JPWO2002024228A1 - How to control osteoclast formation - Google Patents

Abstract

The present invention provides a method for controlling an osteoclast-forming signal. It has been found that by activating type II IFN signaling, the process of osteoclast formation can be suppressed. Activation of the type II IFN signal down-regulated the expression of TRAF6 protein induced by RANKL and suppressed osteoclast formation. Type II IFN (IFN-γ) showed an effect of suppressing osteoclast formation in a dose-dependent manner. It has been found that a signal via Stat1 is important for suppression of osteoclast formation by type II IFN. The present invention provides a number of new therapeutic strategies for diseases involving pathological bone destruction, including autoimmune arthritis.

Description

Technical field
The present invention relates to a method for controlling an osteoclast-forming signal. More specifically, it relates to a method of controlling osteoclast formation.
Background art
Bone mass is maintained on a coordinated balance between osteoblastic bone formation and osteoblastic bone resorption. Abnormal bone resorption has been implicated in a variety of bone metabolic disorders, including osteoporosis, osteoarthritis, metastatic bone cancer, and inflammatory bone diseases such as rheumatoid arthritis (RA) and periodontal disease. Therefore, it is extremely important to effectively regulate bone resorption by osteoclasts. However, no effective treatment has been established to control pathological bone resorption.
Although the immune response is an important defense mechanism for the host, the immune system is also involved in bone resorption, and prolonged abnormal activation, such as in certain autoimmune conditions, causes tissue destruction by effector cells (Rodman, GD, Exp. Hematol. 27, 1229-41 (1999); Suda, T. et al., Endocrine Rev. 20, 345-57 (1999)). For example, in autoimmune arthritis, severe bone destruction progresses due to enhancement of bone resorption by osteoclasts, resulting in progressive joint destruction (Kong, YY et al., Nature 402, 304-9 ( Takayanagi, H. et al., J. Clin. Invest. 104, 137-46 (1999)). In such joint tissues, the expression of RANKL in T cells plays an important role. RANKL (receptor activator ofnuclear factorkB ligand, also known as TRANCE / OPGL / ODF) is a TNF family factor essential for osteoclast formation, and activated T cells produce RANKL to induce osteoclast formation in vitro. (Kong, YY et al., Nature 397, 315-23 (1999); Kong, YY et al., Nature 402, 304-9 (1999); Horwood, NJ et al., Biochem. Biophys. Res. Commun. 265, 144-50 (1999)). However, it is not known that activated T cells have an action involved in maintaining bone homeostasis by offsetting the action of RANKL. In addition to RANKL, other immunomodulators expressed on T cells also regulate osteoclastogenesis (Roodman, GD, Exp. Hematol. 27, 1229-141 (1999); Suda, T. et al. et al., Endocrine Rev. 20, 345-57 (1999); Takahashi, N. et al., J. Immunol. 137, 3544-9 (1986)), and the molecular mechanism of its regulation is unknown.
Disclosure of the invention
An object of the present invention is to provide a method for controlling an osteoclast-forming signal. By the method of the present invention, osteoclast formation can be controlled. Specifically, the present invention provides a method for controlling the action of RANKL signaling involved in osteoclastogenesis. Further, the present invention provides a method for controlling the expression of TRAF6 involved in osteoclastogenesis, its function, or both. According to the present invention, it is possible to control bone resorption and bone destruction. Another object of the present invention is to provide a drug for use in these controls. Another object of the present invention is to provide a medicament for controlling osteoclast formation or bone destruction.
The present inventors investigated the possibility that signaling through type II interferon (IFN) is involved in the regulation mechanism of osteoclastogenesis. A mouse lacking one of the components, IFNGR1 (IFN-γR^{− / −}Bone resorption was induced by endotoxin in mice) to examine the effect of type II IFN signal deficiency on osteoclast formation. As a result, IFN-γR^{− / −}Mice were found to have enhanced osteoclast formation and markedly worsened bone destruction as compared to wild-type mice. Recently, it has been reported that in autoimmune arthritis, activated T cells promote osteoclast formation through RANKL expression and regulate bone loss (Kong, YY et al., Nature). 402, 304-9 (1999)). In contrast, the findings obtained by the present inventors indicate that activated T cells also retain the ability to negatively regulate osteoclastogenesis through the production of IFN-γ. That is, it was found that the balance between the expression of RANKL and IFN-γ by T cells is important for regulating osteoclast formation and maintaining bone tissue.
To investigate the effect of IFN-γ producing T cells on osteoclastogenesis, the effect of activated T cells on the in vitro osteoclastogenesis system was next examined. Specifically, bone marrow monocyte / macrophage progenitor cells (bone marrow monocyte / macrophage precursor cells; BMMs), which are osteoclast precursor cells in vitro, are stimulated with RANKL in the presence of M-CSF to form osteoclasts. An experiment was conducted in which BMM was co-cultured with activated T cells in a system for inducing. As a result, it was found that when BMM was co-cultured with T cells activated with an anti-CD3 antibody, BMM osteoclast formation was strongly inhibited. IFN-γR^{− / −}When derived BMM was used, the inhibitory effect of activated T cells on osteoclast formation was lost. In addition, the culture supernatant of activated T cells showed an effect of inhibiting BMM osteoclast formation, but it was confirmed that this inhibitory effect was neutralized by anti-IFN-γ antibodies. These results revealed that IFN-γ suppressed RANKL-induced osteoclast formation. RANKL-induced osteoclastogenesis of BMM is inhibited even when recombinant IFN-γ is used in place of activated T cells, and extremely low levels of IFN-γ cause osteoclastogenesis even in the presence of excess RANKL. Was strongly inhibited.
To examine the signaling pathway of inhibition of osteoclastogenesis by IFN-γ in more detail, IFN-γR^{− / −}, Stat1^{− / −}Or IRF-1^{− / −}The effect of IFN-γ on RANKL-induced osteoclast formation was examined using mouse-derived BMM. As a result, IFN-γR^{− / −}Mouse or Stat1^{− / −}In mice, the ability of IFN-γ to suppress osteoclast formation was markedly reduced, but IRF-1^{− / −}In mice, the same inhibitory effect of IFN-γ as in wild-type mice was observed. This fact indicates that the activation of signal transduction via Stat1 (signal @ transducer @ and @ activator @ of @ transscription-1) plays an essential role in suppressing osteoclast formation. This indicates that the suppression of the RANKL signaling pathway by IFN-γ requires a Stat1 (GAF) -independent IRF-1 independent gene induction pathway.
When RANKL stimulates its receptor, RANK, it induces the adapter protein TRAF (tumor, factor, receptor, associated-factor) family, and NF (nuclear factor) -κB and JNK / SAPK (c-Jun). Terminal kinase / stress-activated protein kinase) (Dougall, WC et al, Genes Dev. 13, 2412-24 (1999); Hsu, H. et al., Proc. Natl. Acad. Sci.U S A 96, 3540-5 (1999)). When the activation of NF-κB and JNK induced by RANKL was examined in BMM treated with IFN-γ, it was found that the activation of both NF-κB and JNK was inhibited by IFN-γ. It was also found that the expression of TRAF6, an effector molecule downstream of the RANK signaling pathway, was significantly reduced by IFN-γ. When the TRAF6 gene was exogenously introduced into BMM and forcedly expressed, this BMM was resistant to inhibition of osteoclast formation by IFN-γ, and activation of NF-κB and JNK was stimulated by RAINKL. The differentiation into osteoclasts was induced. These differentiated cells expressed calcitonin receptor and exhibited bone resorption activity on dentin sections. These results indicate that IFN-γ inhibits osteoclastogenesis at least in part through a decrease in TRAF6 expression.
