for screening a substance affecting osteoclast formation, wherein (a) signaling of L-serine and / or L-glycine in osteoclast precursor cells in the presence of a sample containing a test substance A step of detecting suppression or promotion, and (b) suppression of the signal transduction of the L-serine and / or L-glycine for suppression of osteoclast formation, or signal transduction of the L-serine and / or L-glycine Associating promotion of osteoclast formation with promotion of osteoclasts,
(14) A method for screening a substance that antagonizes L-serine involved in osteoclast formation, and (a) culturing osteoclast precursor cells in a medium containing a test substance, L-serine and RANKL And (b) detecting the suppression of L-serine and / or L-glycine signal transduction in osteoclast precursor cells, and (15) L-serine involved in osteoclast formation and / or Or a method for screening a substance having an L-glycine-like action, wherein (a) culturing osteoclast precursor cells in a medium containing a test substance and RANKL but not containing L-serine and / or L-glycine And (b) detecting the promotion of L-serine and / or L-glycine signaling in osteoclast precursor cells,
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Furthermore, the present invention provides
(16) Use of a substance that antagonizes L-serine for producing a medicament for preventing and / or treating an NFAT2-related disease,
(17) The use according to (16) above, wherein the substance that antagonizes L-serine is D-serine, L-homoserine, a salt thereof or an ester thereof, and (18) an NFAT2-related disease The use according to (16) or (17) above, which is osteoporosis,
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  According to the present invention, NFAT2 expression, particularly NFAT2 gene expression, can be suppressed, so that differentiation from osteoclast precursor cells to osteoclasts can be suppressed. As a result, bone destruction by osteoclasts can be effectively suppressed. In addition, a drug that controls NFAT2 expression, particularly NFAT2 gene expression by a signal pathway that targets NFAT2 of the present invention, particularly NFAT2 gene, is quantitatively associated with diseases related to NFAT2, such as normally mineralized bone matrix. Osteoporosis, autoimmune arthritis, Paget's disease or rheumatoid arthritis, bone osteogenesis and bone sclerosis, or bone sclerosis or immunity in which osteoclast dysfunction results in abnormal bone structure and remodeling It is useful as a drug for sexual diseases. In addition, the present invention can provide a screening system for a novel NFAT2 expression control method by measuring the effective concentration of L-glycine and / or L-serine, and thus is useful for searching for a novel NFAT2 expression regulator. .

It is a figure which shows the differentiation from the mouse | mouth RAW264 cell to an osteoclast in the in vitro experiment. In the figure, MN cells indicate multinucleated cells. It is a figure which shows the result of the western blotting of a RAW264 cell extract. In the figure, F in the medium indicates a fresh medium, and C indicates a conditioned medium (the same applies to the following figures). It is a figure which shows the result of the western blotting of the RAW264 cell extract after RANKL stimulation. A) is a figure which shows the result of the western blotting of NFAT2-siRNA expression cell extract, B) is a figure which shows the NFAT2-siRNA expression cell after RANKL stimulation. It is a figure which shows the relationship between the seeding cell density of RAW264 cell, and a polygonal cell formation rate. In the figure, a cell density of 1 indicates 2.5 × 10 ⁴ cells / well. It is a figure which shows the result of the western blotting of the RAW264 cell extract after RANKL stimulation in the low density seeding | inoculation of RAW264 cell or high density seeding | inoculation. It is a figure which shows the result of the western blotting of the RAW264 cell extract after seed | inoculating the RAW264 cell in a different cell density and RANKL stimulation. In the figure, a cell density of 1 indicates 2.5 × 10 ⁴ cells / well. It is a figure which shows the differentiation induction efficiency of the RAW264 cell at the time of carrying out differentiation induction in a different culture medium, and this cell. It is a figure which shows the change of the culture medium component in NFAT2 expression in RAW264 cell, and the influence of NEAA. It is a figure which shows the influence of NEAA in NFAT2 expression in a low density seeding | inoculation or a high density seeding | inoculation RAW264 cell. In the figure, L indicates low density seeding, and H indicates high density seeding. It is a figure which shows the influence of NEAA in NFAT2 expression in RAW264 cell. It is a figure which shows the influence of NEAA with respect to RAW264 cell proliferation. It is a figure which shows the influence of the amino acid in NFAT2 expression in RAW264 cell using Conditioned medium. In the figure, A is alanine, D is aspartic acid, E is glutamic acid, G is glycine, N is asparagine, P is proline, and S is serine (same in the following figures). It is a figure which shows the influence of the amino acid in NFAT2 expression in RAW264 cell using Fresh medium. It is a figure which shows the influence of a serine analog in NFAT2 expression in RAW264 cell using Fresh medium. In the figure, Ser represents serine (same in the following figures), D represents D-serine, and L-Homo represents L-homoserine. It is a figure which shows the influence of D-serine in NFAT2 expression in RAW264 cell. It is a figure which shows the cell density-dependent multinucleated cell formation of the osteoclast precursor cell. A) The result of inducing differentiation of osteoclast precursor cells obtained from mouse bone marrow cells at different cell densities into osteoclasts is shown in relation to the number of TRAP-positive cells and the cell concentration. B) shows the morphology of multinucleated cells. It is a figure which shows the expression of NFAT2 depending on the reduction | decrease of L-serine in the culture medium in a high-density culture, and L-serine concentration. A) Changes in the amount of L-serine in the medium when RAW264 cells are seeded at low density or high density and cultured in the presence or absence of RANKL. B) The expression of NFAT2 in RAW264 cells after culturing RAW264 cells in media supplemented with different concentrations of L-serine is shown by the results of Western blotting. It is a figure which shows the influence of the cell density and L-serine with respect to the expression of c-Fos. A) The expression of c-Fos when RAW264 cells seeded at low honeyness were cultured in the presence of RANKL is shown by the results of Western blotting. B) RAW264 cells are cultured by low density seeding and high density seeding, and the expression of c-Fos is shown by the results of Western blotting. In the figure, Low indicates low density seeding, and High indicates high density seeding. c) Western blotting shows the expression of c-Fos and NFAT2 after RAW264 cells were seeded at low density and cultured in a medium supplemented with NEAA, L-serine or L-glycine. The numerical value of each lane shows the following. 1: RAW264 cells cultured in medium without NEAA; 2: RAW264 cells cultured in medium with L-serine; 3: RAW264 cells cultured in medium with L-glycine; 4: L-serine and L -RAW264 cells cultured in medium supplemented with glycine; 5: RAW264 cells cultured in medium supplemented with NEAA without L-serine; 6: RAW264 cells cultured in medium supplemented with NEAA without L-glycine; 7: RAW264 cells cultured in a medium supplemented with NEAA not containing L-serine and L-glycine; 8: RAW264 cells cultured in a medium supplemented with NEAA. It is a figure which shows the influence of L-serine in the differentiation to the osteoclast of a bone marrow cell. A) The influence of L-serine on the proliferation of bone marrow cells is shown. A vertical axis | shaft shows the result of having measured the degree of cell proliferation and cell death by MTT activity. The serine concentration x1 on the horizontal axis is equal to the normal amount (0.1 mM). B) shows the effect of L-serine on multinucleated cell formation of bone marrow-derived osteoclast precursor cells. The left figure shows the amount of multinucleated cells formed, and the right figure shows the cell morphology. * Indicates that there is a significant difference (P <0.05) by Student's t-test. C) Western blotting results of cell lysates cultured for 48 hours after stimulation of osteoclast precursor cells with RANKL and M-CSF. D) shows the effect of L-serine on multinucleated cell formation in bone marrow cells in which NFAT2 was forcibly expressed via retroviral infection. The figure on the left shows the amount of multinucleated cells formed, and the figure on the right shows the morphology of GFP-expressing cells and NFAT2-expressing cells (TRAP staining).

  The present invention relates to a method for suppressing the expression of NFAT2. More specifically, the osteoclast formation is suppressed by suppressing the signal transduction pathway of L-serine and / or L-glycine in osteoclast precursor cells and suppressing the expression of NFAT2, particularly the expression of the NFAT2 gene. Furthermore, the present invention relates to a preventive and / or therapeutic drug for NFAT2-related diseases by controlling the expression of NFAT2, and a screening method thereof. The present invention will be described below.

  “L-serine and / or L-glycine signal transduction pathway” refers to a signal transduction pathway essential for osteoclast formation from osteoclast progenitor cells along with RANKL (receptor activator of NFκB ligand) pathway. The route may be a route that responds to another route. Further, the signal transduction pathway of L-serine and / or L-glycine is a transduction pathway different from the signal transduction pathway, and L-serine and / or L-glycine is involved in a part of the different transduction pathway, and NFAT2 Also includes a transduction pathway in which the NFAT2 gene is expressed. Accordingly, by promoting L-serine and / or L-glycine signaling in osteoclast precursor cells, differentiation of the cells into osteoclasts can be promoted, and conversely, L-serine and / or By blocking L-glycine signaling, differentiation of the progenitor cells into osteoclasts can be inhibited.

The “osteoblast” refers to a fusion of mononuclear osteoclast precursor cells differentiated from monocyte / macrophage cells (hereinafter abbreviated as CFU-M) originating from hematopoietic stem cells. It refers to the formed multinucleated giant cells (Suda, T. et al., Endocr., Rev. 20, p. 345, 1999). Osteoclasts secrete collagenase that digests the bone matrix and show bone resorption ability. In general, mature osteoclasts express tartrate-resistant acid phosphatase (hereinafter abbreviated as TRAP), calcitonin receptor, and vitronectin receptor. For this reason, collagenase, TRAP or calcitonin receptor, and vitronectin receptor can be used as an index of osteoclast formation (bone resorption marker). Therefore, osteoclast formation is measured by at least one of these indices. It can be judged based on. Osteoclasts are preferably TRAP-positive and have bone resorption activity. Osteoclasts differentiate from CFU-M as described above. Specifically, for example, the bone marrow monocyte / macrophage progenitor cells (BMMs) and the like are expressed by macrophage colony-stimulating factor (M-CSF), interferon, and the like. Eleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), 1,25-dihydroxyvitamin D3 (1,25 (OH) ₂ D ₃ ), etc. In the presence of RANKL.
Osteoclast formation can be observed, for example, according to the literature (Yasuda, H. et al., Proc. Natl. Acad. Sci. USA, Vol. 95, p. 3597-602 (1998)). Specifically, for example, in the case of an individual, a bone slice can be created and bone resorption can be observed. Also, multinucleated giant cells can be identified by microscopic observation, and osteoclast formation can be detected both in vivo and in vitro by known detection methods such as TRAP staining and detection of calcitonin receptor or vitronectin receptor expression. can do. The bone resorbability can be evaluated by a known method, for example, the Pit formation assay method for measuring the pit formation area on the ivory slice.
The “osteoblast precursor cell” includes monocyte / macrophage cells that can differentiate into osteoclasts, and more specifically, cells whose proliferation is promoted in response to M-CSF or the like. preferable. Osteoclast precursor cells may be cells contained in bone marrow or spleen, or may be immortalized cell lines. Osteoclast precursor cells can also be differentiated in vitro from hematopoietic stem cells.

