WO2016027990A1 - Pharmaceutical composition containing dusp5 as active ingredient for preventing or treating bone metabolic diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating bone metabolic diseases and a pharmaceutical composition for preventing or treating STAT3-mediated diseases, which contain DUSP5 as an active ingredient, and to a method for inhibiting STAT3 activity using DUSP5. The present invention can be favorably used as a novel therapeutic agent capable of treating bone metabolic diseases and STAT3-mediated diseases since DUSP5 can effectively inhibit expression or activity of RANKL, RANK, NFATc1, and TRAP, which are associated with differentiation of osteoclasts; can simultaneously inhibit or reduce activity of Th17 and promote or increase activity of regulatory T cells (Treg); and can inhibit activity of STAT3 through the inhibition of phosphorylation of STAT3.

Description

Pharmaceutical composition for the prophylaxis or treatment of bone metabolic diseases, which contains both DVPS5 as an active ingredient

The present invention relates to a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, including DUSP5 as an active ingredient, a pharmaceutical composition for the prevention or treatment of STAT3-mediated diseases, and a method for inhibiting the activity of STAT3 using DUSP5.

Bone is bone cells (osteocytes), osteoclasts (osteoclasts), osteoblasts (osteoblasts), hydroxyapatite crystals, collagenous fibers, glycosaminoglycans It consists of spaces such as bone matrix and bone marrow cavity, vascular canals, canaliculi and lacunae (Stavros CM, Endocrine Reveiws, 21). 2), 115-137 (2000)). Bones serve to mechanically support the body, protect vital organs, provide the microenvironment needed for hemopoiesis, and store calcium and other minerals.

Bone growth, development, and maintenance occur continuously throughout life. Aged bone is destroyed and new bone is regenerated instead. This turnover occurs mainly in basic multicellular units (BMUs), which are composed of osteoclasts and osteoblasts, which restore the bone's microscopic damage caused by growth and stress and maintain bone function. The destruction or absorption of aged bone is performed by osteoclasts. On the other hand, osteoblasts are responsible for the formation of new bone. Osteoclasts are attached to the surface of the bone to secrete acids and degrading enzymes to remove bone matrix such as apatite crystals and collagen that make up the bone and destroy the bone. Osteoblasts synthesize and release the bone matrix Skeletal formation is achieved by controlling the concentration of and phosphorus (Stavros CM, Endocrine Reviews, 21 (2), 115-137 (2000)).

Bone metabolic disease results from the disruption of the balance between osteoclasts and osteoblasts in vivo. Representative examples of bone metabolic diseases include osteoporosis. Osteoporosis refers to a condition in which total bone mass decreases due to an increase in osteoclast activity compared to osteoblasts. When osteoporosis occurs, the width of the cortical bone is reduced, the cavity of the bone marrow is enlarged, the reticulum is lowered, and the bone continues to be porous. As osteoporosis progresses, the bone's physical strength decreases, causing low back pain and joint pain, and the bone easily breaks down even with a slight impact. Bone metabolic diseases include, but are not limited to, bone metastasis lesions in which tumors such as breast cancer and prostate cancer have metastasized to bone, primary tumors (such as multiple myeloma) in bone, rheumatoid or degenerative arthritis, and bacteria that cause periodontal disease. Periodontal disease caused by destruction of alveolar bone, inflammatory alveolar bone resorption disease after implantation of dental implants, inflammatory bone resorption disease caused by implants implanted to fix bone in orthopedic areas, and various genetic Paget's disease caused by predisposition.

In addition, myeloma is a disease in which bone is easily fractured with severe pain, and is caused by tumor cells promoting osteoclast activity. Breast and prostate cancers easily metastasize to bones, which also promote osteoclast activity and destroy bones. Even in rheumatoid arthritis or degenerative arthritis, tumor necrosis factor (TNF), interleukin-1, and interleukin-6 produced by immune response enhance the activity of osteoclasts in the joint cavity by Local bone breakdown occurs. When the bacteria causing the periodontal disease are infected and inflamed, the immune response results in the production of inflammatory cytokines such as TNF, interleukin-1, and interleukin-6, which promote osteoclast differentiation and destroy the alveolar bone that supports the teeth.

Recently, as the molecular biological researches for the treatment of such metabolic diseases including osteoporosis have been actively conducted, bone formation promoters and osteoclast inhibitors have been developed, and fluoride preparations, parathyroid hormones, T GF-beta (TGF-β), bone morphogenetic protein (insulin-like growth factor), and the like. As osteoclast inhibitors, estrogen, calcitonin, vitamin D and its analogues (Vitamin analogues), bisphosphonates and the like are known (Jardine et al., Annual Reports in Medicinal Chemistry, 31, 211 (1996). )).