The present invention reveals a new biological function of IFN-γ that protects the destruction of calcified tissue in the activation of T cells. The findings obtained according to the present invention indicate that osteoclast formation is regulated by the balance between RANKL and IFN-γ. Findings in collagen-induced arthritis (CIA) observed in IFN-γ receptor deficient mice (Manoury-Schwartz, B. et al., J. Immunol. 158, 5501-6 (1997); Vermeire, K. et al. 158, 5507-13 (1997)) can be at least partially explained by this inhibitory effect of IFN-γ on osteoclast differentiation. For example, as seen in an endotoxin-induced bone resorption model, it is thought that IFN-γ production is enhanced during the acute immune response phase, and that abnormal osteoblast formation is suppressed by offsetting the increased expression of RANKL. . In contrast, in chronic synovitis in rheumatoid arthritis, this balance effect appears to lean toward RANKL expression. In this regard, the arthritic joints have suppressed IFN-γ expression despite significant T cell infiltration (Firestein, GS & @Zvaifler, NJ, Arthritis). Rheum. 33, 768-73 (1990); Kinne, RW et al., Biochim. Biophys. Acta 1360, 109-41 (1997)), while RANKL expression is enhanced (Takayanagi, H.). It is noteworthy that Gra et al., Arthritis Rheum. 43, 259-69 (2000); Gravallese, EM et al., Arthritis Rheum. 43, 250-8 (2000)). That is, it is considered that IFN-γ deficiency and RANKL expression enhancement contribute to activation of osteoclast formation in arthritis. Therefore, by promoting type II IFN signaling in the osteoclast precursor cells of this joint, abnormal formation of osteoclasts can be suppressed, and bone destruction can be prevented.
Further, according to the present invention, it was shown that TRAF6 is an important target molecule in suppressing osteoclast formation via IFN-γ. This indicates that developing a drug targeting TRAF6 is extremely important for the development of a specific therapeutic drug for pathological bone resorption including inflammatory bone disease.
The present invention enables a new therapeutic approach to the following bone metabolic diseases.
1) Control of osteoclast generation and activity is effective for prevention and treatment of bone metabolic diseases. Particularly effective for autoimmune arthritis.
2) Inhibition of the RANKL signaling pathway is an effective therapeutic strategy for bone metabolic diseases including autoimmune arthritis.
3) TRAF6 is an effective target molecule in the prevention and treatment of bone metabolic diseases including autoimmune arthritis.
4) Type II IFN (eg, IFN-γ) is effective in preventing and treating bone metabolic diseases including autoimmune arthritis.
Thus, the present inventors have elucidated a novel regulatory mechanism of osteoclast formation, and have developed a method for controlling abnormal bone resorption by osteoclasts. That is, the present invention relates to a method for controlling an osteoclast-forming signal, and drugs and medicaments for use in the control, more specifically,
(1) a method of controlling an osteoclast-forming signal, comprising a step of promoting or suppressing type II IFN signaling in osteoclast precursor cells;
(2) a method for controlling any of the following (a) to (d), comprising a step of promoting or suppressing type II IFN signaling in osteoclast precursor cells:
(A) Osteoclast formation
(B) RANK signaling
(C) TRAF6 expression or its function
(D) bone resorption
(3) a method for suppressing inflammatory bone destruction, comprising a step of promoting type II IFN signaling in osteoclast precursor cells;
(4) the method according to any one of (1) to (3), wherein the type II IFN signaling is Stat1-mediated signaling;
(5) The method according to any one of (1) to (3), wherein the promotion or suppression of type II IFN signaling is the promotion or suppression of the interaction between a type II IFN receptor and its ligand.
(6) The method according to (5), wherein the promotion of the interaction between the type II IFN receptor and its ligand is carried out by the following steps (a) or (b):
(A) Administration of type II IFN receptor ligand
(B) Administration of vector expressing type II IFN receptor ligand
(7) The method according to (5) or (6), wherein the ligand is IFN-γ.
(8) A method for detecting an effect of a compound on (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or its function, or (4) bone resorption,
(A) promoting RANK signaling in osteoclast precursor cells and type II IFN signaling in the presence of a sample containing the test compound;
(B) detecting (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or function, or (4) detecting bone resorption;
(9) A method for screening a compound that regulates (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or function thereof, or (4) bone resorption,
(A) promoting RANK signaling in osteoclast precursor cells and type II IFN signaling in the presence of a sample containing the test compound;
(B) detecting (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or function, or (4) bone resorption,
(C) selecting a compound that modulates (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or function, or (4) bone resorption compared to control conditions; Including the method,
(10) The method according to (8) or (9), wherein the type II IFN signaling is Stat1-mediated signaling.
(11) The method according to (8) or (9), wherein the promotion of type II IFN signaling is the promotion of interaction between a type II IFN receptor and its ligand.
(12) The method according to (11), wherein the promotion of the interaction between the type II IFN receptor and its ligand is performed by the following steps (a) or (b):
(A) Administration of type II IFN receptor ligand
(B) Administration of vector expressing type II IFN receptor ligand
(13) the method according to (11) or (12), wherein the ligand is IFN-γ; (14) the following (a) to (d) comprising a type II IFN receptor ligand or a vector expressing the ligand. The drug according to any of the above,
(A) Osteoclast formation inhibitor
(B) RANK signaling inhibitor
(C) TRAF6 inhibitor
(D) Bone resorption inhibitor
(15) the agent according to (14), wherein the type II IFN receptor ligand is IFN-γ;
(16) a pharmaceutical composition for suppressing bone destruction, comprising a type II IFN receptor ligand or a vector expressing the ligand;
(17) The pharmaceutical composition according to (16), which is used for prevention or treatment of bone destruction in autoimmune arthritis.
The present invention relates to a method for controlling osteoclast-forming signals by promoting or suppressing type II IFN signaling. In the present invention, the “osteoclast-forming signal” refers to an action or a part of the action in a process of inducing osteoclast formation and a process induced by osteoclast formation. These effects include phenotypic changes, including changes in gene expression, cell morphology and function, and metabolic activities caused by these phenotypic changes. By promoting type II IFN signaling, osteoclastogenic signals are inhibited and bone resorption is suppressed. Conversely, by inhibiting type II IFN signaling, inhibition of osteoclastogenic signals is reduced and bone resorption is promoted.
Osteoclasts are multinucleated giant cells formed by fusing mononuclear osteoclast precursor cells differentiated from macrophage / monocytic cells (CFU-M) originating from hematopoietic stem cells (Udagawa, N.C.). et al., Proc. Natl. Acad. Sci. USA {87: 7260, 1990). Generally, mature osteoclasts express tartrate-resistant acid phosphatase (TRAP), calcitonin receptor, vitronectin receptor, and M-CSF receptor (c-Fms), and exhibit bone resorption ability. Osteoclast formation can be determined based on at least one of these indicators. In an in vitro culture system, bone marrow monocyte / macrophage progenitor cells (BMMs) are transformed into M-CSF (macrophage-colony stimulating factor under the presence of macrophage-stimulating factor L under macrophage-stimulating factor K). Differentiates into osteocytes. In addition to these factors, for bone destruction in inflammatory arthritis and the like, IL (interleukin) -1, IL-6, TNF (tumour necrosis factor) -α, FGF (fibroblast growth factor) -2, and PDGF (platelet) -Derived {growth factor) and growth factors are involved. When osteoclasts perform bone resorption, they come into contact with the bone at a site rich in actin called the clear zone, forming a membrane invaginating structure called a ruffled border, and acid-induced by a proton pump. The bone matrix is degraded by producing matrix degrading enzymes such as matrix metalloprotease (MMP) and cathepsin in the environment. According to the present invention, an osteoclast-forming signal that induces such osteoclast differentiation and bone resorption can be controlled by regulating type II IFN signaling. That is, bone destruction can be suppressed by promoting type II IFN signaling, and conversely, bone destruction can be promoted by suppressing type II IFN signaling.
As described above, osteoclast formation is generally induced by RANKL in the presence of M-CSF. RANKL is a ligand of RANK (receptor / activator / of / nuclear / factor / κB) expressed on the cell surface, and induces RANK-mediated signal transduction. Activation of RANK causes signals to be transmitted downstream via effector molecules, including TRAF2, TRAF6, and other TRAF family factors, to promote osteoclast differentiation and activation to form osteoclasts. In addition to RANKL, TNFα and IL-1 are involved in the formation of osteoclasts, and TNFα is mediated by type I and type II TNF receptors, and IL-1 is mediated by IL-1 receptors. Transmits osteogenic signals. TRAF family factors are also involved in signals mediated by TNF receptor and IL-1 receptor. For example, TRAF6 also functions as an IL-1 receptor adapter molecule. By promoting or suppressing type II IFN signaling, it is possible to suppress or promote these osteoclast-forming signals, respectively.