  In the present specification, “suppressing L-serine and / or L-glycine signal transduction pathway” means a signal transduction pathway that transmits the concentration of L-serine and / or L-glycine and their functional information. Thus, it refers to suppressing a signal transduction pathway involved in the expression of NFAT2 (also referred to as NFATc1, hereinafter unified with NFAT2), particularly the expression of the NFAT2 gene, and includes inhibiting or suppressing the signal transduction itself. In order to suppress the signal transduction pathway, L-serine and / or L-glycine antagonist is administered to reduce the effective concentration of L-serine and / or L-glycine, L-serine and / or L -Substantially reducing the concentration of L-serine and / or L-glycine by promoting metabolism, modification and / or degradation of glycine, and accepting L-serine and / or L-glycine receptors by antibodies This includes, but is not limited to, blocking. In addition, any part of the signal transduction pathway may be suppressed. For example, the expression of a binding protein such as a receptor or a transporter that senses the concentration of L-serine, L-glycine, or a derivative thereof may be suppressed. Inhibition of NFAT2 expression from the binding protein, particularly suppression of a signal transduction pathway leading to expression of the NFAT2 gene. In short, if a signal indicating that the concentration or activity of L-serine and / or L-glycine is substantially reduced can be transmitted to suppress the expression of NFAT2, particularly the expression of the gene of NFAT2, it is possible to suppress any part of the signal transduction pathway. There may be. In this case, via a substance synthesized from L-serine and / or L-glycine or a metabolite generated from L-serine and / or L-glycine (including lipids, sugars, etc. to which serine is added). It also includes the case where the suppression of NFAT2 expression, particularly the expression of the NFAT2 gene is suppressed by signal transduction. Moreover, not only one place of a signal transmission path | route but a several location may be suppressed. In addition, in order to suppress the signal transduction pathway of L-serine and / or L-glycine, the signal transduction pathway is different from the signal transduction pathway of L-serine and / or L-glycine, and at least of the different transduction pathways. In part, L-serine and / or L-glycine is involved and NFAT2 is expressed. In particular, when NFAT2 gene is expressed, it also includes suppressing the engagement (action) of L-serine and / or L-glycine. .

NFAT2 is one of transcription factors activated by dephosphorylation in a Ca ²⁺ -calcineurin-dependent manner, translocating into the nucleus, and being activated. NFAT2 was discovered as a factor that regulates cytokine production in T cells, but plays an important role in the function or differentiation of many cells (Crabtree, GR and Olson, EN, Cell, 109). Volume: S67-S79, 2000). Therefore, such functions and differentiation can be suppressed by suppressing the expression of NFAT2 by the method or agent of the present invention. As a specific example of NFAT2, for example, the base sequence of human NFAT2 mRNA is shown in Accession number NM_006162, and the amino acid sequence of protein is shown in NP_006153. In addition, NFAT2 includes not only NFAT2 represented by the above specific amino acid sequence, but also fragments, homologues, derivatives, and mutants thereof as long as the biological function is equivalent to NFAT2. Is done. Here, examples of homologues include NFAT2 of other biological species such as mouse and rat corresponding to human NFAT2, and these include HomoLomoGene (http://www.ncbi.nlm.nih.gov/HomoloGene/). It can be identified a priori from the base sequence of the gene identified by the above. For example, the base sequence of mouse NFAT2 mRNA is shown in Accession number AF239169, AF087434 (isoform a), and AF049606 (isoform b), and the amino acid sequences of the NFAT2 proteins encoded by these are shown in AAC36725, AAC36725, and AAC0550, respectively. Yes.
Variants include naturally occurring allelic variants, non-naturally occurring variants, and variants having amino acid sequences modified by artificial deletions, substitutions, additions and insertions. . In addition, as the above-mentioned mutant, an amino acid sequence is at least about 70%, preferably about 80%, more preferably about 95%, and still more preferably about 97% homologous to a non-mutated protein or (poly) peptide. Can be mentioned.

Suppression of NFAT2 expression includes suppression of NFAT2 gene expression, suppression of NFAT2 gene transcription and / or translation, and suppression of transcription through NFAT1 expression and activity suppression (Zhou B et. Al., J. Biol. Chem). , 277, p. 10704, 2002), promotion of destabilization of NFAT2 mRNA, promotion of degradation of NFAT2 protein, exogenous antisense nucleic acid against NFAT2 mRNA or ribozyme or RNAi that cleaves NFAT2 mRNA Expression inhibition of NFAT2 by expression is included. The preferred suppression of NFAT2 expression is suppression of NFAT2 gene expression.
In the present specification, a gene (or DNA) is used in the meaning including not only double-stranded DNA but also each single-stranded DNA such as a sense strand and an antisense strand constituting the DNA. The length is not particularly limited. Therefore, in this specification, unless otherwise specified, a gene (DNA) is a double-stranded DNA containing human genomic DNA, a single-stranded DNA containing cDNA (positive strand), and a sequence having a sequence complementary to the positive strand. Both double-stranded DNA (complementary strand) and fragments thereof are included. The gene or DNA includes not only a gene or DNA represented by a specific base sequence, but also a protein having a biological function equivalent to the protein encoded by these (for example, a homolog, homolog, mutant, and A gene or DNA encoding a derivative or the like) is included. Examples of the gene or DNA encoding the homologue, mutant or derivative include a gene or DNA having a base sequence that hybridizes with a complementary sequence of a specific base sequence under stringent conditions.
Here, stringent conditions are taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques in Enzymology, Vol. 152, Academic Press, San Diego CA) It can be determined based on temperature (Tm). For example, as washing conditions after hybridization, the conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” can be mentioned. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although not particularly limited, as stricter hybridization conditions (washing conditions after hybridization), about “0.5 × SSC, 0.1% SDS, 42 ° C.”, more severe hybridization conditions (washing conditions after hybridization) As the cleaning condition, “0.1 × SSC, 0.1% SDS, 65 ° C.” can be mentioned. Specifically, as such a complementary strand, a strand consisting of a base sequence that is completely complementary to the base sequence of the target positive strand, and at least about 90%, preferably about 95% homology with the strand Examples thereof include a chain consisting of a base sequence having a property.

  The “substance that antagonizes L-serine” refers to a substance that can convey the concentration or activity of L-serine lower than the actual level in the L-serine and / or L-glycine signal transduction pathway. Examples of such substances include, for example, amino acid analogs of L-serine and / or L-glycine. Typical examples include, but are not limited to, D-serine and L-homoserine, which are amino acid analogs that can suppress L-serine and / or L-glycine signal transduction pathways, and preferably express NFAT2 (preferably Any one can be used as long as it can suppress the expression of the NFAT2 gene. The expression of NFAT2 can be observed by Western blotting, for example, by adding a substance that antagonizes L-serine to cultured cells as in Example 10. Differentiation into osteoclasts can be determined by using any one of polynuclearization (cell fusion), TRAP, calcitonin receptor, vitronectin receptor, collagenase expression or bone resorption ability as an index. It is also possible to measure the effect of a substance that antagonizes L-serine and / or L-glycine using the above as an index.

The “L-serine analog” typically refers to an amino acid analog of L-serine, but is not limited thereto. The L-serine analog may be any substance that is structurally or functionally similar to all or part of L-serine and that acts in competition with L-serine in vivo, for example.
In addition, L-serine analogs are synthesized using optical isomers of L-serine, derivatives having side chains in L-serine, pharmaceutically acceptable salts or esters of L-serine, and L-serine as raw materials. And a metabolite produced from L-serine (including lipids added with serine, sugars, etc.), and substances that antagonize L-serine are also included, but are not limited thereto. Moreover, it is a substance designed by drug design from the L-serine receptor, and may be a substance that antagonizes L-serine. Examples of pharmaceutically acceptable salts of L-serine include alkali metal (potassium, sodium, lithium, etc.) salts, alkaline earth metal (calcium, magnesium, etc.) salts, ammonium salts (tetramethylammonium salts, tetra Butylammonium salt, etc.), organic amine (triethylamine, methylamine, dimethylamine, etc.) salt, acid adduct salt [inorganic acid salt (hydrochloride, hydrobromide, hydroiodide, sulfate, phosphoric acid) Salt, nitrate, etc.) or organic acid salt (acetate, lactate, tartrate, oxalate, fumarate, maleate, benzoate, citrate, methanesulfonate, glucuronate, gluconic acid Salt, etc.)]. Examples of the L-serine ester include esters of lower alkyl groups such as methyl, ethyl, and propyl.
Further, the L-serine analog may be a hydrate or a solvate. The hydrate or solvate is preferably non-toxic and water-soluble. Examples of the hydrate include monohydrate, dihydrate, 1/2 hydrate, and 4/3 hydrate. Examples of the solvate include alcohol solvents such as an adduct of ethanol. Japanese products are listed.

  Preferred examples of the L-serine analog include D-serine and L-homoserine. D-serine or L-homoserine may be a salt thereof or an ester thereof. The salts include all pharmaceutically acceptable ones. Examples of the salt include the above-described pharmaceutically acceptable salts of L-serine. As an ester body, the above-mentioned ester body of L-serine is mentioned. Specific examples of D-serine, L-homoserine, their salts, or esters thereof include D-serine methyl ester hydrochloride and L-O-caffeoyl homoserine.