However, all of these treatments are ineffective because they are ineffective and difficult to treat.

Therefore, it is urgent to develop a new therapeutic agent that can improve a more fundamental cause and have fewer side effects.

Therefore, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, including both DUSP5 as an active ingredient.

In addition, another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of STAT3-mediated diseases, including both DUSP5 as an active ingredient.

In addition, another object of the present invention includes the step of transforming the cells with an expression vector comprising a promoter and a DUSP5 nucleotide sequence of SEQ ID NO: 2 operably linked to the cell in vitro, to express the DUSP5 gene, in vitro It is to provide a method for reducing the activity or expression of STAT3 within.

The present invention for achieving the above object provides a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, including both DUSP5 as an active ingredient.

In one embodiment of the present invention, the DUSP5 may be composed of the peptide of SEQ ID NO: 1.

In one embodiment of the present invention, the peptide of DUSP5 may be encoded by the nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the present invention, the DUSP5 may inhibit osteoclast differentiation by inhibiting the expression or activity of RANKL, RANK, NFATc1 and TRAP.

In one embodiment of the present invention, the bone metabolic disease is osteoprosis (osteoprosis), Paget disease (paget disease), metastatic cancer (metastatic cancer) or rheumatoid arthiritis (rheumatoid arthiritis), bone metastatic cancer (bone metastatic cancer) , Solid cancer bone metastasis, musculoskeletal complications due to solid cancer bone metastasis, hypercalcemia due to malignant tumors, multiple myeloma, primary bone tumor, degenerative arthritis, periodontal disease, inflammatory alveolar bone disease and inflammatory bone resorption disease It may be selected.

In one embodiment of the present invention, the DUSP5 may be to inhibit or reduce the activity of Th17, and to promote or increase the activity of Regulatory T cells (Treg).

In one embodiment of the present invention, the DUSP5 may have an activity of inhibiting phosphorylation of STAT3.

In one embodiment of the present invention, the DUSP5 may be contained in the composition in the form of an expression vector comprising a promoter and the DUSP5 base sequence of SEQ ID NO: 2 operably linked thereto.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of STAT3-mediated diseases, including both DUSP5 as an active ingredient.

In one embodiment of the present invention, the STAT3-mediated disease is selected from the group consisting of arthritis, peritonitis, multiple sclerosis, psoriasis, asthma, edema, bronchial spasms, allergies, inflammatory diseases, autoimmune diseases, infectious diseases and necrotic diseases It may be.

In one embodiment of the present invention, the DUSP5 may have an activity of inhibiting phosphorylation of STAT3.

In one embodiment of the present invention, the DUSP5 may be composed of the peptide of SEQ ID NO: 1.

In one embodiment of the present invention, the peptide of DUSP5 may be encoded by the nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the present invention, the DUSP5 may be to inhibit or reduce the activity of Th17, and to promote or increase the activity of Regulatory T cells (Treg).

In addition, the present invention includes the step of transforming the cells with an expression vector comprising a promoter and a DUSP5 nucleotide sequence of SEQ ID NO: 2 operably linked thereto in vitro, expressing the DUSP5 gene, STAT3 in vitro It provides a method for reducing the activity or expression of.

The present invention relates to a pharmaceutical composition for preventing or treating bone metabolic diseases, including DUSP5 as an active ingredient, a pharmaceutical composition for preventing or treating STAT3 mediated diseases, and a method for inhibiting STAT3 activity using DUSP5. DUSP5 can effectively inhibit the expression or activity of RANKL, RANK, NFATc1 and TRAP associated with osteoclast differentiation, inhibit or reduce the activity of Th17 and promote or increase the activity of Regulatory T cells (Tregs). It is possible to simultaneously act and inhibit the STAT3 activity by inhibiting the phosphorylation of STAT3, and thus will be useful as a new therapeutic agent for treating bone metabolic diseases and STAT3-mediated diseases.

Figure 1 confirms the effect of arthritis treatment by DUSP5, the results of analyzing the arthritis index and the incidence of arthritis by DUSP5 administration in the mouse group induced arthritis with collagen (Fig. The result confirmed by the crude staining method (Fig. 1b) and the result of analyzing the degree of reduction of immunoglobulin in the mouse serum is shown (Fig. 1c).

Figure 2 shows the results of analyzing the anti-inflammatory activity by DUSP5, after killing the arthritis-induced mouse group, and shows the result of observing the expression level of inflammatory factors in the joint tissue by immunostaining method (Fig. 2a), Fig. 2b is a blood vessel The result of observing the expression level of the formation factor by immunostaining method is shown.