As shown in Example 2, by promoting or suppressing type II IFN signaling, RANK signaling in particular can be effectively controlled. IFN-γ has a relatively modest inhibitory effect on the proliferation of BMM dependent on M-CSF, whereas osteoclastogenesis induced by RANK signal is strongly inhibited even at very low levels of IFN-γ The effect was shown (FIG. 3). Furthermore, IFN-γ not only inhibits osteoclast formation of BMMs that differentiate depending on M-CSF and RANKL, but also RANKL-induced osteoclast formation of M-CSF-independent macrophage cell line RAW264.7 Also inhibits. These findings indicate that by promoting type II IFN signaling, RANK signaling can be particularly selectively suppressed. Conversely, by inhibiting type II IFN signaling, inhibition of RANK signaling can be reduced. “RANK signal transduction” refers to a series of signal transduction via RANK or a part thereof, and is caused by activation or increase of expression of a signal molecule downstream of RANK, induction of target gene expression, activation of RANK. Includes changes in cell characteristics. These include increased expression of TRAF6, activation of TRAF6, signal transduction via TRAF6, activation of NF-κB, and activation of MAPK signaling including JNK activation (phosphorylation). These include formation of osteoclasts, induction of expression of genes expressed in osteoclasts such as TRAP, calcitonin receptor and vitronectin receptor, and acquisition of bone resorption ability.
Further, as shown in Example 3, it was found that promotion of type II IFN signaling significantly inhibited the expression of TRAF6. The results of the immunoblot analysis indicate that promotion of type II IFN signaling significantly reduces TRAF6 protein levels (FIG. 6c). Therefore, it is possible to suppress the expression of TRAF6 by the RANK signal by promoting the type II IFN signaling. By suppressing the expression of TRAF6, the function of TRAF6 is suppressed. The function of TRAF6 includes, for example, signal transmission involving TRAF6. That is, by promoting type II IFN signaling, TRAF6-related signaling can be suppressed. In addition to RANK signaling, TRAF6 has been suggested to be involved in CD40, p75NTR, and Toll / IL-1 receptor family signaling. For example, TRAF6 is also involved in LPS / endotoxin signals including TLR (Toll-like receptor) -2 and TLR-4. By controlling TRAF6 expression using the method of the present invention, it is possible to control signaling through these TRAF6. It is suggested that a method of inhibiting the expression or function of TRAF6 is also an alternative strategy for drug discovery to suppress osteoclasts.
In the present invention, “type II IFN signaling” refers to signaling induced by type II IFN or a part thereof. That is, signaling induced by type II IFN, signaling induced by activation of type II IFN receptor, signaling induced by effector molecules downstream of type II IFN receptor, etc. Included in signaling. In the present invention, type II IFN signaling preferably refers to signaling induced by activation of type II IFN receptor.
Type II IFNs include IFN-γ, which functions as a ligand for type II IFN receptor (IFN-γR) to activate type II IFN signaling. When a ligand such as IFN-γ binds to the type II IFN receptor, the type II IFN receptor phosphorylates the Stat family (such as Stat1) through the activation of tyrosine kinases of the JAK family (such as JAK1 and JAK2). The Stat1 dimer is also referred to as GAF (IFN-γ activated 、 factor), and the phosphorylated dimer translocates to the nucleus and binds to GAS (IFN-γ activated sequence) to promote transcription of a target gene group. As described above, the type II IFN signaling includes signaling via Stat1 (GAF). IRF-1 also enhances the expression of IFN-γ target genes.
To activate type II IFN signaling, for example, a ligand of type II IFN receptor is acted on. Type II IFN receptor ligands include IFN-γ. Further, IFN-γ expression may be induced by expression of IRF-1. It can also activate signaling downstream of the type II IFN receptor. Especially, it is preferable to promote Stat1-mediated signal transmission. For example, by promoting GAF activation, type II IFN signaling can be activated. Further, by promoting the expression of a group of GAS-responsive genes, type II IFN signaling can be activated.
In order to suppress the type II IFN signaling, for example, the ligand of the type II IFN receptor is removed or the binding between the ligand and the receptor is inhibited. For example, the ligand can be neutralized by administering an antibody against the ligand. Alternatively, IFN-γ expression may be suppressed by a factor that suppresses IFN-γ expression, that is, other cytokines (including IL-4 and IL-10) or a transcriptional repressor. In addition, signaling downstream of the type II IFN receptor may be suppressed.
It is also possible to promote Stat1 dimerization to promote type II IFN signal by increasing intracellular tyrosine kinase activity of the JAK family, for example, by expressing activated JAK in the cell. is there. Conversely, if JAK is inhibited, the type II IFN signal can be suppressed. In addition, Stat1 binds to a transcription coactivator such as CBP / p300 in the nucleus to promote transcription activity. Therefore, if CBP / p300 or the like is overexpressed or the binding ability of these to Stat1 is increased, the type II IFN signal is enhanced. Conversely, if the binding of Stat1 to CBP / p300 or the like is inhibited, the type II IFN signal can be inhibited (Horvai, AE et al., Proc. Natl. Acad. Sci. USA # 94: 1074). -1079 (1997)).
Target cells that control type II IFN signaling are osteoclast precursor cells. In the present invention, “osteoclastic precursor cells” refer to cells that can differentiate into osteoclasts. Such cells include macrophages and monocyte cells that can differentiate into osteoclasts. Specific examples include macrophage cells (eg, macrophage / monocyte precursor cells) present in osteochondral tissue, such as bone marrow macrophage cells and synovial macrophages. Hematopoietic stem cells are also included. The species from which the cell is derived is not particularly limited. The RANKL signaling system and the type II IFN signaling system are widely conserved in mammals. In human cells and the like, these signal molecule groups are common to mouse cells, and their functions are the same. It has been known. Therefore, for example, by controlling type II IFN signaling on osteoclast precursor cells derived from primates including humans and monkeys according to the present invention, it is possible to control osteoclast formation of the cells. It is.
For example, to examine the effect of IFN-γ on in vitro osteoclastogenesis from human peripheral blood mononuclear cells, peripheral blood was collected from a healthy subject, diluted 1: 1 with RPMI 1640, and diluted with Ficoll / plaque. Overlay and centrifuge at 400 g for 30 minutes. The isolated peripheral blood mononuclear cells (10⁶Cells / well) to a 24-well multiwell plate. These cells were cultured in α-MEM containing 20% horse serum in the presence of 100 ng / ml @ RANKL and 10 ng / ml @ M-CSF and in the presence or absence of recombinant human IFN-γ for 10 days. It can be performed by detecting osteoclast formation.
A ligand for a type II IFN receptor refers to a molecule that binds to and activates the type II IFN receptor. When signal transduction is promoted using a ligand of the type II IFN receptor, the ligand of the type II IFN receptor used is not particularly limited as long as it binds to the receptor and activates the receptor. Ligands include natural ligands, derivatives of natural ligands, other proteins that act as ligands, artificial ligands, and the like. The ligand may be a protein or peptide or a non-peptidic compound. The peptidic ligand may be recombinant or modified. Further, the ligand may be formulated. Further, a compound conjugated with another compound such as a glycol conjugate (glycol-conjugated @IFN) may be used.
Examples of ligands for type II IFN receptor include IFN-γ. In addition, derivatives thereof are also included. In the present invention, IFN-γ includes not only wild-type IFN-γ but also derivatives of wild-type molecules. For example, a protein in which one or more amino acids have been added, deleted, substituted, and / or inserted into wild-type IFN-γ, a protein having a modified main chain or side chain, and the like are also included in IFN-γ in the present invention. It is. IFN-γ includes all IFN-γ subtypes, derivatives exhibiting IFN-γ-like activity, and the like.
By administering these type II IFN receptor ligands and allowing the ligand to interact with the type II IFN receptor, type II IFN signaling can be promoted. The administration of the ligand may be appropriately performed according to a known administration method so that the ligand can bind to the receptor of the target cell.
Instead of using the ligand itself, it is conceivable to use a vector that expresses the ligand. A peptidic ligand can be produced by expressing a nucleic acid encoding the ligand. For example, when a vector expressing IFN-γ or a derivative thereof is introduced into a cell, these molecules produced from the introduced cell bind to the type II IFN receptor and activate the type II IFN signal. In the present invention, the vector is not limited as long as it can express the type II IFN receptor ligand, and may be in the form of a polynucleotide containing DNA and RNA itself, or a complex containing the polynucleotide. Known vector systems, including plasmid vectors and viral vectors, can be used to prepare the vector that expresses the ligand. Examples of the virus vector include a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, and the like. The administration of the vector to the living body can be performed by an in vivo method of directly administering the vector, or an ex @ vivo method of administering cells into which the vector has been introduced.