  Examples of a method for suppressing the expression of NFAT2, particularly for suppressing the expression of the NFAT2 gene, include bringing a substance that antagonizes L-serine into contact with the signal transduction pathway of L-serine and / or L-glycine. The contact includes contact with a protein to which L-serine and / or L-glycine acts or associates, or contact with, for example, a cell, tissue or individual having the signal transduction pathway. As a result of the contact, in the L-serine and / or L-glycine signal transduction pathway, for example, a substance that antagonizes L-serine competes with L-serine and / or L-glycine, for example, L-serine and Inhibits or suppresses L-glycine signal transmission.

  According to the method of the present invention, by suppressing the signal transduction pathway of L-serine and / or L-glycine, it is possible to suppress the expression of NFAT2, particularly the expression of the NFAT2 gene. When the expression of NFAT2 is suppressed, osteoclast formation is inhibited. Osteoclasts are cells that absorb bone, and abnormalities in the cells cause NFAT2-related diseases such as bone diseases such as osteoporosis and marble bone disease, and immune diseases. Therefore, the method of the present invention can be used in a method for preventing or treating NFAT2-related diseases, for example, bone diseases such as osteoporosis and marble bone disease, and immune diseases. In addition to osteoclast maturation, NFAT2 is known to have an inducing activity of cytokines that activate immune responses such as interleukin-2 (IL-2). Can also be used for immunosuppression

In the method of the present invention, it is preferable to contact and / or introduce a substance that antagonizes L-serine, such as D-serine, into osteoclast precursor cells. By contacting osteoclast progenitor cells with a substance that antagonizes L-serine, such as D-serine or L-homoserine, efficiently through the suppression of NFAT2 expression, particularly NFAT2 gene expression, in osteoclast progenitor cells Osteoclast formation can be inhibited.
The method of the present invention comprises administration of a substance that antagonizes an effective amount of L-serine, such as D-serine, L-homoserine, a salt thereof, or an ester thereof, to a mammal. The administration may be either oral or parenteral as long as the signal transduction pathway of L-serine and / or L-glycine in vivo can be suppressed.

Substances that antagonize L-serine used in the method for suppressing the expression of the NFAT2 gene of the present invention, such as D-serine and L-homoserine, can suppress the formation of osteoclasts. It can be used as a preventive or therapeutic agent for autoimmune arthritis such as rheumatoid arthritis and osteopenic diseases such as osteoporosis, or as an immunosuppressive agent such as immune diseases.
One embodiment of the present invention includes administration of D-serine, for example. Administration of D-serine suppresses NFAT2 expression, particularly NFAT2 gene expression. The concentration of D-serine necessary for this suppression is about 5 to 20 times, preferably about 10 times that of L-serine.

  In addition, the method of suppressing NFAT2 expression of the present invention, particularly the method of suppressing NFAT2 gene expression, is preferably used in combination with a drug or the like that inhibits osteoclast formation. Examples of agents that inhibit osteoclast formation include calcineurin inhibitors. Examples of inhibitors of calcineurin include FK506, cyclosporin A (CyA / CsA), and analogs thereof. By simultaneously administering these calcineurin inhibitors and a substance that antagonizes L-serine according to the present invention, such as D-serine or L-homoserine, it becomes possible to more efficiently suppress bone resorption.

The screening method of the present invention can be performed using, for example, an osteoclast differentiation induction system. That is, the screening method of the present invention includes the following steps (a) and (b): (a) L-serine and / or L-glycine in osteoclast precursor cells in the presence of a sample containing a test substance. Detecting the suppression or promotion of signal transduction,
(B) Inhibiting the signal transduction of the L-serine and / or L-glycine for suppressing osteoclast formation, or promoting the signal transduction of the L-serine and / or L-glycine to promote osteoclast formation. The process of associating with

  Examples of cells used in such screening methods include osteoclast precursor cells that can be differentiated into osteoclasts. Any cell can be used without limitation as long as it is an osteoclast precursor cell that can differentiate into osteoclasts. For example, bone marrow macrophage cells are preferably used. More specifically, BMMs, RAW264.7 cells (macrophage-like cells; sometimes abbreviated as RAW264 cells), embryonic stem cells (ES cells), and the like can be mentioned. RAW264 cells are preferred. In particular, mouse RAW264 cells (RIKEN Cell Bank; HYPERLINK HYPERLINK "http://www.rtc.riken.go.jp/CELL/HTML/RIKEN" http: //www.rtc.riken.L.TM.JL/go. / RIKEN, http://www.rtc.riken.go.Jp/CELL/HTML/RIKEN Cell Bank. Html) is known to differentiate into osteoclasts upon addition of RANKL, a differentiation inducer. (Biochem. Biophys. Res. Commun. 282, 278-283 (2001)).

  In the screening method of the present invention, for osteoclast formation from osteoclast precursor cells, it is usually preferable to stimulate osteoclast precursor cells with RANKL in the presence of M-CSF. Specifically, for example, in order to form osteoclasts from BMM or ES cells, RANKL stimulation is performed in the presence of M-CSF. The concentrations of M-CSF and RANKL can be adjusted as appropriate. For example, the concentration of M-CSF is about 1 to 20 ng / mL, preferably about 5 to 15 ng / mL, and more preferably about 8 to 12 ng / mL. The concentration of RANKL is about 10 to 200 ng / mL, preferably about 50 to 150 ng / mL, more preferably about 80 to 120 ng / mL. In addition, cells that differentiate into osteoclasts in response to RANKL in an M-CSF-independent manner, such as RAW264 cells, can differentiate into osteoclasts even in the absence of M-CSF in osteoclast formation. Only RANKL stimulation needs to be performed. In this case, the concentration of RNAKL is the same as that described above. In the screening method of the present invention, for example, an osteoclast culture in which a frozen rat osteoclast precursor cell (derived from bone marrow), a dedicated medium containing M-CSF and RANKL, and a dentin section for Pit formation assay are set. Kits (manufactured by Hokudo Co., Ltd.) and the like can also be preferably used.

  The sample containing the test substance is not particularly limited. For example, a library of amino acid analogs, a library of synthetic low molecular substances, a purified protein, an expression product of a gene library, a library of synthetic peptides, a cell extract, Examples include cell culture supernatants, plant extracts, and microorganism-derived substances.

Detection of L-serine and / or L-glycine signal transduction can be performed using NFAT2 expression as an indicator. The expression of NFAT2 can be detected using a known method such as Western blot, dot blot, or slot blot, for example, using an antibody that specifically binds to NFAT2. Specifically, for example, Western blotting uses NFAT2 antibody as a primary antibody, followed by a chemiluminescent reagent such as HRP (horseradish peroxidase) as a secondary antibody, a radioisotope such as ¹²⁵ I, a fluorescent substance, etc. Using the labeled antibody labeled with (the antibody that binds to the primary antibody), the signal derived from the chemiluminescence, radioisotope, fluorescent substance, etc. of the labeled compound is obtained with a chemiluminescence detector, radiation measuring instrument, fluorescence detector, etc. This can be done by detecting and measuring.

  In addition, the expression of NFAT2 is preferably performed using the detection of gene expression level as an index, in addition to the protein as described above. Detection of the NFAT2 gene at the gene expression level can be performed by using RNA prepared from the cells or a polynucleotide complementary to the RNA transcribed from the RNA and, for example, a promoter region of the NFAT2 gene or the NFAT2 gene. In addition to known methods such as Northern blotting and RT-PCR (Reverse Transcription Polymerase Chain Reaction), it can be carried out using a DNA chip or the like. Examples of the gene include NFAT2 binding sequences [GGAAAA and similar sequences (eg, GGAAAAN, etc., N is A, T, C, or G), Macian, F. et al. et al. Oncogene, Vol. 20, p. 2476-2489, 2001], preferably NFAT2 and transcription factor AP-1 (activator protein-1) binding sequence [TGA [G / C] TCA and its similar sequences (eg, TGNNTCA, etc., where N is A , T, C, or G), Bio Science new term library “transcription factor”, experimental medicine separate volume, Yodosha, pp. 204-205] is preferred. Moreover, the thing which combined the desired reporter gene downstream from the said DNA can be used preferably. By binding the reporter gene, transcription from the NFAT2 promoter can be monitored by quantifying the expression level of the reporter gene using reporter activity or the like as an index. Specific examples of the promoter sequence include TRAP promoter, calcitonin promoter (P3 promoter), and the like. Reporter genes include luciferase genes, fluorescent protein genes (GFP, YFP, BFP, etc.), chloramphenicol acetyltransferase genes, β-galactosidase genes, and the like. The gene induced by NFAT2 is dependent on NFAT2, for example, TRAP, calcitonin receptor, cathepsin K, carbonic anhydrase II (CA II), or Examples thereof include genes such as matrix metalloproteinase (MMP) -9. A promoter sequence containing the NFAT2 binding site of these genes can be suitably used as a promoter linked to the reporter. These genes are induced in terminal osteoclast differentiation and contain multiple NFAT2 and AP-1 binding sites (Anusaksathien, O. et al., J Biol. Chem., 276, p. 22663-22674). 2001; David, JP et al., J. Cell Physiol., 88, p.89-97, 2001; Motycova, G. et al., Proc. Natl. Acad. Sci. 98, p. 5798-5803, 2001; Reddy, SV et al., J. Bone Miner Res., 10, 601-606, 1995).

  The expression level of the NFAT2 gene is detected and quantified using a cell line in which a fusion gene in which a marker gene such as a luciferase gene is connected to a gene region (expression control region) that controls the expression of the NFAT2 gene is used. It can also be carried out by measuring the activity of a gene-derived protein. The screening method for an NFAT2 expression control substance of the present invention includes a method for searching for a target substance using the expression level of the marker gene as an index. In this sense, the concept of “NFAT2 gene” includes a fusion gene of an expression control region of NFAT2 gene and a marker gene. The marker gene is preferably a structural gene of an enzyme that catalyzes a luminescence reaction or a color reaction. Specifically, in addition to the luciferase gene, the chloramphenicol / acetyltransferase gene, β-glucuronidase gene, β-galactosidase Genes and reporter genes such as the aequorin gene can also be used. Here, as the expression control region of the NFAT2 gene, for example, about 1 kb, preferably about 2 kb upstream of the transcription start site of the gene can be used. Creation of the fusion gene and measurement of the activity derived from the marker gene can be performed by known methods.