3 is a result of analyzing the effects on the differentiation of Th17 cells and Treg cells by DUSP5, and shows the results of analysis of Th17 cells and Treg cell differentiation according to pcDNA-DUSP5 and MOCK vector injection by flow cytometry (FIG. 3A). ), FIG. 3b shows the results of immunofluorescence microscopy using antibodies to IL-17, Foxp3, CD4, and CD25, and FIG. 3c shows the results of analyzing the expression level of each factor by RT-PCR. It is shown.

4 is a result of analyzing the regulatory activity of ERK, Th17 and Tregs by DUSP5, 4a transduced CD4 + T cells with pcDNA-DUSP5 and MOCK vectors, respectively, DUSP5, IL-17, Gene expression levels of ERK and Foxp3 were analyzed by RT-PCR, and FIG. 4B is a result of analysis of the production amount of IL-17, IL-21 and TNF-a in cell culture by ELISA. 4C shows the results of RT-PCR analysis of the expression level of DUSP5, IL-17, ERK and Foxp3 in cells after DUSP5 siRNA treatment, and FIG. 4D shows IL-17, IL-21 and TNF-a. The result of analyzing the amount of production is shown.

5 is a result of analyzing the effect on the activity of regulating STAT3 and STAT5 by DUSP5, 5a obtained spleen from mice immunized with CII and immunized the degree of phosphorylation of STAT3 and STAT5 by DUSP5 treatment in spleen cells 5 shows the results of observing the degree of phosphorylation through Western blot, and FIG. 5 c shows the result of analyzing the phosphorylation of ERK by immunofluorescence staining.

6 is a result of analyzing the osteoclast differentiation inhibitory activity by DUSP5, 6a is a result showing the expression of RANKL, RANK, NFATc1 in the tissue of the disease model through tissue staining, 6b is a disease mouse model After induction of osteoclast differentiation from bone marrow, the decrease of TRAP-positive cells by DUSP5 treatment was analyzed, and 6c shows the expression of factors related to osteoclast differentiation in cells upon overexpression and suppression of DUSP5. The results of the analysis on RT-PCR are shown.

The present invention is characterized in that it provides a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, including both DUSP5 as an active ingredient.

 Dual-specificity phosphatase 5 (DUSP5) is known to act as an inhibitor of MAPKs, and unlike DUSP1 and DUSP4, it acts to inhibit the activity of ERK, while JNK and p38 MAPK do not inhibit activity.

Therefore, although it is expected to play various roles in DUSP5 intracellular signaling, the research on this molecule is not progressed yet.

 Some prior art discloses that DUSP5 can be used as a marker for diagnosing cancer (KR 20110078453A), and only discloses that it can be used for diagnosing obesity (KR 20090031400A). It has not been reported that metabolic diseases can be treated and STAT3-mediated diseases including autoimmune diseases can be prevented and treated.

 In this regard, the present invention is characterized by identifying a new use in terms of the medical use of DUSP5, in particular, the pharmaceutical use of DUSP5 identified in the present invention can be used for the prevention or treatment of bone metabolic diseases, STAT3 It can be used as a pharmaceutical composition for the prevention or treatment of mediated diseases, it was confirmed that it can be used to reduce the activity or expression of STAT3.

According to one embodiment of the present invention, it was confirmed through experiments that DUSP5 according to the present invention is excellent in inhibiting the differentiation of osteoclasts, in particular of the factors involved in osteoclast differentiation RANKL, RANK, NFATc1 and TRAP It was confirmed that there is an action that inhibits expression or activity.

Osteoclasts are known to be differentiated by various differentiation-inducing factors in macrophage family progenitor cells. In particular, RANKL (receptor activator of nuclear factor kappa B ligand) secreted from osteoblasts binds to osteoclast precursor cells and RANK (receptor activator of nuclear factor kappa B) present on the surface of osteoclasts. Causes differentiation and activation. Differentiated osteoclasts were activated by NF-kB, c-Fos, c-jun, AP-1, NFATc1, MAPK, ERK, JNK, p38 activation process, Src, Akt MITF activation, etc. It is known to promote the expression of osteoclast specific proteins such as cathepsin K and calcitonin receptor.

In this regard, DUSP5 of the present invention has an effect of effectively inhibiting the expression of osteoclast specific proteins and osteoclast differentiation related factors, thereby preventing or treating osteo metabolic diseases caused by osteoclast differentiation.