When a gene is introduced by the ex @ vivo method, for example, a retrovirus vector can be suitably used. Since retrovirus vectors integrate a transgene into the host chromosome, stable expression can be expected for a long period of time. For example, when introducing a gene into a joint, it is conceivable to introduce the gene into synovial cells taken out of the body and transplant the cell into which the gene has been introduced again into the joint. When a gene is introduced by the in vivo method, for example, an adenovirus vector can be used. Adenovirus can be concentrated at a high concentration and is highly safe because the transgene is not integrated into the host genome. In fact, it has been reported that the gene transfer efficiency of an adenovirus vector to synovial cells is high, and it can be used for gene transfer to joints. In addition, gene transfer into a cell or a living body using a non-viral vector can be performed by administration of naked DNA or a method using Sendai virus liposome. The cells to which the vector is administered are not particularly limited. The vector or cells into which the vector has been introduced are administered so that the produced ligand can act on osteoclast precursor cells. For example, it can be locally administered to an osteochondral site.
In addition, it is conceivable to administer not only cells into which the vector has been introduced but also any cells that produce a ligand of type II IFN receptor. Administration of a type II IFN receptor ligand in the present invention also includes indirect administration of the ligand, such as administration of a cell that produces the ligand. Cells that naturally produce IFN include lymphocytes such as T cells, NK cells, and NKT cells in the case of IFN-γ.
Further, by promoting type II IFN production from type II IFN-producing cells in the body, type II IFN signaling can also be promoted. For this purpose, a stimulus for promoting IFN production from type II IFN-producing cells may be given. As shown in the Examples, activated T cells released IFN-γ and suppressed the formation of osteoclasts. Therefore, IFN-γ production can be promoted, for example, by activating T cells. In general, type II IFN production is induced by viral or microbial infection, endotoxin, synthetic small molecules, antibiotics, polysaccharides, nucleic acids, mitogens, administration of immunopotentiators, etc. (Mayer, ED et al., "Interferons and Other Regulatory Cytokines" (interferons and other regulatory cytokines), 1998, John Wiley & Sons.
When the type II IFN receptor ligand interacts with the receptor by the above method, osteoclast formation induced by RANKL or the like in osteoclast precursor cells is inhibited and bone resorption is suppressed. it can. According to the method of the present invention, for example, bone destruction in autoimmune arthritis can be suppressed. There are no particular limitations on the individual to which bone destruction is to be suppressed, and examples include humans and non-human mammals such as mice, rats, rabbits and monkeys. The application to non-human mammals is also useful as a model for developing preventive or therapeutic methods for human bone destructive disease. This allows for the development of new methods for preventing bone destruction, for example in autoimmune arthritis.
For example, to analyze the effect of IFN-γ on inhibiting osteoclast formation using an individual, an endotoxin-induced model of inflammatory bone destruction as described in Example 1 can be used. Specifically, lipopolysaccharide (LPS) (Sigma) was locally administered at a dose of 25 mg / kg-body weight to the calvaria of 7-8 week-old mice (n = 20), and the recombinant was treated for 4 days following the same site. The therapeutic effect of local infusion of IFN-γ can be tested by subcutaneous administration of IFN-γ (eg, 10,000 U / day) (n = 10) or control saline (n = 10). Five days after LPS injection, the number of osteoclasts per millimeter of bone surface of the trabecular bone of the trabecular bone and the percentage of bone surface covered by osteoclasts (eroded surface) are analyzed as previously described (Chiang, C. et al. Y. et al., Infect. Immun. 67, 4231-6 (1999)).
For example, by administering IFN-γ to the site of inflammation daily or at intervals, inhibition of osteoclast formation and bone resorption can be examined. Thereby, it is possible to examine the conditions of clinical application of IFN-γ in bone destruction diseases in more detail. Because bone destruction is often associated with tumors, including multiple myeloma and metastatic bone cancers, and inflammatory conditions such as autoimmune arthritis, IFN-γ is a therapeutic for cancer and arthritis-induced bone destruction. May have a particular effect. Local delivery of IFN-γ to the site of bone destruction can reduce the dose, thereby reducing side effects.
In addition, in order to examine the effect of type II IFN on pathological bone resorption in human diseases, a system of in vitro osteoclast formation from cultured synovial cells derived from patients with rheumatoid arthritis may be used. This system is an ex @ vivo model of pathological osteoclastogenesis observed in the rheumatoid synovium (Takayanagi, H. et. Al. Arthritis @ Rheum., 43, 259 (2000)). The synovial tissue is dissected, RPMI @ 1640 containing 1 mg / ml collagenase (Wako pure Chemicals, Osaka, Japan) and 0.15 mg / ml DNase I (Sigma, St. Louis, MO) is added and mixed by 20 times, and shaken. While incubating at 37 ° C for 90 minutes. The cell suspension is filtered through a 70 mm cell strainer and the cells are finally suspended in α-MEM containing 20% horse serum (Gibco BRL). The isolated synovial cells (10⁶Cells / well) were spread on a 24-well multiwell plate, and 1,25-dihydroxyvitamine @ D3 [1,25 (OH)₂D₃] (10^-7M) and 2 ng / ml in the presence of recombinant human M-CSF and in the presence or absence of recombinant human IFN-γ for 20 days. Change the medium half every three days. In this ex @ vivo osteoclast formation model, the inhibitory effect of recombinant IFN-γ on osteoclast formation can be detected. By such a method, it is possible to develop a new strategy for inhibiting bone resorption in human bone diseases by local or systemic administration of type II IFN.
Recently, antibody induction therapy and antibody induction therapy using a DNA vaccine have been developed as one method of suppressing protein function. Using these immunological techniques, it is possible to control bone resorption and bone destruction by blocking the function of molecules involved in osteoclast-forming signals. The present invention provides methods for modulating osteoclastogenesis by inducing antibodies against molecules involved in osteoclastogenesis signals. In a preferred embodiment, osteoclastogenesis is regulated by inducing an immune response to a protein involved in RANKL signaling. For example, an antibody-induced therapy for suppressing bone destruction can be performed by inducing an immune response to a protein that performs RANKL signaling for pathological bone destruction diseases such as cancer bone metastasis and rheumatism. . Examples of proteins involved in RANKL signaling include, but are not limited to, RANKL, RANK, TRAF6, c-Fos, NF-κB, JNK, Fra-1, and Fra-2. Particularly preferred target proteins include RANKL and RANK.
In another preferred embodiment, osteoclast formation is regulated by inducing an immune response to a protein involved in type II IFN signaling. Since the type II IFN signal suppressively acts on osteoclast formation, suppressing this signal transmission can promote osteoclast formation. By performing immunotherapy against a protein involved in type II IFN signaling for a pathological condition in which bone formation is enhanced, it is possible to promote osteoclast formation and suppress an increase in bone mass. Proteins involved in type II IFN signaling include, but are not limited to, type II IFN such as IFN-γ, its receptor, and Stat1. In particular, IFN-γ and IFN-γ receptor are preferred as targets for inducing an immune response.
An immune response targeting proteins involved in the osteoclast-forming signal is induced by administering these proteins or partial proteins having antigenicity as antigens. For example, RANKL or RANK or a partial protein thereof is administered as an antigen to induce an antibody. The antigen protein can be appropriately administered in combination with an adjuvant. Suppression of RANKL-RANK binding (including anti-RANKL antibody and anti-RANK antibody induction therapy) using a method of inducing an immune response to RANKL or RANK suppresses osteoclast formation and reduces bone resorption. Can be reduced. In addition, the binding of type II IFN-II type IFN receptor is suppressed using a method for inducing an immune response to type II IFN or its receptor (anti-type II IFN antibody, anti-type II IFN receptor antibody). Induction therapy), the suppression of osteoclast formation is released, and bone resorption can be promoted. The present invention provides a method for treating a disease having pathological bone destruction or bone formation by controlling bone resorption by such an inhibitory antibody induction method.