Inhibition or promotion of NFAT2 protein or NFAT2 gene expression as an indicator is the expression level of the reporter gene by the NFAT2 protein or NFAT2 gene or NFAT2 inducible promoter in a control cell to which no test substance is added, and the cell to which the test substance is added Can be carried out by comparing the expression levels of the NFAT2 protein or the above-mentioned gene. For example, the expression level of the NFAT2 protein or the gene in the cell to which the test substance is added is about 90% or less, preferably 40% or less of the expression level of the NFAT2 protein or the gene in the control cell to which the test substance is not added, Preferably, the test substance suppresses signal transduction of L-serine and / or L-glycine in osteoclast precursor cells when it is preferably 30% or less, particularly preferably 20% or less, and particularly preferably 10% or less. Can be selected as a candidate substance. Conversely, for example, the expression level of the NFAT2 protein or the gene in the cell to which the test substance is added is about 110% or more, preferably 120% of the expression level of the NFAT2 protein or the gene in the control cell to which the test substance is not added. %, More preferably 130% or more, particularly preferably 150% or more, the test substance is a candidate substance that promotes L-serine and / or L-glycine signaling in osteoclast precursor cells Can be selected.
In addition, the association between the suppression or promotion of NFAT2 protein or NFAT2 gene expression as an index and the suppression or promotion of osteoclast formation is related to the formation of osteoclasts when the expression of NFAT2 protein or NFAT2 gene as an index is suppressed. When the expression of NFAT2 protein or NFAT2 gene as an index is promoted, osteoclast formation is promoted to the same extent.

In general, bone tissue of a patient suffering from a bone metabolic disorder such as osteoporosis specifically shows increased osteoclast differentiation / activation and NFAT2 (NFAT2 gene) is expressed. That is, the screening method of the present invention provides a candidate substance that becomes an active ingredient of a preventive or therapeutic agent for bone metabolic abnormalities related to osteoclast formation by searching for a substance that suppresses the expression of NFAT2 or NFAT2 gene. Is.
Selection of candidate substances can be performed using as an indicator that the expression of NFAT2 or the expression of the NFAT2 gene induced by the expression inducer is suppressed by the presence of the test substance. That is, the expression of NFAT2 or the expression of the NFAT2 gene in a cell contacted with a test substance in the presence of an expression inducer is lower than that of a control cell not contacted with the test substance in the presence of an expression inducer. A test substance can be selected as a candidate substance. Specifically, signal transduction of L-serine and / or L-glycine is measured by the above-described method in the presence of a sample containing the test substance, and a substance that promotes or suppresses the signal transduction as compared with the control is selected. Control conditions include the absence of the test substance or a lower dose, typically in the absence of the test substance (eg, only the carrier used to add the test substance). (Addition). Moreover, if the case where the test substance is contained at a lower dose is used as a control condition, the dose dependency of the substance becomes clear. In addition, NFAT2 expression or NFAT2 gene expression in cells contacted with a test substance in the presence of an expression inducer is lower than that in positive control cells contacted with a known compound that regulates osteoclast formation. Alternatively, the test substance can be selected as a candidate substance by increasing the value. In this case, a substance that promotes or suppresses signal transduction of the L-serine and / or L-glycine can be selected as compared with the case in the presence of the compound used as a positive control. In such screening, a substance having a higher effect than that of the compound used as a positive control can be obtained.

In addition, patients suffering from abnormal bone metabolism such as marble bone disease have osteoclast hypoplasia and the like, and the expression of NFAT2 (NFAT2 gene) is decreased. That is, the screening method of the present invention searches for a substance that promotes the expression of NFAT2 or the NFAT2 gene, so that a candidate substance that becomes an active ingredient of a preventive and / or therapeutic drug for bone metabolic disorders related to osteoclast formation is obtained. It is to provide.
Selection of candidate substances can be performed using as an indicator that the expression of NFAT2 or the expression of the NFAT2 gene induced by the expression inducer is promoted by the presence of the test substance. That is, the expression of NFAT2 or the expression of the NFAT2 gene in a cell contacted with a test substance in the presence of an expression inducer is higher than that of a control cell not contacted with the test substance in the presence of an expression inducer. A test substance can be selected as a candidate substance. Specifically, signal transduction of L-serine and / or L-glycine is measured by the above-described method in the presence of a sample containing the test substance, and a substance that promotes or suppresses the signal transduction as compared with the control is selected. Control conditions and the like are the same as in the search for a substance that suppresses the expression of the NFAT2 and / or NFAT2 gene.

  Examples of the medium used in the screening method of the present invention include natural medium, semi-synthetic medium, synthetic medium, solid medium, semi-solid medium, and liquid medium. In order to differentiate osteoclast precursor cells into osteoclasts. Any nutrient medium that is used for culturing animal cells, particularly hematopoietic stem cells, can be preferably used. Examples of such a medium include Dulbecco's Modified Eagles' Medium (DMEM), Ham's F12 medium (Ham's Nutrient Mixture F12), McCoy's 5A medium, and McCoy's 5A medium. Medium (Minimum Essential Medium; MEM), α-MEM medium (α-modified Minimum Medium; α-MEM), RPMI 1640 medium, Iscove's Modified Dulbecco medium (Isocove's Modified DubM); ), X-VIVO 10 (Cambrex), X-VIVO 5 (Cambrex), HPGM (Cambrex), StemSpan H3000 (Stem Cell Technologies), StemlineII (Sigma-Aldrich) or QBSF-60 (Quality Biological, Inc.) and the like.

More specifically, the screening method of the present invention can be performed using, for example, an in vitro assay system for osteoclast formation. Specifically, for example, non-contact bone marrow cells (5 × 10 ⁵ cells per well of a 24-well plate) are cultured for 2 days in, for example, α-MEM containing about 10 ng / mL of M-CSF, and about 100 ng / A system in which osteoclasts are formed by culturing for an additional 3 days in the presence of mL of soluble RANKL and about 10 ng / mL of M-CSF can be utilized. A sample containing a test substance is added here and cultured. Examples of the in vivo system include a system using an osteoporosis model mouse or an endotoxin-induced bone resorption animal model by ovariectomy or the like. Furthermore, an osteoclast-forming system from ES cells can be used. In a screening system using ES cells, cells lacking a desired gene involved in osteoclast formation can be used. If an osteoclast-forming system from gene-deficient ES cells is used, useful information can be obtained as to which signal pathway of the osteoclast-forming signal is involved in the test substance.
As a result of this screening, if the addition of a sample containing a test substance significantly promotes or suppresses signal transduction of L-serine and / or L-glycine as compared to the control condition, the test sample used Is a candidate for a substance that regulates osteoclastogenesis. Substances isolated by this screening method are useful for controlling osteoclast formation, and are also useful in the development of preventive and therapeutic agents for bone metabolic diseases.

In addition, since the screening method of the present invention is also a method for examining the effect of a substance on osteoclast formation, a substance that suppresses osteoclast formation can be identified by this method.
In addition, a substance that antagonizes L-serine involved in osteoclast formation can also be screened using the screening method of the present invention. In this case, a test substance is added to a medium containing L-serine and RANKL, and osteoclast precursor cells are cultured. Similarly to the above, L-serine and / or L-glycine in osteoclast precursor cells is cultured. It is preferred to measure signal transduction. As a result of the measurement, if the expression of NFAT2 or the suppression of the expression of the NFAT2 gene is observed or the expression is not observed, the test substance can be identified as an antagonist of L-serine. The identified antagonist of L-serine can be preferably used as a substance that suppresses osteoclast formation. The degree of suppression is such that the expression level of the NFAT2 protein or the gene in the cell to which the test substance is added is the same as that of the NFAT2 protein or the gene in the osteoclast precursor cell (control cell) cultured without adding the test substance. The expression level is about 90% or less, preferably 40% or less, more preferably 30% or less, particularly preferably 20% or less, and particularly preferably 10% or less. In this case, in order to exclude the case where NFAT2 or NFAT2 gene is expressed via L-glycine, it is preferable to use a medium that does not contain L-glycine. Specifically, for example, a medium obtained by adding L-serine to a medium obtained by removing L-glycine from α-MEM is prepared, and for example, BMM or ES cells are present in the presence of M-CSF and RANKL using the prepared medium. Culturing is carried out under the condition, or, for example, RAW264 cells are cultured in the presence of RANKL. The concentrations of M-CSF and RANKL are the same as those described above.

The screening method of the present invention is also a method for screening L-serine and / or L-glycine-like substances involved in osteoclast formation. In this case, the test substance is added to a medium obtained by removing L-serine and L-glycine from the components of the medium described above, and osteoclast precursor cells are cultured. -It is preferable to measure the signal transduction of serine and / or L-glycine. If the NFAT2 expression or the NFAT2 gene expression is promoted as a result of the measurement, the test substance can be identified as L-serine and / or L-glycine-like substance. The identified L-serine and / or L-glycine-like substance can be preferably used as a substance that promotes osteoclast formation. The degree of the promotion is such that the expression level of the NFAT2 protein or the gene in the cell to which the test substance is added is equal to that of the NFAT2 protein or the gene in the osteoclast precursor cell (control cell) cultured without adding the test substance. About 110% or more of the expression level, preferably 120% or more, more preferably 130% or more, and particularly preferably 150% or more. Specifically, for example, a medium in which L-serine and L-glycine are removed from α-MEM is prepared, and for example, BMM or ES cells are cultured in the presence of M-CS and RANKL using the prepared medium. Alternatively, for example, RAW264 cells are cultured in the presence of RANKL. The concentrations of M-CSF and RANKL are the same as those described above.