The bone metabolic disease of the present invention is not limited thereto, but osteoporosis (osteoprosis), Paget disease, (metastatic cancer) or rheumatoid arthiritis (rheumatoid arthiritis), bone metastatic cancer (bone metastatic cancer) , Solid cancer bone metastasis, musculoskeletal complications due to solid cancer bone metastasis, hypercalcemia due to malignant tumors, multiple myeloma, primary bone tumor, degenerative arthritis, periodontal disease, inflammatory alveolar bone disease and inflammatory bone resorption disease Can be selected.

In addition, DUSP5 according to the present invention is characterized by inhibiting or reducing the activity of Th17 and simultaneously inducing an action of promoting or increasing the activity of Regulatory T cells (Treg).

T cells are one of a group of cells that play a central role in the immune system as a biological defense system against various pathogens. T cells are produced in the thymus of the human body and undergo a series of differentiation processes to differentiate into T cells with unique characteristics.T cells, which have completed differentiation, are largely divided into type 1 helper cells (Th1) and type 2 according to their function. It is divided into helper cells (Th2). Among them, the main function of Th1 cells is involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations are balanced with each other so that they are not activated with each other.

Therefore, most of the immune diseases can be attributed to the imbalance between these two immune cells, for example, abnormally increased activity of Th1 cells can cause autoimmune diseases, and abnormally increased activity of Th2 cells It is known that immune diseases occur due to hypersensitivity reactions.

Meanwhile, according to a recent study on the differentiation of Th1 cells, the existence of a new group of immunoregulatory T cells (Tregs), which can control Th1 cell activity, is known, and studies on the treatment of immune diseases using the same are emerging. In addition, since Treg cells inhibit the function of abnormally activated immune cells to control the inflammatory response, many experiments have been reported to treat immune diseases through the action of increasing the activity of Treg cells.

In addition to the Treg cells, another group made during the differentiation is Th17 cells, which are known to be formed through a process similar to the differentiation of Treg cells during the differentiation of undifferentiated T cells. That is, differentiation of Treg cells and Th17 cells is performed in the presence of TGF-β in common but does not require IL-6 in Treg cells, whereas IL-6 is present in combination with TGF-β in Th17 cells. Differentiate in situations In addition, differentiated Th17 cells are characterized by the secretion of IL-17.

Unlike Treg cells, Th17 cells have been found to be involved in the forefront of inflammatory reactions seen in immune diseases, maximizing the signal of inflammatory responses and accelerating disease progression. Therefore, in the case of autoimmune diseases which are not controlled by Treg cells among autoimmune diseases, development of therapeutic agents for autoimmune diseases that target the inhibition of Th17 cell activity has been highlighted.

On the other hand, STAT (Signal transducers and activators of transcription) is a signal transduction and transcription regulator protein, is activated by extracellular stimulation of cytokines, hormones, growth factors, etc., phosphorylation of tyrosine residues, dimers by the interaction of the SH2 domain A dimer is formed that enters the nucleus and binds to a specific promoter. The signaling system of these STAT proteins can be inhibited by dephosphorylation and protein degradation.

In particular, recently, active forms of STAT1, STAT3 and STAT5 have been found in various carcinomas. STAT3 is not only a hematological cancer such as leukemia, but also breast cancer, head and neck cancer, melanoma, ovarian cancer, lung cancer, pancreatic cancer and prostate cancer. It is active in various solid cancers and has become an important anticancer target (Hua Yu and Richard Jove, Nature Review Cancer., 2004, 8, 945).

In addition, the activity of STAT3 has been known to inhibit apoptosis, induce angiogenesis, and induce immune evasion (Wang T. et al., Nature Medicine., 2004, 10, 48). Inhibition of STAT3 activity is a complex anticancer mechanism that can control tumors, and since STAT3 protein is involved in various intracellular functions as well as tumors, its inhibitor discovery can be developed as an immunosuppressive agent.

In general, the immune system controls specific immune responses to autoantigens in a normal state, and also suppresses immune responses to external antigens. For example, a pregnant woman's response to an unborn baby and a chronically infected microorganism Reaction. These phenomena are known to be induced by clonal deletion, clone anergy and active control by immunoregulatory T cells (Treg) as a mechanism by which antigen specific immunotolerance can be induced. Investigating some patients who accidentally acquired immunotolerance against transplanted antigens or experimentally induced animal models showed that all three of these mechanisms are involved in immunological tolerance. Is attracting attention as an important cell involved in controlling almost all immune responses of living body such as autoimmune, tumoral immunity, infectious immune response as well as transplantation immune response.