It is also possible to utilize soluble factors other than antibodies to regulate osteoclast formation. For example, OPG, known as a decoy receptor for RANKL, blocks RANKL signaling by inhibiting the binding of RANKL to RANK. OPG's therapeutic effects on arthritis have been demonstrated in vitro (Takayanagi, H. et al., Arthritis Rheum. 43, 259-269 (2000)) and in vivo (Kong, YY et al., Nature $ 402: 304-309 (1999)). The use of these decoys can inhibit the action of ligands such as RANK and type II IFN involved in osteoclast formation. As a protein that functions as a decoy, a natural soluble receptor or the like can be used, or a partial protein of the extracellular region of a membrane-bound receptor can be produced and used. For example, if RANKL is captured by a partial peptide containing a ligand binding region of RANKL, it is possible to inhibit the binding of RANKL to RANK and suppress osteoclast formation. When a decoy that binds to type II IFN is used, it is possible to block the type II IFN signal and promote osteoclast formation. Alternatively, the signal can be blocked by using a factor that binds to the receptor but does not activate the receptor, instead of a protein that binds to the ligand. Such a factor may be a mutant of a ligand, a partial peptide containing a receptor binding site, or a non-peptidic compound. The present invention relates to a bone disease in which pathological bone destruction or bone formation is caused by inhibiting bone resorption by inhibiting the binding between RANK-RANKL or between the type II IFN-II type IFN receptor using a decoy or the like. And a method for performing a treatment for In addition, if an inhibitory antibody against decoy intrinsic to the living body is induced as described above, it is considered that bone resorption can be more variously controlled.
If a method of regulating osteoclast-forming signals by controlling type II IFN signaling is used, it is possible to construct an assay system or a screening system for compounds that can affect the signal. In these systems, the effects of the compounds are verified using, for example, regulation on osteoclastogenesis, RANK signaling, TRAF6 expression or its function, or bone resorption as an index. For example, by examining how the above-mentioned regulatory effect by type II IFN signaling changes in the presence of a test compound, it is possible to find a compound that promotes or suppresses these controls by type II IFN signaling. it can.
Specifically, methods for detecting the effects of a compound on (1) osteoclastogenesis, (2) RANK signaling, (3) TRAF6 expression or its function, or (4) bone resorption, include:
(A) promoting type II IFN signaling in osteoclast precursor cells under conditions that can induce osteoclast formation in the presence of a sample containing the test compound;
(B) (1) Osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or function, or (4) a step of detecting bone resorption.
“Conditions that can induce osteoclasts” in this detection refers to conditions under which osteoclast formation is induced unless type II IFN signaling is activated. Such conditions are, for example, conditions that promote RANK signaling in osteoclast precursor cells.
This detection method is particularly useful for promoting a compound that suppresses (1) osteoclastogenesis, (2) RANK signaling, (3) TRAF6 expression or its function, or (4) bone resorption by type II IFN signaling. Or it is useful for detecting the suppression effect. For example, if (1) osteoclastogenesis, (2) RANK signaling, (3) TRAF6 expression or function, or (4) bone resorption is induced even under conditions that promote type II IFN signaling For example, it is determined that the test compound used has an activity of reducing these suppressive actions by type II IFN signaling. In addition, under the conditions that promote type II IFN signaling, the test compound is (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or its function, or (4) bone resorption. If the inhibition is further increased, the test compound used may have an activity to increase these inhibitory effects by type II IFN signaling. Further, if the same detection is performed under the condition that the type II IFN signaling is inhibited, it is possible to know whether the effect of the compound is dependent on the type II IFN signal. For such detection, for example, IFN is neutralized by a neutralizing antibody for type II IFN, or IFN-γR as used in the Examples.^{− / −}Of individuals and cells can be used.
The cells used are not limited as long as they can differentiate into osteoclasts. For example, bone marrow macrophage cells and the like are used. To promote RANK signaling, for example, RANKL, a ligand of RANK, may be administered. If necessary, M-CSF and other cytokines / growth factors are administered so that osteoclast formation is induced unless the IFN signal is activated. The method for promoting type II IFN signaling may be performed by the method described above. Specifically, for example, a type II IFN receptor ligand such as INF-γ can be administered. The sample containing the test compound is not particularly limited, and examples thereof include a library of synthetic low-molecular compounds, a purified protein, an expression product of a gene library, a library of synthetic peptides, a cell extract, and a cell culture supernatant. Can be
By using the detection method of the present invention, a compound that regulates (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or its function, or (4) bone resorption can be screened. . This screening method
(A) promoting type II IFN signaling in osteoclast precursor cells under conditions that can induce osteoclast formation in the presence of a sample containing the test compound;
(B) detecting (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or function, or (4) bone resorption,
(C) selecting a compound that modulates (1) osteoclastogenesis, (2) RANK signaling, (3) TRAF6 expression or function, or (4) bone resorption compared to control conditions; It is a method including.
Similarly to the above detection method, the “conditions that can induce osteoclasts” refer to conditions under which osteoclast formation is induced unless type II IFN signaling is activated. Such conditions are, for example, conditions that promote RANK signaling in osteoclast precursor cells. In addition, as in the above-described detection method, this screening method comprises (1) osteoclast formation by type II IFN signaling, (2) RANK signaling, (3) TRAF6 expression or its function, or (4) bone It can be used to screen for compounds that increase or decrease the inhibitory effect of absorption.
The control conditions are not particularly limited, and include, for example, the case in the absence of a sample containing the test compound. That is, in the step (c), (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or its function, as compared to the case where no sample containing the test compound is present. Or (4) by selecting compounds that increase or decrease bone resorption, compounds that regulate these can be obtained. Control conditions can also include cases in the presence of other compounds that can modulate osteoclastogenesis, RANK signaling, TRAF6 expression or function, or bone resorption. Such an embodiment is also included in the case where the sample containing the test compound in step (a) is absent. In this case, in the above step (c), (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or its function, or (4) ) Select compounds that increase or decrease bone resorption. In such a screening, a compound having a higher effect than a certain compound can be obtained. Further, in the step (c), a case where a lower dose of the test compound than in the step (a) is used can also be mentioned. This reveals the dose dependence of the compound.
The detection method and screening method of the present invention can be performed using, for example, an in vitro assay system for osteoclast formation as described in Example 2. Specifically, for example, non-contact bone marrow cells (5 × 10 5 per well of a 24-well plate)⁵Cells) were cultured for 2 days in α minimum essential medium (α-MEM) containing 10 ng / ml M-CSF, 100 ng / ml soluble RANKL, 10 ng / ml M-CSF, and an appropriate concentration of IFN-γ Can be used to form osteoclasts after further culturing for 3 days in the presence of. Here, a sample containing the test compound is added and cultured. In addition, in the case of an in vivo system, for example, a system using an endotoxin-induced bone resorption animal model as used in the examples may be mentioned.
Osteoclast formation can be observed, for example, according to the literature (Yasuda, H. et al., Proc. Natl. Acad. Sci. U.S.A. 95, 3597-602 (1998)). Specifically, for example, in the case of an individual, a bone slice can be prepared and bone resorption can be observed. In addition, osteoclast formation can be identified in both in vivo and in vitro by known micronucleated giant cells by microscopic observation, TRAP staining, and the detection of calcitonin receptor or vitronectin receptor expression by known methods. Can be detected. In the case of RANK signaling, for example, activation of NF-κB or JNK can be detected by the methods described in the Examples and the like. In the case of TRAF6 expression, the expression of TRAF6Δ mRNA can be detected by Northern blot, RT-PCR, or the like, and the TRAF6 protein can be detected by immunoblot, immunoprecipitation, ELISA, or the like. If it is a function of TRAF6, it can be detected by, for example, activation of NF-κB or JNK as in the case of the above-mentioned RANK signaling, and as described above, CD40, p75NTR, or Toll / IL-1 It can also be assessed by detecting the activation of other signaling through TRAF6, such as the receptor family. For example, phosphorylation of not only JNK but also other signal molecules of the MAP kinase pathway may be used as an index, and binding or modification with a molecule such as TAK1 or ECSIT (evolutionary conserved signaling intermediate in toll pathways) that binds to TRAF6. It can also be used as an indicator. In the case of bone resorption, for example, the bone resorption activity on an ivory section can be detected by a known method.
As a result of this detection, the addition of the sample containing the test compound allows (1) osteoclast formation, (2) RANK signal transduction, as compared to control conditions (for example, detection in the absence of the sample). If (3) TRAF6 expression or its function, or (4) bone resorption is significantly promoted or suppressed, the test sample used is a candidate for a compound that regulates these controls by type II IFN signaling. The compound isolated by this screening method is useful for controlling osteoclast formation and is also useful for developing preventive and therapeutic drugs for bone metabolic diseases.