  The substance obtained by the screening of the present invention can be used as an inhibitor or promoter of osteoclast formation. The conditions of administration of the substance for clinical application can be determined using the osteoclastogenic system described herein. That is, administration conditions including dosage, administration interval, and administration route can be examined using the above-described test method, and conditions under which appropriate preventive or therapeutic effects can be obtained can be determined. Such a substance becomes a medicament for prevention or treatment of NFAT2-related diseases and the like. The substance may be used as it is or in a known pharmaceutically acceptable additive, for example, excipients (for example, lactose, sucrose, glucose, starch, crystalline cellulose, etc.), binders (for example, starch paste, hydroxypropyl cellulose, carmellose). Liquid, gum arabic liquid, gelatin liquid, sodium alginate liquid, etc.), lubricant (eg magnesium stearate, talc, stearic acid, calcium stearate etc.), disintegrant (eg starch, carmellose sodium, calcium carbonate etc.), solvent (For example, water for injection, sterilized purified water, physiological saline, buffer, vegetable oil, etc.), emulsifier (for example, polyoxyl 40 stearate, sorbitan sesquioleate, polysorbate 80, sodium lauryl sulfate, lauromacrogol, gum arabic, popidone, etc.) , Suspensions (eg Carmellose, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, popidone, etc.), stabilizers (eg sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene etc.), preservatives (eg Methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax, etc.), surfactants (eg polysorbate 80, Polyoxyethylene hydrogenated castor oil or the like) and the like can be prepared as a pharmaceutical composition. The pharmaceutical composition is prepared in a dosage form (tablet, pill, capsule, powder, granule, syrup or other oral administration agent; injection, infusion, external preparation, suppository, etc.) ) And the like can be administered orally or parenterally. In addition, the pharmaceutical composition may be a sustained-release preparation or a DDS (drug delivery) preparation together with a release controlling substance. Examples of the release controlling substance include α-hydroxycarboxylic acids known per se (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.), hydroxydicarboxylic acids (eg, malic acid, etc.), hydroxytricarboxylic acids (eg, citric acid, etc.). A polymer, copolymer or mixture thereof synthesized from one or more of the above by non-catalytic dehydration polycondensation, poly-α-cyanoacrylate, polyamino acid (eg, poly-γ-benzyl-L-glutamic acid, etc.) ), Biodegradable polymer materials such as maleic anhydride copolymers (eg, styrene-maleic acid copolymers, etc.).

  Administration to a patient depends on the nature of the active ingredient and the dosage form to be prepared, eg percutaneous, intranasal, transbronchial, intramuscular, intraperitoneal, intravenous, intraarticular, subcutaneous, spinal cavity It can be performed internally, intraventricularly, or orally, but is not limited thereto. Administration can be systemic or local, but when side effects due to systemic administration are problematic, local administration to the lesion site is preferred. The dose and administration method vary depending on the tissue transferability of the active ingredient of the pharmaceutical composition, the purpose of treatment, the weight and age of the patient, symptoms, etc., but can be appropriately selected by those skilled in the art.

  The pharmaceutical composition can be used as a preventive and / or therapeutic agent for NFAT2-related diseases. Examples of NFAT2-related diseases include osteomalacious diseases in which bone mass is reduced by osteoclasts, or osteosclerotic diseases caused by osteoclast formation failure or dysfunction. Examples of osteomalablative diseases include primary osteoporosis, age-related (senile) osteoporosis, secondary osteoporosis and the like. Secondary osteoporosis is caused by endocrine / metabolic abnormalities (for example, hyperparathyroidism, gonadal dysfunction, Turner syndrome, anorexia nervosa syndrome, Klinefelter syndrome, etc.), inflammation (for example, rheumatoid arthritis, etc.) , Based on congenital diseases (eg osteogenesis imperfecta, homocystinuria, mastocytosis, mastocytoma, mastocytosis, etc.), based on blood diseases (eg multiple myeloma, etc.), based on drugs (eg Steroid osteoporosis and the like). Examples of osteosclerotic diseases include marble bone disease.

  The pharmaceutical composition of the present invention usually contains about 0.1 to 90% by mass of a therapeutic drug (the above substance) by weight of the total composition. The dosage varies depending on the type of active ingredient, administration route, administration subject or patient's age, body weight, symptoms, etc., and cannot be defined unconditionally, but for parenteral administration, about 0.0001 to 1000 mg per kg body weight per day, preferably The amount is about 0.001 to 300 mg, more preferably about 0.01 to 100 mg. However, depending on the disease state, body weight, individual patient response to treatment, the type of composition to which the therapeutic drug is administered, and the mode of administration, the stage of the course of the disease, it is preferable to adjust these administration rates as appropriate. . Therefore, it is appropriate to use less than the above minimum dosage, while in other cases the upper limit must be exceeded to obtain a therapeutic effect. Administration can be divided into 1 to several times, and can be administered about 1 to 5 times a day. Examples of the subject individuals include humans or non-human mammals such as mice, rats, rabbits, dogs or monkeys, and other vertebrates. The application to non-human mammals is also useful for making a model for developing preventive or therapeutic methods for human bone destructive diseases or osteosclerotic diseases. This makes it possible to develop a new treatment protocol for preventing osteoclast formation failure and dysfunction in NFAT2-related diseases such as osteoporosis and bone destruction in autoimmune arthritis and marble bone disease.

The pharmaceutical composition of the present invention is useful for the treatment and prevention of NFAT2-related diseases. In particular, the pharmaceutical composition containing a substance that suppresses L-serine and / or L-glycine signal transduction in osteoclast precursor cells of the present invention is used for the treatment and prevention of osteopenic diseases involving bone resorption by osteoclasts. And / or useful for prevention. Specific diseases that are the main treatment targets include autoimmune arthritis, particularly including rheumatoid arthritis (RA). Rheumatoid arthritis is an autoimmune chronic inflammatory disease, in which the proliferated synovium actively invades osteochondral, resulting in multiple joint destruction. The preventive and / or therapeutic agent for NFAT2-related diseases of the present invention is suitably used for preventing this joint bone destruction.
Moreover, periodontal disease is mentioned as another NFAT2-related disease to which the prevention and / or treatment agent of NFAT2-related disease of this invention can be applied. In addition, the preventive and / or therapeutic agent for NFAT2-related diseases of the present invention enhances bone resorption by osteoclasts, including giant cell tumor, cancer bone metastasis, and pigmented villonodular synovitis (PVS). The present invention can also be applied to various diseases having a pathological condition. Moreover, application to the treatment of osteoporosis and hypercalcemia of cancer is also expected. Furthermore, it can also be applied to the prevention and / or treatment of Paget's disease, bone loss associated with hepatitis and AIDS, bone resorption / bone destruction associated with leukemia and multiple myeloma, and bone resorption (loosening) around artificial joints ( Rodan, GA & Martin, TJ, Science, 289, p. 1508-1514 (2000); Kong, Y. Y. et al., Nature, 402, 304-309. (1999); Takayanagi, H. et al., Arthritis Rheum., 43, p. 259-269 (2000); Honore, P. et al., Nat. Med., 6, p. 528 (2000)). The present invention enables new therapeutic intervention by controlling osteoclast formation and function.
In addition, the pharmaceutical composition containing a substance that promotes L-serine and / or L-glycine signaling in osteoclast precursor cells of the present invention is an osteosclerotic disease or bone caused by osteoclast formation or dysfunction. It is useful for the prevention and / or treatment of absorption disorders. Therefore, the preventive and / or therapeutic agent for NFAT2-related diseases of the present invention can be used for diseases based on osteoclast formation failure or dysfunction such as osteopetrosis.

  The present invention can also be used in combination with other NFAT2 expression suppressing (or promoting) agents and osteoclast formation inhibiting (or promoting) agents. As such a combination example, for example, a kit for treatment of osteopenic disease, which includes a calcineurin inhibitor and D-serine, can be mentioned. By using a calcineurin inhibitor and D-serine in combination, a synergistic inhibitory effect on osteoclast formation can be expected. Preferred examples of the calcineurin inhibitor include cyclosporin A and FK506.

  The invention described in the present specification can be easily devised by those skilled in the art, but such embodiments are included in the scope of the present invention. References cited in this specification are incorporated as a part of this specification. EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.

[1] Preparation of medium 9.4 g of Eagle's MEM (manufactured by Nissui) was dissolved in 1 L of distilled water for culture and autoclaved at 120 ° C. for 15 minutes. After autoclave sterilization, 0.292 g of L-glutamine (Nissui) dissolved in 10 mL of sterilized distilled water for culture, 25 mL of 7.5% sodium bicarbonate, 10 mL Essential amino acid mixture (non-essential amino acid: NEAA; L-serine, L-alanine, L-glycine, L-aspartic acid, L-glutamic acid, L-proline and L-asparagine 10 mM each) [GIBCO-BRL ], 100 mL of fetal bovine serum (FBS) was added. Each component of the non-essential amino acid was purchased from Nacalai Tesque (Kyoto), and L-homoserine and D-serine were purchased from Sigma (USA). [ ³ H] -L-serine was purchased from Amersham Bioscience. When various non-essential amino acids were added individually, they were added so as to have the same concentration as the final concentration when the above mixed solution was used. Further, L-homoserine and D-serine will be described each time. Spectra / Por® 6 regenerated nitrocellulose dialysis membrane (MWCO1000) [Spectrum Laboratories Inc. Made] was used. First, the dialysis membrane was cut to about 10 cm and rinsed easily with distilled water for culture. Next, the dialysis membrane was immersed in a 5% sodium hydrogen carbonate solution containing 10 mM EDTA, washed at 60 ° C. for 1 hour, and then washed with 1 L of distilled water for culture for 5 minutes. This washing with distilled water for culture was repeated three times. After washing, serum was put into a dialysis membrane and dialyzed overnight at 4 ° C.
As a medium for inducing osteoclast precursor cells from mouse bone marrow cells, α-MEM (manufactured by Sigma) was dissolved in 1 L of distilled water for culture and autoclaved at 120 ° C. for 15 minutes. After autoclaving, 100 mL of fetal bovine serum (FBS) was added to the medium cooled to room temperature, and M-CSF (manufactured by Sigma) was added to a final concentration of 5 ng / mL.