In particular, immunoregulatory T cells, ie immunoregulatory T lymphocytes (Tregs), whose presence has recently been identified, can be largely divided into natural and adaptive Treg cells, and CD4 + CD25 + T cells, which are natural Tregs, are cells. Is newly immunized from the thymus and is present at 5-10% of the peripheral CD4 + T lymphocytes in normal individuals. The mechanism of immunosuppression of this cell is not yet known, but it has recently been discovered that the expression control factor of the gene, Foxp3, plays an important role in the differentiation and activity of the cell. In addition, peripheral natural T cells can be differentiated into cells that exhibit immunosuppressive effects upon stimulation of autologous or external antigens under certain circumstances, which are called adaptive or inducible Tregs and secrete IL-10. These include Tr1, Th3 and CD8 Ts that secrete TGF-β.

In addition, T cells are differentiated into Th17 cells through differentiation in addition to Treg cells. Th17 cells are formed in the presence of TGF-β in common with Treg cells, but Treg cells do not require IL-6, Th17 cells are characterized by differentiating in the presence of IL-6 with TGF-β and secreting IL-17.

Th17 cells are characterized by having cytotoxicity that maximizes the signal of the inflammatory response and accelerates disease progression. Therefore, inhibition of differentiation or activity into Th17 cells is one of the ways to treat immune diseases.

Therefore, DUSP5 of the present invention can prevent and treat not only bone metabolic diseases, but also immune diseases, and can also prevent and treat diseases caused by STAT3 mediation.

STAT3-mediated disease refers to a disease caused by activation of the STAT3 pathway, which is known in the art to indicate that overexpression, hypersecretion or overactivity of interleukin-1β and / or interleukin-6 induces activation of the STAT3 pathway. It is known that interleukin-1β and / or interleukin-6 are known to increase in expression, secretion or activity by inflammatory diseases, autoimmune diseases, destructive bone diseases, infectious diseases, degenerative diseases and necrotic diseases, etc. It is known to be due to STAT3 pathway activation induced by interleukin-1β and / or interleukin-6. Therefore, in the development of a therapeutic agent for the disease, it is obvious that the substance that inhibits the activation of STAT3 is one of the methods for treating these diseases.

Thus, non-limiting examples of STAT3-mediated diseases that can be prevented or treated by the compositions of the present invention include arthritis, peritonitis, multiple sclerosis, psoriasis, asthma, edema, rheumatoid arthritis, degenerative arthritis, bronchial spasms, allergies, inflammatory diseases, Autoimmune diseases, destructive bone diseases, infectious diseases, degenerative diseases, necrotic diseases and inflammatory periodontal diseases.

In particular, it was confirmed that DUSP5 of the present invention inhibits activity through the inhibition of phosphorylation of STAT3. Thus, the present invention provides an expression vector comprising a DUSP5 nucleotide sequence of SEQ ID NO: 2 operably linked thereto to a cell in vitro. There is also a feature that can provide a method of reducing the activity or expression of STAT3 in vitro, comprising transforming a cell to express the DUSP5 gene.

DUSP5, an active ingredient contained in the composition provided in the present invention, may be a functional equivalent to a polypeptide having an amino acid sequence represented by SEQ ID NO: 1. The term 'functional equivalent' refers to a sequence homology with at least 60%, preferably 70%, more preferably 80% or more of the amino acid sequence represented by SEQ ID NO. 1 as a result of the addition, substitution or deletion of amino acids. It refers to a polypeptide that exhibits substantially homogeneous activity with DUSP5 of the present invention. Here, "substantially homogeneous activity" refers to the activity of DUSP5 described above. Such functional equivalents may include, for example, amino acid sequence variants in which some of the amino acids of the amino acid sequence represented by SEQ ID NO: 1 are substituted, deleted or added. Substitution of amino acids may preferably be conservative substitutions, examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids may preferably be located at portions not directly involved in the activity of DUSP5 of the invention. The functional equivalent may also include polypeptide derivatives in which some chemical structures of the polypeptide are modified while maintaining the basic backbone of DUSP5 and its physiological activity. For example, fusion proteins made by fusion with other proteins while maintaining structural changes and physiological activities for altering the stability, storage, volatility or solubility of the polypeptide of the present invention may be included.

DUSP5 protein can also be obtained using genetic recombination techniques. When using a recombinant technique, a polynucleotide encoding a DUSP5 protein is inserted into an appropriate expression vector, the vector is transformed into a host cell, the host cell is cultured so that the DUSP5 protein is expressed, and the DUSP5 protein is recovered from the host cell. It can be obtained by the process. Proteins are expressed in selected host cells and then subjected to conventional biochemical separation techniques, such as treatment with protein precipitants (salting), centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography for isolation and purification. (Gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like can be used. In general, these are used in combination to separate proteins of high purity.