The drug candidate compound selected by the screening can be used to determine detailed administration conditions for clinical application using the above-described test method of the present invention. That is, by using the above-described test method, administration conditions including administration amount, administration interval, and administration route can be examined, and conditions for obtaining appropriate preventive or therapeutic effects can be determined. Similarly, to determine detailed administration conditions for clinical application of a type II IFN receptor ligand, it is necessary to: (a) add a type II IFN receptor ligand to an osteoclast precursor cell under conditions capable of inducing osteoclastogenesis; (B) (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or function, or (4) detecting bone resorption. A method for detecting the effect of a body ligand can be used. Specifically, it can be performed using the method described in the present specification.
In addition, the present invention relates to an agent for controlling an osteoclast-forming signal, including a type II IFN receptor ligand or a vector expressing the ligand. The present invention provides the following agents comprising a type II IFN receptor ligand or a vector expressing the ligand.
(A) Osteoclast formation inhibitor
(B) RANKL signaling inhibitor
(C) TRAF6 inhibitor
(D) Bone destruction inhibitor
As the type II IFN receptor ligand and the vector expressing the ligand, those described above can be used. In the present invention, the drug includes a reagent and a medicine. These drugs use ligands and vectors themselves, as well as sterile water, physiological saline, buffers, salts, stabilizers, preservatives, surfactants, other proteins (such as BSA), transfection reagents (lipofection reagents, (Including liposomes) and the like as appropriate. These may be mixed or separated until mixed at the time of use. The agent of the present invention has an effect of suppressing osteoclast formation, an effect of suppressing RANKL signaling, an effect of suppressing the expression or activity of TRAF6 protein, or suppressing the suppression of bone destruction It is preferable that the drug has the action described in a container, a packaging box, an attached instruction, or the like of the drug, or is associated with the drug. The term “associated” means that information that the drug has the above-mentioned effect can be obtained directly or indirectly from the drug, its container, packaging box, or accompanying instructions. For example, there are cases where the action or use of these drugs is described in a separately issued printed matter, or is disclosed on the web. The agent of the present invention can be applied to cultured cell lines and individuals. Target individuals include mammals such as humans, monkeys, mice, rats, rabbits, and other vertebrates. In particular, the pharmaceutical composition of the present invention containing a type II IFN receptor ligand or a vector expressing the ligand is useful as a prophylactic and therapeutic agent for human bone destruction disease.
When a type II IFN receptor ligand, a vector expressing the ligand, or the above drug is used as a pharmaceutical composition, the ligand or vector itself can be formulated by a known pharmaceutical method in addition to directly administering to the patient or individual. It is also possible. For example, pharmacologically acceptable carriers or vehicles, specifically, sterile water or physiological saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, formulated in appropriate combination with a sustained release agent and the like It is conceivable to administer. The pharmaceutical compositions can be in the form of aqueous solutions, tablets, capsules, troches, buccal tablets, elixirs, suspensions, syrups, nasal drops, or inhalants. The content of the ligand or vector in the composition may be appropriately determined.
Administration to patients depends on the nature of the active ingredient, e.g., transdermal, intranasal, transbronchial, intramuscular, intraperitoneal, intravenous, intraarticular, subcutaneous, intraspinal, intraventricular, or It can be performed orally, but not limited to. It can also be administered systemically or locally. When systemic side effects due to administration of a drug such as IFN poses a problem, the dose can be reduced by local administration. 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, and the like, but can be appropriately selected by those skilled in the art. As shown in the Examples, 1 U / ml of IFN-γ almost completely suppressed osteoclast formation in the in vitro system. For example, even in in vivo, the dosage of the drug can be determined so as to maintain such an effective concentration around the target osteoclast precursor cell. Administration can be carried out once to several times.
The pharmaceutical composition of the present invention is particularly useful for preventing inflammatory bone resorption and inflammatory bone destruction. "Inflammatory bone resorption and destruction" refers to bone resorption and destruction with inflammation. For example, in arthritis, osteoclast formation from synovial macrophages contained in the synovium is promoted, and active bone resorption occurs in the frontal region where pannus enters bone. Particularly in chronic arthritis, long-term joint destruction proceeds due to this bone resorption. This pathological bone resorption can be suppressed by administering the pharmaceutical composition of the present invention. Such arthritis with inflammatory bone resorption includes, in particular, autoimmune arthritis including rheumatoid arthritis (RA). Rheumatoid arthritis is a chronic, autoimmune, inflammatory disease in which the proliferating synovium actively invades osteochondral, resulting in multiple joint destruction. The pharmaceutical composition of the present invention is suitably used for preventing this arthritic bone destruction. Another inflammatory bone resorption to which the pharmaceutical composition of the present invention can be applied includes periodontal disease.
In addition, the pharmaceutical composition of the present invention can be used for various diseases in which bone resorption is promoted by osteoclasts, including giant cell tumor, cancer bone metastasis, and pigmented villous nodular synovitis (PVS). May also be applied. It is also expected to be applied to the treatment of osteoporosis and hypercalcemia of cancer. Further, the present invention can be applied to prevention and treatment of bone loss associated with Paget disease, hepatitis and AIDS, bone resorption / destruction associated with leukemia and multiple myeloma, and bone resorption (loosening) around artificial joints. The pharmaceutical composition of the present invention is described in a container, a packaging box, an attached instruction, or the like that the composition is applicable to at least one of the above-mentioned diseases, or the composition is Is preferably associated with The present invention allows new therapeutic interventions by controlling osteoclast formation and function.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples. All documents cited in this specification are incorporated as a part of this specification.
IFN-γR used in the experiment^{− / −}Mouse and IRF-1^{− / −}The production of mice has been described previously (Huang, S. et al., Science 259, 1742-5 (1993); Matsuyama, T. et al., Cell 75, 83-97 (1993)). C57BL / 6 mice with a genetically identical background to these mutant mice were used as wild-type controls. Stat1^{− / −}Mice (Meraz, MA et al., Cell 84, 431-42 (1996)) were purchased from Taconic.
[Example 1] Regulation of osteoclast formation via T cells by IFN-γ
Induction of bone resorption by endotoxin has been established as a bone resorption model system, and it has been shown that T cells play an important role in this system (Ukai, T. et al., J. 31. 414-22 (1996); Chiang, CY et al., Infect. Immun. 67, 4231-6 (1999)). Using this bone resorption model system, mice lacking IFNGR1 which is one of the components of IFN-γ receptor (IFN-γR) (IFN-γR^{− / −}Osteoclast formation in mice (Huang, S. et al., Science 259, 1742-5 (1993)) was analyzed. 7-8 week old IFN-γR^{− / −}Mouse and IFN-γR^+/-Lipopolysaccharide (LPS) (Sigma) was locally administered at 25 mg / kg-body weight to the calvaria of mice (n = 10), euthanized 5 days later, and described previously (Chiang, CY et al. Infect, Immun. 67, 4231-6 (1999)), it was found that osteoclast formation was enhanced and bone destruction was markedly worsened (FIG. 1). These observations suggested that IFN-γ has an important function in maintaining bone tissue involving a process mediated by T cells.
In order to investigate the effect of IFN-γ producing T cells on osteoclast formation, activated T cells or resting spleen T cells and bone marrow monocyte / macrophage precursor cells (bone @ marrow @ monocytochrome / macrophage @ precursor @ cells; BMMs) In an in vitro co-culture system, an experiment was performed in which RANKL was stimulated in the presence of M-CSF.
To observe in vitro osteoclast formation, non-contact bone marrow cells (5 × 10 5 per well of a 24-well plate)⁵The cells were cultured in α-minimum essential medium (α-MEM) containing 10 ng / ml of M-CSF (R & D) for 2 days and used as BMM. These BMMs were cultured for another 3 days in the presence of 100 ng / ml of soluble RANKL (Peprotech) and 10 ng / ml of M-CSF to form osteoclasts. Observation of multinucleated cells was performed as previously described (Yasuda, H. et al., Proc. Natl. Acad. Sci. U.S.A. 95, 3597-602 (1998)). For activation of T cells, purified CD3⁺Spleen cells (> 93%) were cultured in RPMI 1640 and stimulated with 5 μg / ml anti-CD3ε antibody (PharMingen) for 1 day. The addition of activated T cells or T cell culture supernatant was performed simultaneously with the addition of RANKL to the BMM culture. The medium was changed every 48 hours.