[2] Cell Culture and Maintenance Mouse monocyte macrophage-derived cell line RAW264 was seeded on a 10 cm plate and cultured in 10 mL of the above medium at 37 ° C. and CO ₂ concentration of 5%. When the number of cells increased on one side of the plate, 1/10 of the proliferated cells were passaged, and then passage was performed every 3 days.
Mouse bone marrow cells 1 × 10 ⁶ cells / well are seeded in a 24-well plate, cultured in α-MEM with 10% FBS, 5 ng / mL M-CSF at 37 ° C. and CO ₂ concentration of 5% for one day. Only adherent cells were collected. This was further cultured in the presence of 100 ng / mL M-CSF, and after removing the floating cells after 3 days of culture, osteoclast precursor cells were obtained.

[3] Preparation of soluble RANKL RNA is extracted from mouse stromal cells ST2 having the RANKL gene, and then 1 μg of cDNA prepared from 3 μg of extracted RNA is used as a template, and the sequence shown in SEQ ID NO: 1 (5′-CAATTTGCGGATCTCAACAGAATATCAG-3 ′) The RANKL gene was amplified by PCR using the oligonucleotide having a sense primer and the oligonucleotide having the sequence shown in SEQ ID NO: 2 (5′-CAATTGGGAAATGGATCTCAGTCAATG-3 ′) as an antisense primer. The PCR method uses Pfx DNA polymerase (manufactured by GIBCO-BRL), and after performing 5 cycles of 94 ° C. for 15 seconds, 52 ° C. for 30 seconds, and 68 ° C. for 1 minute in a 50 μL system according to the attached protocol, It was carried out by performing 15 cycles of 94 ° C. for 15 seconds, 56 ° C. for 30 seconds, and 68 ° C. for 1 minute. The RANKL gene obtained by amplification by this PCR method is cleaved with the restriction enzyme MnuI, and the C-terminal region 244 amino acids of RANKL is encoded by the GST (glutathione S-transfer) coding region of pGEX-2TK vector (Amersham Pharmacia Biotech). The vector pGEX-2TK-RANKL was constructed by inserting it into the BamHI and EcoRI sites located downstream of. The pGEX-2TK-RANKL [soluble GST-RANKL] and the standard vector pGEX-2TK [GST protein] were introduced into Escherichia coli JM109 and used for expression of the recombinant protein. Super Broth (Tryptone Peptone [manufactured by DIFCO) 2.5 g, Bact Yeast Extract [manufactured by DIFCO]] 15 g, NaCl [manufactured by Nacalai Tesque] 5 g / 1 L H ₂ O) medium and pre-cultured overnight at 37 ° C., followed by addition of 5 mL of the pre-cultured solution per 500 mL of OD ₆₀₀ Super Broth medium, and the absorbance (wavelength: 600 nm) of the medium was 0. The main culture was performed until the range of 6 to 0.8 was reached. Thereafter, IPTG (Isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.25 mM, protein expression induction was started, and the cells were cultured at 18 ° C. for about 12 hours.
After collecting E. coli cultured above, the supernatant was discarded, and the cells were washed twice with NET buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA [manufactured by Nacalai Tesque)]. Discard the supernatant again, and add 1/250 volume of NETN buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40 [manufactured by Sigma]) on ice. E. coli was suspended and sonicated (30 seconds, twice) to destroy E. coli. Glutathione Sepharose (registered trademark) 4B (50% slurry [Amersham Pharmacia Biotech AB Co., Ltd.) of 1/1000 of the main culture volume was equilibrated in advance with the NETN buffer, added to the previous supernatant at 4 ° C. The reaction was allowed to proceed for 8 hours, washed 3 times with Wash buffer (20 mM NaCl, 4 mM MgCl ₂ .6H ₂ O, 1 mM 2-ME, 20 mM HEPES pH 7.4 [manufactured by Nacalai Tesque)], and 1.6 mL of Glutathione per 1 mL of beads. -NaCl buffer (100 mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM glutatione (manufactured by Nacalai Tesque); confirm that the pH is on the alkali side) was added and allowed to react for 1 hour or longer. Soluble RANKL was eluted and passed through an endotoxin removal column (Detoxi-Gel Endotoxin Removing Gel [PIERCE]) to prevent the effect of LPS on osteoclast differentiation induction as much as possible. It was frozen with liquid nitrogen and stored at −80 ° C. When differentiation induction was performed, it was appropriately thawed and used.

^[4] 2.5 × 10 4 cells in differentiation induction RAW264 cells RAW264 cells 24-well plates, 10 × 10 ⁴ cells in 3.5cm plate, 20 × 10 ⁴ cells in 6cm plate, the 10cm plate The seeds were seeded at 50 × 10 ⁴ cells and cultured at 37 ° C. and CO ₂ concentration of 5% for 24 hours. Thereafter, the medium was replaced with a medium containing soluble RANKL (0 hour) and cultured at 37 ° C. and CO ₂ concentration of 5%. After 72 hours, the medium was replaced again with freshly prepared fresh medium containing soluble GST-RANKL, and cultured at 37 ° C. and CO ₂ concentration of 5% for 24 hours. By this series of operations, the formation of osteoclasts can be confirmed on the entire surface of the plate 96 hours after the addition of RANKL (FIG. 1).
[5] Induction of osteoclast differentiation from bone marrow cells Induction of osteoclast differentiation is described in Takahashi, N .; et al. , Generating murine osteoblasts form bone marrow Methods Mol. Med. (2003), pp. 129-144. The method shown in was followed.
Osteoclast precursor cells obtained from bone marrow cells by the method shown in [Culture and maintenance of cells] were seeded in a 24-well plate at 1 × 10 ⁴ cells / well and cultured for one day. Thereafter, 500 ng / mL RANKL and 100 ng / mL M-CSF were added for induction. Every 3 days, the medium was replaced with a fresh medium containing the aforementioned amounts of RANKL and M-CSF.
[6] Forced expression of NFAT2 by retrovirus The NFAT2 gene or a GFP gene as a comparison target was incorporated into a retrovirus vector pCX4puro, and each expression vector was obtained by drug selection with puromycin. These expression vectors were introduced into a packaging cell line Platinum-E (Plat-E) together with a pE-eco vector and a pGP vector (manufactured by Takara), and the cells were cultured for 48 hours. The supernatant was filtered with a 0.22 μm pore filter to obtain a stock of retovirus. Osteoclast precursor cells were cultured overnight in the presence of retrovirus, followed by induction of osteoclast differentiation as described above.

[7] Cell fixation and TRAP staining Remove the medium of the plate on which osteoclasts were formed according to [4] above by suction, wash the plate by adding 500 μL of phosphate buffered saline (PBS), and completely remove PBS. remove. After repeating this operation once more, 500 μL of methanol was gently added, and after 30 seconds, methanol was removed and the plate was thoroughly dried.
FAST was mixed with a solution prepared by mixing 0.026 M tartaric acid containing 0.1 M acetic acid (pH 5.2) and 12.5 mg / mL naphthol AS-BI phosphate containing 0.1 M acetic acid (pH 5.2) at a ratio of 24: 1. An appropriate amount of GARNET GBC was added, and a solution passed through a 0.8 μm filter was used as a TRAP staining solution. 500 μL of TRAP staining solution was added to the cell-fixed plate and reacted at 37 ° C. for 1 hour or longer. At this point, cells with TRAP activity are reddish purple. After the reaction, the TRAP staining solution was removed, and the plate was washed with ultrapure water (FIG. 1; TRAP-stained MN cells).

[8] Bone resorption activity It was confirmed that the formed multinucleated cells had bone resorption activity. Differentiation was induced on a hydroxyapatite plate [BD Bio Coat (BD Bioscience)], and after the formation of multinucleated cells, decomposed calcium phosphate was measured by von Kossa staining method (FIG. 1; HA plates). The decomposed area appears white.

[9] Preparation of cell extract Unless otherwise specified, the culture plate was transferred to ice after a certain time, and the culture supernatant was removed. After washing the cells twice with PBS cooled to 4 ° C., an appropriate amount of EBC Lysis buffer (50 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.5% NP-40, 1 mM EDTA, 0.5 mM PMSF, 100 KIU / mL) Aprotinine, 20 mM NaF, 2 mM Na ₃ VO ₄ ) was added to scrape the cells, and the mixture was allowed to stand on ice for 30 minutes. Thereafter, the cells were disrupted by pipetting, centrifuged (15,000 rpm, 4 ° C., 10 minutes), and only the supernatant was transferred to an Eppendorf tube on ice, which was used as a cell extract. The cell extract was frozen with liquid nitrogen and stored at −80 ° C.
In an experiment to examine the effect of addition of NEAA, 1 × 10 ⁵ cells were seeded on a 35 mm diameter plate, and RANKL stimulation was performed overnight to 24 hours later. After a certain time (9-24 hours), the medium was removed, and the remaining medium was removed by inclining for about 5 seconds. The cells were washed (rinse) with 30 μL of EBC Lysis buffer without washing with PBS, and collected with a scraper. Then, it heated at 100 degreeC for 5 minute (s), and processed 5 times for 10 second with the ultrasonic crusher (Tomy), and destroyed the cell. After centrifugation (15,000 rpm, 4 ° C., 10 minutes), only the supernatant was transferred to an Eppendorf tube on ice, which was used as a cell extract. The cell extract was frozen with liquid nitrogen and stored at −80 ° C.

[10] Western blotting The protein mass of the prepared cell extract was measured by the Bradford method, and the protein mass of each sample was made the same, and then separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE). After the electrophoresis, the protein was transferred to a PVDF membrane [PolyVinylene Fluoride; manufactured by Millipore] by wet blotting using a blotting tank. After transfer, the PVDF membrane was washed with PBS for 5 minutes and blocked with 5% Skim Milk / PBS for 1 hour. After the PVDF membrane was washed in the order of 5 minutes with PBS, 10 minutes with PBST, 5 minutes with PBS, and 5 minutes with PBS, the primary antibody diluted with 5% BSA / PBS was reacted for 2 hours or more. After the reaction, the PVDF membrane was washed in the order of 5 minutes with PBS, 10 minutes with PBST, and 5 minutes with PBS. Next, the PVDF membrane was reacted with a secondary antibody diluted with 5% Skim Milk / PBS for 45 minutes at room temperature, then 5 minutes with PBS, 10 minutes with PBST (Phosphate Buffered Saline / Tween), and 5 minutes with PBS. The PVDF membrane was washed in this order, and the proteins separated by ECL (Amersham Bioscience) or ECL Plus (Amersham Bioscience) were detected.
The results of Western blotting using these protocols under the following conditions are shown in FIG.
Antibodies used for Western blotting:
・ Anti-mouse NFAT2 antibody [manufactured by Santa Cruz] (1/500 dilution)
Anti-mouse c-Fos antibody (Santa Cruz) (1/5000 dilution)
Anti-mouse β-actin antibody [manufactured by Sigma] (diluted 1/8000)
As a secondary antibody,
-Bovine anti-goat IgG HRP [manufactured by Santa Cruz] against goat antibody
・ For mouse antibody, sheep anti-mouse IgG HRP conjugated [manufactured by Amersham Bioscience]
-Protein A-HRP [manufactured by Amersham Bioscience] for rabbit antibody
Sample volume used:
NFAT2: 10-20 μL / lane → Detected with ECL plus (1-5 minutes)
actin: ~ 5 μL / lane → detected with ECL (less than 1 minute)
In Western blotting in the following examples, the conditions are the same as described above.