Accordingly, the present invention can provide a pharmaceutical composition comprising a vector comprising a promoter and a polynucleotide encoding DUSP5 operably linked thereto.

In the present invention, the polynucleotide encoding the DUSP5 may be isolated or artificially modified in nature, the base sequence encoding the DUSP5 protein may be modified by substitution, deletion or insertion of one or more nucleic acid bases, such The protein expressed by the modification should not contain significant changes in its biological functionality. Such modifications include modifications to heterologous homologous genes.

Therefore, the polynucleotide encoding the DUSP5 protein is preferably characterized by being represented by the nucleotide sequence of SEQ ID NO: 2, but not limited to the above polynucleotide, that is, the nucleic acid sequence and 70% or more, preferably 80% or more More preferably, it is represented by the base sequence which has 90% or more homology.

Preferably, the polynucleotide encoding the DUSP5 protein of the present invention may be provided in the form of a recombinant expression vector operably linked to the vector expressing it. Expression vectors comprising DUSP5 polynucleotides include, but are not limited to, plasmids, phages, cosmids, viral vectors or other mediators known in the art. Vectors can self replicate or integrate into host DNA.

Polynucleotides encoding the DUSP5 protein can be combined with expression control sequences such as promoter / enhancer sequences and other sequences required for transcription, translation or processing. Regulatory sequences include tissue-specific regulatory and / or inducible sequences as well as direct constitutive expression of nucleotides. The design of the expression vector can be determined by factors such as the host cell to be transfected, the level of expression desired, and the like.

As the expression vector expressing the DUSP5 protein included in the composition of the present invention, a non-viral vector or a viral vector may be used. Viral vectors include, for example, replication defective retroviruses, adenoviruses, adenovirus-associated viruses, and the like. Viral vectors must meet the following criteria: (1) be able to infect the cells of interest, and thus a viral vector with an appropriate host range must be selected, and (2) the delivered genes will be preserved in the cells for an appropriate period of time. Must be able to be expressed and expressed, and (3) the vector must be safe for the host. Other viral vectors that can be used for gene transfer into cells include murine leukemia virus (MLV), JC, SV40, polyoma, Epstein-Barr virus papilloma virus, vaccinia, poliovirus, herpes virus, sindbis virus, lenti Viruses, and other human and animal viruses.

The expression vector according to the present invention can be introduced into cells using methods known in the art. For example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran- DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene guns and other known methods for introducing nucleic acids into cells Can be introduced into cells by the method of (Wu et al., J. Bio. Chem., 267: 963-967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988). .

In addition, in the present invention, the meaning of including the polynucleotide encoding the DUSP5 protein as an active ingredient includes the polynucleotide itself encoding the DUSP5 protein as an active ingredient, and the expression vector into which the polynucleotide encoding the DUSP5 protein is inserted. Means to include as an active ingredient.

In addition, unless otherwise indicated, the term 'treatment' reverses, alleviates, inhibits, or prevents the disease or condition to which the term applies, or one or more symptoms of the disease or condition. As used herein, the term 'treatment' refers to the act of treating when 'treating' is defined as above. Thus, 'treatment' or 'therapy' of an immune disease in a mammal may include one or more of the following:

(1) inhibits the growth of the target disease, ie inhibits its development,

(2) preventing the spread of the target disease, ie preventing metastasis,

(3) Alleviate the target disease.

(4) prevent the recurrence of the target disease, and

(5) alleviating the symptoms of the target disease (palliating)

The composition according to the invention may comprise a pharmaceutically effective amount of DUSP5 protein alone or may comprise one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat the symptoms of an immune disease.

The pharmaceutically effective amount of DUSP5 protein according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed according to the degree of symptoms of immune disease, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.

In addition, the pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorders, dizziness or the like when administered to humans. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

In addition, the composition according to the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient is determined by the route of administration, the age, sex, weight and severity of the patient. It may be appropriately selected depending on several factors, and the composition according to the present invention may be administered in parallel with known compounds having the effect of preventing, ameliorating or treating the symptoms of the disease.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Example 1>

Treatment Effect of Rheumatoid Arthritis by Dusp5

The present inventors prepared a collagen-induced arthritis mouse model to determine whether Dusp5 has a therapeutic effect on rheumatoid arthritis and performed the following experiment. At this time, the weight (weight) of each mouse used in the experiment was used a mouse 300g.