As shown in FIG. 2a, when co-cultured with T cells activated with anti-CD3 antibody, BMM osteoclast formation was strongly inhibited but not resting T cells. Under these conditions, most BMMs differentiated into mature macrophages. Activated T cells are isolated from IFN-γR^{− / −}When co-cultured with BMM and stimulated with RANKL, the inhibitory effect of activated T cells was completely lost (FIG. 2b). These IFN-γRs^{− / −}Since BMM differentiated into osteoclasts with low efficiency in the absence of recombinant RANKL in co-culture of activated T cells, the effect of T cells on osteoclast formation was determined by the balance of RANKL and IFN-γ. Is suggested. The culture supernatant of the activated T cells showed an action of suppressing the formation of osteoclasts by the recombinant RANKL, and it was confirmed that this suppressive effect was neutralized by an antibody against IFN-γ. These results indicated that IFN-γ is essential for T cell suppression of RANKL-induced osteoclastogenesis.
[Example 2] Inhibition of IFN-γ in osteoclastogenesis induced by RANKL via Stat1 activation pathway
Recombinant mouse IFN-γ (Genzyme) was added to the above in vitro assay system of BMM osteoclast formation by RANKL stimulation, and the effect was verified. The addition of the recombinant IFN-γ was performed simultaneously with the addition of RANKL to the BMM culture. As a result, a remarkable inhibitory effect of IFN-γ on BMM osteoclast formation was observed (FIGS. 3 and 4). In the presence of excess RANKL, very low levels of IFN-γ strongly inhibited osteoclast formation (FIG. 3). On the other hand, IFN-γ showed a relatively slight inhibitory effect on the proliferation of BMM that depends not on RANKL but on M-CSF (FIG. 3). Furthermore, IFN-γ was also found to inhibit osteoclast formation by RANKL in the M-CSF-independent macrophage cell line RAW264.7. These results suggest that IFN-γ selectively suppresses the RANKL signaling pathway. In cells, the signal of IFN-γ is transmitted via activation of the transcription factor Stat1 (Stark, GR et al., Annu. Rev. Biochem. 67, 227-64 (1998)). Activated Stat1 is also called IFN-γ activated activated factor (GAF) and directly or via a transcription factor IRF-1 (Taniguchi, T. et al., Biochim. Biophys. Acta 1333, M9-17 (1997)). Induces expression of target genes of IFN-γ. As shown in FIG. 5, the inhibitory effect of IFN-γ on osteoclastogenesis was^{− / −}Mouse or Stat1^{− / −}Although completely lost in mice, IRF-1^{− / −}It was not lost in mice. In addition, similarly, IRF-9^{− / −}When the effect of IFN-γ on the formation of osteoclasts from BMMs induced by RANKL / M-CSF was examined using mice, the inhibitory effect of IFN-γ was observed. It was found not necessary for inhibition by γ (FIG. 5b). From this, it was shown that a gene induction pathway independent of IRF-1 and IRF-9 via Stat1 (GAF) is important for suppression of the RANKL signaling pathway.
[Example 3] Inhibition of RANKL signaling by IFN-γ is mediated by suppression of TRAF6 protein expression
When RANKL stimulates its receptor, RANK, it induces the adapter protein TRAF family and activates the NF-κB and JNK pathways (Dougall, WC et al., Genes Dev. 13, 2412-24 (1999); Hsu, H. et al., Proc. Natl. Acad. Sci. US A 96, 3540-5 (1999)). Among the TRAF family, TRAF6 is known to be important for RANKL signaling.^{− / −}Mice lack osteoclasts that undergo bone resorption and exhibit a phenotype of osteopetrosis (Lomaga, MA et al., Genes Dev. 13, 1015-24 (1999); Naito, A. et al. , Genes @ Cells 4,353-62 (1999)). To investigate the target of IFN-γ suppression of osteoclastogenesis, RANKL-induced activation of NF-κB and JNK was examined in IFN-γ treated BMM. As a result of a gel shift assay (electrophoretic mobility shift assay; EMSA), RANKL-induced activation of NF-κB was significantly inhibited in IFN-γ-treated BMM (FIG. 6a). Furthermore, RANKL-induced activation of JNK was also inhibited by IFN-γ (FIG. 6b). In BMM stimulated with RANKL and M-CSF, analysis of effector molecules downstream of the RANKL signaling pathway by immunoblot revealed that IFN-γ specifically and markedly inhibited TRAF6 expression (FIG. 6c). This result indicates that induction of the proteolytic system by IFN-γ is involved in TRAF6 degradation.
[Example 4] Effect of TRAF6 gene expression on osteoclast formation
To examine whether inhibition of TRAF6 expression is important for suppression of IFN-γ-mediated osteoclastogenesis, BMM expressing TRAF6 exogenously was stimulated with RANKL, and osteoclastogenesis by IFN-γ was performed. An experiment was performed to observe the inhibition of the enzyme.
A retroviral vector (pMX-TRAF6-IRES-EGFP) encoding an internal ribosomal entry site (IRES) -enhanced green fluorescent protein (EGFP) downstream of the TRAF6 cDNA is a 2.0 kb EcoRI-StuI fragment of the mouse full-length TRAF6 gene. Inserted into the same site of pMX-IRES-EGFP (Nosaka, T. et al., EMBO J. 18, 4754-65 (1999)) and described previously (Nosaka, T. et al., EMBO J. 18, 4754-65 (1999)). Retrovirus packaging was performed by co-transfecting these pMX vectors and pPAMpsi2 (Sato, M. et al., FEBS @ Lett. 441: 106-110 (1998)) into 293T cells. The prepared retrovirus vector was infected to BMM and analyzed by flow cytometry and immunoblot. The TRAF6-expressing virus and the virus expressing only EGFP used for control (pMX-IRES-EGFP) showed high efficiency. It was found that the gene could be successfully introduced into BMM (FIG. 7).
Next, the effect of TRAF6 expression on the inhibition of osteoclast formation by IFN-γ was examined. Retroviruses were infected with BMM and RANKL / M-CSF and IFN-γ were added to the medium 48 hours later. Three days after RANKL addition, osteoclastogenesis was assessed by TRAP staining and bone resorption assays (Yasuda, H. et al., Proc. Natl. Acad. Sci. U.S.A. 95, 3597-602 (1998)). .
Interestingly, overexpression of TRAF6 in BMM was resistant to suppression of osteoclast formation by IFN-γ (FIG. 8). In these cells, activation of NF-κB and JNK induced by RANKL was observed. Even in the presence of IFN-γ, about 50% of GFP-positive cells are positive for tartrate-resistant acid phosphatase (TRAP⁺It is noteworthy that the cells were differentiated into multinucleated cells (Fig. 8). These differentiated cells expressed calcitonin receptor and showed bone resorption activity on ivory sections. These results indicate that the suppression of osteoclast formation by IFN-γ can be explained at least in part by a decrease in TRAF6 expression.
Industrial potential
The present invention has revealed a previously unknown link between IFN-γ and bone homeostasis, that is, maintenance of bone mass by inhibiting osteoclast formation. Destruction of IFN-γR by gene targeting enhances osteoclast formation during inflammation, causing exacerbation of bone destruction associated with inflammation. This is an interesting example of the physiological function of cytokines whose effects have been originally established on antiviral responses. The present invention has provided a method for suppressing osteoclast formation via a type II IFN signal. According to the method of the present invention, osteoclast formation can be suppressed by activating type II IFN signaling such as administration of IFN-γ. By controlling osteoclast formation, abnormal bone metabolism can be controlled. The method of the present invention is useful for preventing or treating diseases associated with pathological bone destruction, including autoimmune arthritis.
[Brief description of the drawings]
FIG. 1 shows IFN-γR^+/-Mouse or IFN-γR^{− / −}FIG. 2 is a photograph showing a tissue section (stained with tartrate-resistant acid phosphatase [TRAP] and hematoxylin) of a skull injected with saline or LPS in a mouse. Note the enhancement of osteoclast formation as shown by staining with the osteoclast-specific enzyme TRAP. IFN-γR without LPS administration^+/-Only a small number of TRAP-positive cells are found in mice. IFN-γR^{− / −}Only a small number of TRAP-positive cells are observed in mice. In the case of LPS administration, IFN-γR^+/-In mice, bone resorption is induced, and TRAP-positive cells are observed on the contact surface of the resorption cavity with bone. IFN-γR^{− / −}IFN-γR in mice^+/-Bone resorption is significantly worse than in mice, and osteoclasts are eroded over a wide range of bone surfaces. The number of osteoclasts per mm of the surface of the trabecular bone (trabecular bone) and the ratio of the bone surface (eroded surface) covered with osteoclasts were measured (Chiang, CY et al., Infect. Immun. .67, 4231-6 (1999)). By this morphological measurement, IFN-γR^{− / −}In mice, it was found that the number of osteoclasts and the eroded surface increased more than 2-fold.