[11] Preparation method of conditioned medium 5 × 10 ⁵ RAW264 cells were seeded on a 10 cm plate and cultured overnight at 37 ° C. and CO ₂ concentration of 5%. On the next day, after removing the medium from the plate and washing twice with PBS, 10 mL of the medium was injected and cultured at 37 ° C. and CO ₂ concentration of 5% for 24 hours. Thereafter, the medium was recovered from the plate and passed through a 0.2 μm filter to remove cells. This was used as a low density recovery medium. In addition, 40 × 10 ⁵ RAW264 cells were seeded on a 10 cm plate, and a medium recovered by the same method was used as a high-density recovery medium. In addition, a medium obtained by culturing the medium for 24 hours at 37 ° C. and a CO ₂ concentration of 5% was designated as Fresh medium. When these media were stored at 4 ° C. and osteoclast differentiation was induced, a necessary amount of soluble RANKL was added to these media.

Design of NFAT2 siRNA and introduction into RAW 264 cells, isolation of expression clones From the NFAT2 gene sequence, a target sequence (5′-GGTACGTACGGATATCAGG-3 ′; SEQ ID NO: 3) is selected, and the corresponding DNA is designated as a pSUPER vector (Oligo Engine, US). This vector was introduced into RAW264 cells together with a neomycin resistance cassette, and an NFAT2-siRNA expression cell line was selected.

Induction of NFAT2 Differentiation was induced by seeding RAW264 cells under the conditions indicated in “Materials and Techniques” and applying RANKL stimulation. After RANKL stimulation (= 0 hour), a cell extract was prepared over time, and expression of NFAT2 was confirmed by Western blotting using an anti-NFAT2 antibody (FIG. 3). The expression of NFAT2 protein was confirmed at least 12 hours after RANKL stimulation, and the expression was confirmed after 48 hours. Β-actin is for confirming the amount of protein used (the same applies to the following experiments).

[1] Effect of NFAT2 expression on multinucleated cell formation In order to confirm the role of NFAT2 in the process of forming multinucleated cells from RAW264 cells by RANKL stimulation, the NFAT2-siRNA expression cell line prepared in Example 2 was used. did. Here, two clones, KD-1 and KD-4, are shown as representative examples of the NFAT2-siRNA-expressing cell line. In these strains, it can be confirmed that the expression level of the NFAT2 protein is markedly reduced. In addition, MOCK is a strain into which the vector itself used for expressing siRNA is introduced (FIG. 4A). When the RAW 264 parent cell line was stimulated with RANKL, multinucleated cells were formed after 96 hours, and mononuclear cells were observed with GST stimulation, whereas KD-1 or KD-4 was induced to differentiate under the same conditions. However, the formation of multinucleated cells is not observed (FIG. 4B). This indicates that NFAT2 expression is essential for multinucleated cell formation.

[2] Effect of Cell Seeding Density on Multinucleated Cell Formation Efficiency When RAW264 cells are seeded on a dish, the cell number is set to a cell density (2.5 × 10 ⁴ cells / well) at which normal differentiation induction is performed. Adjustment to 5 times, 1 time, 2 times, 4 times, and 8 times was performed, and differentiation into osteoclasts was induced under the same conditions. Each cell was subjected to TRAP staining after the induction and stained TRAP positive cells. As a result, osteoclast differentiation induction efficiency (multinucleated cell formation rate) decreases as the cell density increases with the cell density peaking under normal conditions, and almost no osteoclast formation occurs at 8 times the cell density. It could not be confirmed (FIG. 5). In this case, the differentiation induction efficiency was reduced to about 60% even with a plate having a cell density of 0.5 times. However, since the absolute number of cells in the plate is small, osteoclasts are inevitably formed less. This is thought to be the cause (Fig. 6).

[3] Effect of cell seeding density on multinucleated cell formation efficiency of bone marrow cells A similar phenomenon was observed in a cell line prepared from mouse bone marrow cells instead of RAW264 cells. When osteoclast precursor cells derived from mouse bone marrow cells are seeded in a 24-well plate, the number of cells is 0.5 times the cell density (1.0 × 10 ⁴ cells / well) for normal differentiation induction, They were prepared in 1 ×, 2 ×, 4 ×, 8 ×, 16 ×, and 32 ×, and differentiation into osteoclasts was induced under the conditions shown in “Example 1”. After the induction of each cell, TRAP positive cells were stained. As a result, it was confirmed that the formation of osteoclasts did not occur as the cell density increased as in the case of RAW264 cell mouse cells (FIGS. 17A and 17B). In this way, a phenomenon similar to that of the RAW264 cell was reproduced even in the bone marrow cell line reflecting the phenomenon in vivo.

Effect of Cell Seeding Density on NFAT2 Expression The results of FIGS. 3 and 4 suggest that NFAT2 expression is important for progression to multinucleated cell formation. Therefore, it was confirmed by Western blotting that the expression of the NFAT2 protein depends on the cell density. The cell density seeded on the plate was adjusted to 1 time (2.5 × 10 ⁴ cells / well), 2 times, 4 times, and 8 times the cell density for inducing differentiation, and after RANKL stimulation, the same conditions were used. The extract of the cells cultured for 24 hours under was used. As a result, it was confirmed that the expression of NFAT2 protein decreased depending on the cell density (inversely proportional) (FIG. 7).

Effects of medium components on the formation of multinucleated cells As described above, when cells are seeded at a high density [8-fold density: (2 × 10 ⁵ cells / well)], differentiation can be induced under normal conditions. The formation efficiency of multinucleated cells is significantly reduced. In order to clarify this cause, a medium (conditioned medium) was collected 24 hours later from a culture plate seeded at high density, and differentiation induction was performed using the medium under normal conditions (low density seeding). After 96 hours, FIG. 8 shows a comparison of the formation efficiency of multinucleated cells between the conditioned medium and the fresh medium.
From this experiment, it can be seen that when the conditioned medium is used, the efficiency of differentiation induction is reduced even if the number of cells is low. From this, as a result of culturing at high density, it was suggested that some change occurred in the medium components, which suppressed the formation of multinucleated cells.

Effect of NEAA on recovery of NFAT2 expression In order to investigate the relationship between changes in medium components, changes in NFAT2 expression, and multinucleated cell formation, factors related to the formation of multinucleated cells were investigated using NFAT2 expression as an index. Possible causes of changes in the medium components were (1) some inhibitory activity factor was secreted under high-density culture, and (2) changes in the medium components under high-density culture. Therefore, in the presence of NEAA [pre-NEAA (+)] and in the absence [pre-NEAA (-)], a conditioned medium is prepared, and then the subsequent culture (low density) is performed with the recovered conditioned medium. The effect of adding NEAA (post-NEAA) was examined. As a result, it was found that the presence or absence of NEAA (pre-NEAA) at the time of preparation of the conditioned medium was not very important, but rather the influence of NEAA (post-NEAA) added later was great (FIG. 9). Therefore, it was suggested that whether NEAA is added at the time of starting culture (at low density) is important for the expression of NFAT2. In order to confirm this, it was examined what effect the expression of NFAT2 would have when NEAA was omitted (FIG. 10). As a result, it was reconfirmed that when post-NEAA was added under low-density culture (L), NFAT2 expression was induced even in conditioned medium. When NEAA is removed when cultivating in a fresh medium under high-density culture conditions (H), the inhibitory effect on NFAT2 increases, so the presence or absence of NEAA is more than the possibility that inhibitory activity exists in the medium. It was more strongly suggested that is associated with the presence or absence of inhibitory activity. In order to further confirm the above, when preparing a high-density medium, the timing for adding FBS, glutamic acid and NEAA was set at the time of conditioned medium preparation (pre-) or RANKL addition (post-), and Similarly, cell culture was performed under low density conditions. In conclusion, when NEAA was added when RANKL was added (= post), it was revealed that the induction level was increased. That is, it was found that the presence or absence of NEAA was closely related to the expression level of NFAT2 (FIG. 11).

Effect of NEAA on the proliferation of RAW264 cells The above experiments suggested that NEAA plays an important role in the expression of NFAT2. However, since there was a concern that NEAA might have any effect on the proliferation of RAW264 cells themselves, in order to confirm that NEAA does not affect the proliferation of cells itself, glutamine (Gln), RAW264 cells were cultured for 24 hours in the presence or absence of NEAA and serum. As a result, it was found that cell proliferation and survival were greatly influenced by the presence or absence of serum and glutamine, but were hardly influenced by the presence or absence of non-essential amino acids (FIG. 12).