<1-1> Experimental Animal Preparation and Arthritis Index Evaluation

The test animals were 6-week-old male DBA / 1J mice, and to prepare an arthritis animal model, type 2 collagen (Cll) was dissolved in 0.1 N acetic acid solution to 4 mg / ml, followed by dialysis buffer. , 50mM Tris, 0.2N Nacl) and mixed in equal amounts with complete Freund's adjuvant (CFA, Chondrex) containing M. tuberculosis and subcutaneously injected at the base of the tail of the mouse to obtain 100 μl of immunogen per animal. (Ie 100 μl / 100 μg). Two weeks later, the same CII was mixed with the same amount of incomplete Freud's adjuvant (IFA, Chondrex) and 100 μl (ie 100 μl / 100 μg) was injected into one hind limb (foot pad).

   Arthritis was induced by intradermal injection of collagen type 2 into DBA1 / J mice, and 100 days after the induction of arthritis, 100 μg of pcDNA-DUSP5 containing the DUSP5 gene and a MOCK vector were injected intravenously with a control group. . After the induction of arthritis, the arthritis index was evaluated for 18 weeks, and the arthritis index was evaluated from 1 to 4 points.

-Evaluation standard-

0 points: There is no edema or swelling.

1 point: mild edema and redness limited to the foot or ankle joint

2 points: Mild swelling and redness from the ankle joint to the metatarsal

3 points: moderate swelling and redness from the ankle joint to the ankle bone

4 points: swelling and redness from ankle to leg

The best arthritis index per head is 4, so the best disease index per mouse is 16.

In addition, the preparation of the pcDNA-DUSP5 vector containing the DUSP5 gene was cut into DUSP5 cDNA clone (GenBank No .: NM_001085390.1) with restriction enzymes HindIII and Xho 1 and inserted into pcDNA3.1 + (Invtrogen) to insert DUSP5 cDNA. Generated recombinant vector (pcDNA-DUSP5), transformed E. coli with the recombinant vector, isolate plasmid DNA with plasmid extraction kit (Qiagan), and analyze the nucleotide sequence of the recombinant gene. The correct recombinant plasmid vector was selected and then cultured to obtain a large amount of plasmid vector DNA.

<1-2> Arthritis destruction inhibition and inflammation inhibition effect analysis

In order to confirm whether DUSP5 has an effect of inhibiting joint destruction and inflammatory cells in the joint tissues in the mouse group used in the experiment of <1-1>, after euthanizing each mouse group, the hind paw of the mouse was 10%. After fixing with formalin, sufficiently fixed tissues were washed with water to remove an excessive amount of fixative reagent, and the tissues were dehydrated using graded alcohol. After descaling the bones, they were buried with paraffin that did not mix with water. Paraffin dissolved in benzene, an organic solvent, was penetrated into the dehydrated tissue, and then completely penetrated by treatment with pure paraffin in liquid state at high temperature (60 ° C.). Paraffin containing tissues were cooled to low temperature to a solid state and then trimmed to a suitable size. Joint sections (5 μm) were prepared and placed on a glass slide treated with adhesive, and paraffin was removed with an organic solvent such as xylene. Since most dyes are aqueous solutions, the tissue-adhered slides were hydrated from a high concentration of alcohol to a low concentration of alcohol solution, and then stained first with an aqueous solution of hematoxylin, followed by secondary dyeing with eosin. The dyes were stained deeply and then subjected to a differentiation process to suitably match the degree of staining of samples overly stained with alcohol solution. In addition, histological examination was performed by toluidine blue and safranin O staining to confirm the degree of cartilage destruction.

In addition, immunohistochemical staining was performed to analyze the effects of VEGF and MMP9 on inflammatory cells such as IL-1β, IL-6, and TNF-α and neovascularization factors in arthritis animal models. Histochemical staining was performed using the Vectastain ABC kit. That is, the primary antibody is reacted at 12 ° C. or more at 12 ° C., washed with PBS, reacted with biotin-conjugated secondary antibody (antirabbit IgG antibody) for 20 minutes, washed with PBS, and then reacted with streptatadine solution containing peroxidase. After reacting for 20 minutes, it was washed with water and developed using DAB chromogen. After counterstaining with Mayer's hematoxylin, the cells were washed with distilled water and embedded with an aqueous adhesive, and the expression levels of IL-1β, IL-6, TNF-α, VEGF and MMP9 were observed by light microscopy. As a control group, a group reacted with PBS instead of the primary antibody was used.

In addition, the expression level analysis of inflammatory cytokines and angiogenesis factors was performed in addition to immunohistochemical staining, mRNA expression levels were analyzed. The cDNAs were isolated from the masons used in the above experiments, and the expression levels of each gene described below were measured. Using the primers, the mRNA expression level of each gene was measured by realtime PCR.