FIG. 2 is a diagram showing the regulation of osteoclast formation via T cells by IFN-γ. a shows suppression of osteoclast formation by activated T cells. Spleen or resting T cells stimulated with anti-CD3 antibody were co-cultured with BMM in the presence of RANKL (100 ng / ml) and M-CSF (10 ng / ml). 3 days later TRAP⁺Cells were counted. b is IFN-γR^{− / −}Figure 2 shows the loss of suppression of T cell-mediated osteoclastogenesis in BMM. IFN-γR^{− / −}BMM was co-cultured with activated or resting T cells as described above.
FIG. 3 shows the effect of IFN-γ on RANKL-induced osteoclastogenesis and BMM proliferation. IFN-γ showed a significant inhibitory effect on osteoclast formation in BMM stimulated with RANKL (100 ng / ml) and M-CSF (10 ng / ml), while M-CSF-dependent It showed a slight inhibitory effect on the proliferation of osteoclast precursor BMM.
FIG. 4 is a photograph showing a microscope image (TRAP staining) of an in vitro osteoclast-forming system. BMM of C57BL / 6 mice was cultured with M-CSF (10 ng / ml) alone (upper panel). Under these conditions, the cells were mononuclear and negative for TRAP. In the presence of RANKL (100 ng / ml) and M-CSF (10 ng / ml), BMM can⁺Differentiated into multinucleated osteoclasts (middle panel). Osteoclast formation was suppressed in the presence of RANKL / M-CSF and IFN-γ (100 U / ml) (lower panel). Cells were unfused, mononuclear and TRAP negative.
FIG. 5 is a diagram and a photograph showing the necessity of IFN-γR and Stat1 in suppressing osteoclast formation by IFN-γ. a, Effect of IFN-γ on osteoclastogenesis of BMM from mice lacking IFN-γR, Stat1, or IRF-1 was determined by the presence of RANKL (100 ng / ml) and M-CSF (10 ng / ml). Checked below. The number of osteoclasts (%) was determined by TRAP in IFN-γ treated cultures.⁺The number of multinucleated cells was determined by dividing the number in the control culture without IFN-γ. b, Comparison of the effects of IFN-γ (10 U / ml) on osteoclast formation from BMMs derived from wild-type mice or Stat1, IRF-9, or IRF-1 deficient mice.
FIG. 6 is a photograph showing the result of analyzing the effect of IFN-γ on effector molecules downstream of RANKL signaling.
a shows EMSA analysis of NF-κB activity. After stimulation with RANKL (100 ng / ml), a nuclear extract was prepared from BMM preincubated for 24 hours in the presence or absence of IFN-γ, and an oligonucleotide containing an NF-κB binding site derived from the immunoglobulin κB enhancer Analysis was performed as previously described using the probe (Lomaga, MA et al., Genes Dev. 13, 1015-24 (1999)). The complex was supershifted by an anti-Rel @ A antibody (Santa @ Cruz) (lane 7), and complex formation was inhibited by an excessive amount of unlabeled probe (lane 8).
b shows a JNK activation inhibitory effect. After stimulation with RANKL (100 ng / ml), BMM preincubated for 24 hours in the presence or absence of IFN-γ was analyzed by immunoblot using anti-phosphorylated JNK antibody and anti-JNK antibody (New England Biomeds). Analyzed.
c shows the effect of IFN-γ on the expression of downstream effector molecules involved in RANKL signaling in osteoclast differentiation. Every 24 hours after the addition of RANKL, the whole cell lysate of BMM was analyzed by immunoblot using a specific antibody (Santa Cruz).
FIG. 7 is a diagram and a photograph showing the efficiency of retroviral gene transfer into primary BMM. Two days after infection, BMM was analyzed by flow cytometry (upper panel) and immunoblot was performed (lower panel). The BAF infected with the TRAF6 virus had a TRAF6 protein level of about 10 times that of the control BMM.
FIG. 8 is a diagram and a photograph showing the effect of TRAF6 overexpression on the inhibition of osteoclast formation by IFN-γ. Two days after retroviral infection, RANKL / M-CSF and IFN-γ (10 U / ml) were added to the BMM culture. Three days later, GFP-positive cells were identified with a fluorescence microscope, and TRAP staining was performed (left panel). The upper row shows cells stained with TRAP. No TRAP-positive osteoclasts are found in virus-uninfected cells and EGFP-expressing virus vector (pMX-IRES-EGFP) infected cells. In contrast, cells infected with the TRAF6-expressing viral vector (pMX-TRAF6-IRES-EGFP) had a large number of TRAPs.⁺Multinucleated cells formed. The lower part shows the result of observing the fluorescence of GFP. Most TRAP⁺Multinucleated cells are GFP-positive, and 50% or more of GFP-positive cells are TRAP even in the presence of IFN-γ.⁺Differentiated into multinucleated cells (right panel).

Claims (17)

A method for controlling an osteoclast-forming signal, comprising the step of promoting or suppressing type II IFN signaling in osteoclast precursor cells. A method for controlling any of the following (a) to (d), comprising a step of promoting or suppressing type II IFN signaling in osteoclast precursor cells.
(A) osteoclast formation (b) RANK signaling (c) TRAF6 expression or function (d) bone resorption A method for suppressing inflammatory bone destruction, comprising a step of promoting type II IFN signaling in osteoclast precursor cells. The method according to any one of claims 1 to 3, wherein the type II IFN signaling is Stat1 mediated signaling. The method according to any one of claims 1 to 3, wherein the promotion or suppression of the type II IFN signaling is the promotion or suppression of the interaction between the type II IFN receptor and its ligand. The method according to claim 5, wherein the promotion of the interaction between the type II IFN receptor and its ligand is carried out by the following steps (a) or (b).
(A) Administration of type II IFN receptor ligand (b) Administration of vector expressing type II IFN receptor ligand The method according to claim 5 or 6, wherein the ligand is IFN-γ. A method for detecting the effects of a compound on (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or function, or (4) bone resorption,
(A) promoting RANK signaling in osteoclast precursor cells and type II IFN signaling in the presence of a sample containing the test compound;
(B) detecting (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or function, or (4) detecting bone resorption. A method for screening for a compound that regulates (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or function, or (4) bone resorption,
(A) promoting RANK signaling in osteoclast precursor cells and type II IFN signaling in the presence of a sample containing the test compound;
(B) detecting (1) osteoclast formation, (2) RANK signaling, (3) TRAF6 expression or function, or (4) detecting bone resorption;
(C) selecting a compound that modulates (1) osteoclastogenesis, (2) RANK signaling, (3) TRAF6 expression or function, or (4) bone resorption compared to control conditions; A method that includes The method according to claim 8 or 9, wherein the type II IFN signaling is Stat1 mediated signaling. 10. The method according to claim 8 or 9, wherein the promotion of type II IFN signaling is the promotion of interaction between a type II IFN receptor and its ligand. The method according to claim 11, wherein the promotion of the interaction between the type II IFN receptor and its ligand is carried out by the following steps (a) or (b).
(A) Administration of type II IFN receptor ligand (b) Administration of vector expressing type II IFN receptor ligand The method according to claim 11 or 12, wherein the ligand is IFF-γ. The agent according to any one of the following (a) to (d), comprising a type II IFN receptor ligand or a vector expressing the ligand.
(A) Osteoclast formation inhibitor (b) RANK signaling inhibitor (c) TRAF6 inhibitor (d) Bone resorption inhibitor The agent according to claim 14, wherein the ligand of the type II IFN receptor is IFN-γ. A pharmaceutical composition for suppressing bone destruction, comprising a type II IFN receptor ligand or a vector expressing the ligand. The pharmaceutical composition according to claim 16, which is used for preventing or treating bone destruction in autoimmune arthritis.

Images