Effects of serine and glycine in NEAA Since previous experiments suggested that NEAA is involved in the expression of NFAT2, in order to determine which of the seven amino acids contained in the NEAA mixture is important (Figure 13). First, serum contains a variety of substances and molecules including amino acids, which may cause background. Therefore, in order to remove low molecular weight molecules including amino acids, serum (F *) that had been dialyzed thereafter was used. Regarding the Conditioned medium, the presence or absence of NEAA (pre-NEAA) at the time of preparation was not important, but it was reconfirmed that the influence of NEAA (post-NEAA) added later was large. In the right 7 lanes of FIG. 13, 7 types of NEAA were prepared by sequentially removing each amino acid one by one and returned to the conditioned medium simultaneously with RANKL. In NEAA from which serine is removed, signal recovery is weak. This experiment was performed using a cell lysate for 12 hours after RANKL stimulation.
The experiment conducted in FIG. 13 suggested the involvement of serine. However, the recovery level is limited even if all types of NEAA are restored under these conditions, and there is a tendency that the dynamic range becomes small. To perform more quantitatively, the fresh medium (F) An experiment using NEAA from which amino acids were sequentially removed was performed (FIG. 14). As a result, it was found that in the absence of serine, the induction of NFAT2 was significantly reduced even in the presence of other NEAA components. Conversely, when only serine was returned in the absence of other NEAA, the expression level of NFAT2 was almost completely restored. Glycine also showed some recovery. From these things, it became clear that serine is a decisive factor among NEAA.

NFAT2 expression by serine analogues, inhibitory effect on multinucleated cell formation From the above results, in the process of multinucleated cell formation from RAW264 cells using MEM medium, when NFAT2 is induced by RANKL stimulation, NEAA is the presence of serine It was suggested that is necessary and sufficient. In addition, the same inducing activity was observed although it was weak against glycine. From these facts, it was considered that the use of a structural isomer of serine (L-serine, D-serine, L-homoserine) could suppress its NFAT2 induction activity in a competitive and inhibitory manner. -The possibility was verified using two kinds of substances, serine and L-homoserine.
First, neither D-serine nor L-homoserine showed activity to induce the expression of NFAT2. On the other hand, when an excess amount of D-serine was added to L-serine, it was confirmed that when 10 times the amount was added, the expression activity of NFAT2 by L-serine was suppressed. In homoserine, weak inhibitory activity was recognized compared with D-serine (FIG. 15).
Next, the influence of D-serine on the formation of multinucleated cells was examined (FIG. 16). As a result, the following was found. (1) No influence from serum dialysis treatment (FIG. 16A), (2) Without L-serine, it progresses to the middle of differentiation, but almost no multinucleated cells are formed (FIG. 16B), (3 ) After 4 days of culturing with D-serine + RANKL, cell viability deteriorates (FIG. 16C), (4) L-serine restores survival (FIG. 16D), (5) Further excess D-serine When acted on, the formation efficiency of multinucleated cells decreases again (FIG. 16E).

[1] Expression of NFAT2 dependent on L-serine From the experimental results of Example 10, it was shown that L-serine is involved in NFAT2 expression and multinucleated cell formation among a plurality of serine structural isomers. In order to confirm that the expression of NFAT2 is dependent on L-serine, the following experiment was performed. RAW264 cells were cultured for 2 hours in a normal medium in the presence of 10% FBS under normal density conditions (2.5 × 10 ⁴ cells / well; hereinafter the same in normal density). At that time, L-serine to be added to the medium was prepared to have a concentration of 0 times, 0.1 times, 0.3 times, and 1 times the normal amount (× 1; 0.1 mM). The expression level of NFAT2 in the cultured cells was confirmed by Western blotting using an anti-NFAT2 antibody (FIG. 18B). As a result, it was shown that the expression level of NFAT2 protein in RAW264 cells increased in proportion to the amount of L-serine present in the medium. Expression of NFAT2 was not observed in the medium containing no L-serine (x0). From this, it was confirmed that L-serine is required for the expression of NFAT2, and that the effect is dose-dependent.

[2] Effect of cell seeding density on abundance of L-serine Since multinucleated cell formation is influenced by cell seeding density, the amount of L-serine in the medium decreases as the density increases. it was thought. Therefore, the concentration of L-serine in the medium when cell seeding at high density was performed was measured. RAW264 cells are seeded at low density (low: 2.5 × 10 ⁴ cells / well) or high density (high: 2 × 10 ⁵ cells / well), and in the presence of [(+) RANKL] or in the absence of RANKL [ (-) RANKL] was cultured for 24 hours in a normal medium containing [ ³ H] -L-serine (10 μCi / mL). At a plurality of time points (0.5, 1, 2, 4, 8, 24 hours), L-serine present in 10 μL of the medium was measured using a scintillation counter with ³ H radioactivity as an index. As a result, it was shown that the abundance of L-serine in the medium decreased in proportion to the culture time, and the decrease rate was higher under high density conditions than under low density conditions (FIG. 18A). Since there was no significant change in the amount of L-serine decreased with or without RANKL, it was revealed that the decrease in the amount of L-serine in the medium was mainly due to the cell seeding density. From the above results, it was confirmed that the inhibition of polynuclear cell formation due to an increase in cell density was caused by a decrease in the expression level of NFAT2 due to a decrease in the abundance of L-serine in the medium.

[1] Effect of L-serine on proliferation of bone marrow cells It became clear that NEAA was present, especially that L-serine played an important role in the expression of NFAT2. Next, instead of RAW264 cells, -The effect of serine was examined. First, in order to examine whether L-serine is involved in proliferation of mouse bone marrow cells, the bone marrow cells were cultured for 3 days using FBS dialyzed. At that time, the concentration of L-serine to be added is 0 times, 0.1 times, 0.3 times, 3 times, 10 times of normal (× 1; 0.1 mM), and the others are described in “Example 1”. As shown. Cell proliferation was assessed by measuring the MTT assay. For the MTT assay, MTT cell growth kit (Chemicon International Inc. Ca, USA) was used. As a result, it was found that L-serine is necessary for the expression of NFAT2, but hardly affects cell proliferation (FIG. 20A). This is the same result as the experiment described in “Example 8” using RAW264 cells.

[2] Effect of L-serine on multinucleated cell formation of bone marrow cells Next, the relationship between multinucleated cell formation and L-serine was confirmed in bone marrow cells. When osteoclast progenitor cells derived from bone marrow cells were seeded at normal density and NEAA without L-serine alone or in the absence of L-serine was added to the medium, each was disrupted by the method shown in “Example 1”. Bone cell induction was performed, and TRAP staining was performed after 5 days of culture. As a result, in the absence of L-serine, the same result as in the case of RAW264 cells was obtained that the formation of multinucleated cells of osteoclast precursor cells was suppressed (FIG. 20B).

[1] Effect of L-serine on c-fos expression It is currently known that the transcription factor c-fos is involved in the expression of NFAT2 (Takayanagi, H et al., 2002, Dev. Cell). , 3, 889-901; Matsuo, K et al., 2004, J. Biol. Chem. 279, 26475-26480). Since the expression of NFAT2 decreases in proportion to the cell density, the expression of c-Fos under the same conditions as in Example 3 was examined. RAW264 cells were cultured at normal density in the presence of RANKL for 48 hours, collected at a plurality of time points, and then c-Fos protein amount was measured by Western blotting using an anti-c-Fos antibody (FIG. 19A). The expression level of c-Fos protein decreased from 12 hours to 24 hours after RANKL stimulation. In addition, after culturing RAW264 cells at low density (normal density) and high density (8 times) for 24 hours, the expression level of c-Fos protein in each cell lysate was determined by Western blotting using an anti-c-Fos antibody. When compared, it was shown that the amount of c-Fos protein decreased under high-density conditions (FIG. 19B).
From the above results, it was shown that the expression of c-Fos is also dependent on cell density. Furthermore, in order to investigate the relationship between c-Fos and L-serine, the following experiment was conducted. RAW264 cells were seeded at low density (normal density), and NEAA and L-serine and / or L-glycine added to the medium in different combinations were cultured for 24 hours. Cells were collected 24 hours later, and Western blotting using anti-NFAT2 antibody and anti-c-Fos antibody was performed (FIG. 19C). As a result, it was shown that both NFAT2 and c-Fos are expressed in an L-serine dependent manner. Therefore, it was shown that L-serine may regulate the expression of NFAT2 through the regulation of c-Fos expression.

[2] Effect of L-serine on c-Fos and NFAT2 expression in bone marrow cells In order to confirm that c-Fos and NFAT2 expression in osteoclasts derived from bone marrow cells occurs in an L-serine dependent manner, Osteoclast precursor cells were cultured at a normal density, and differentiation into osteoclasts was induced in the presence or absence of L-serine. After 48 hours of culture, Western blotting using anti-c-Fos antibody or anti-NFAT2 antibody was performed to detect the protein expression of c-Fos and NFAT2. As a result, the expression of c-Fos and NFAT2 is decreased in the absence of L-serine, and the expression of these two molecules is L-serine dependent. It was confirmed (FIG. 20C).

[3] Effect of NFAT2 on the formation of multinucleated cells of bone marrow cells From the results thus far, it is found that the expression of L-serine and NFAT2 is important for the formation of multinucleated cells in the differentiation of osteoclasts in bone marrow cells. Indicated. Here, in order to examine whether the expression of both L-serine and NFAT2 is essential for the multinucleated cell formation process, osteoclast precursor cells in which NFAT2 was forcibly expressed via retrovirus infection were cultured at a normal density, An experiment for inducing differentiation into osteoclasts was performed in the presence or absence of L-serine. As a result of performing TRAP staining after culturing for 5 days, it was revealed that even in the absence of L-serine, NFAT2 was forced to form almost the same amount of multinucleated cells as in the presence of L-serine (FIG. 20D). . In cells expressing GFP instead of NFAT2, multinucleated cell formation did not occur in the absence of serine. Therefore, NFAT2 is essential for multinucleated cell formation, and even if NFAT2 is expressed even in the absence of L-serine. Osteoclast formation has been shown to occur.

  The method of the present invention is useful as a method for preventing or treating NFAT2-related diseases. In addition, the medicament of the present invention may be used for various diseases related to NFAT2, such as osteoporosis, autoimmune arthritis, Paget's disease or rheumatoid arthritis, various osteopenic diseases and bone cancer, or osteosclerotic diseases such as marble bone disease, Alternatively, it is useful as a drug with few side effects on immune diseases and the like. The screening method of the present invention is useful for searching for a novel NFAT2 expression regulator.

Claims (2)

A prophylactic and / or therapeutic agent for osteoporosis , comprising D-serine, L-homoserine, or a pharmaceutically acceptable salt thereof. Use of D-serine, L-homoserine, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for preventing and / or treating osteoporosis .