Primer sequence Primer Name Primer sequence (5'-3 ') IL-1β forward GGA TGA GGA CAT GAG CAC ATT C IL-1β reverse GGA AGA CAG GCT TGT GCT CTG A IL-6 forward ATG CTC CCT GAA TGA TCA CC IL-6 reverse TTC TTT GCA AAC AGC ACA GC TNF-α forward ATG AGC ACA GAA AGC ATG ATC TNF-α reverse TAC AGG CTT GTC ACT CGA ATT VEGF forward TCT TCA AGC CGT CCT GTG TG VEGF reverse AGG ACC ATT TAC ACG TCT GC MMP9 forward CTG TCC AGA CCA AGG GTA CAG CCT MMP9 reverse GAG GTA TAG TGG GAC ACA TAG TGG β-actin forward GAA ATC GTG CGT GAC ATC AAA G β-actin reverse TGT AGT TTC ATG GAT GCC ACA G

<1-3> Inhibition analysis of inflammatory autoantibody production

In order to determine whether DUSP5 inhibits the production of inflammatory autoantibodies in the arthritis biological model, after injecting a vector expressing DUSP5 into the arthritis-induced mouse group, the blood was collected from each mouse and centrifuged. Serum was separated by using a total IgG and CII specific IgG and IgG2a antibody was performed by enzyme-linked immunoassay (ELISA). It is generally known that when arthritis develops, the overall IgG in the serum is increased and the IgG2a is specifically increased. Therefore, total IgG and antibody specific total IgG, IgG2a, and IgG1 were measured by using a sandwich ELISA of serum of each experimental group. Each monoclonal anti-mouse IgG and CII were reacted at room temperature for 1 hour in a 96 well plate, and then non-specific binding was blocked with a blocking solution (1% BSA / PBST). Mouse contorl serum was serially diluted by 1/2 and used as a standard. The cell culture supernatant was added and reacted at room temperature for 1 hour. Thereafter, anti-mouse IgG-HRP, anti-mouse IgG1-HRP, and anti-mouse IgG2a-HRP were allowed to react at room temperature for 1 hour and washed four times, followed by color development using a TMB system.

<1-4> analysis results

As a result of the experiments as described in <1-1> to <1-3>, first, in the arthritis index analysis, as shown in FIG. 1A, the arthritis index in the arthritis mouse group injected with the recombinant vector overexpressing DUSP5 is MOCK. Arthritis onset analysis results showed that the group of mice injected with the recombinant vector overexpressing DUSP5 significantly inhibited the arthritis onset index compared to the control group injected with the MOCK vector. The results showed that arthritis is rarely developed.

In addition, the safranin O and toluidine blue methods showed a significant decrease in the index of inflammation and joint destruction in the group of mice injected with recombinant vectors overexpressing DUSP5 compared to the control group. It was shown to have about 3-fold inhibition effect (see FIG. 1B).

 In addition, analysis of the effect of DUSP5 on the production of autoantibodies showed that the amount of immunoglobulin in serum was reduced in the group of mice injected with DUSP5, and CII-IgG specific for the type 2 collagen, an arthritis autoantigen. The amounts of, CII-IgG1 and CII-IgG2a were also shown to be significantly inhibited (see FIG. 1C).

Furthermore, as a result of analyzing whether DUSP5 also affects the production of inflammatory factors IL-1β, IL-6, TNF-α and VEGF and MMP9 in the joints of the animal model of arthritis, as shown in FIG. , IL-1β, IL-6, inflammatory cytokines of TNF-α and VEGF and MMP9 all showed a significant inhibitory effect in the group of mice injected with DUSP5 compared to the control group (see FIGS. 2A and 2B). This inhibition was found to result from inhibition of gene expression (see FIG. 2C).

Therefore, through these results, the present inventors can not only suppress the occurrence of arthritis by DUSP5 of the present invention, but also inhibit the destruction of joints, the infiltration of inflammatory cells in joints, the expression of angiogenesis-related factors and the production of inflammatory cytokines. Inflammatory antibodies and inflammatory autoantibodies specific to the CII antigen were also found to have a selective and effective inhibitory activity.

<Example 2>

Co-regulation of Th17 and Tregs by Dusp5

<2-1> T cell isolation

The cells were isolated from the mice used in the above experiments. After the first immunization with CII and the mice were killed 56 days later, the cells and tissues of the spleen and the lymph node were separated. At this time, the experimental group was used as a mouse group injected with a vector expressing DUSP5, the control group was used as a group injected with a MOCK vector.

<2-2> Analysis through flow cytometer

After collecting the cells prepared in the above (2-1) (splenic and lymph node cells) and washing the cells with FACs buffer for flow cytometry used in the art, blocking for 15 minutes at 4 ℃ to inhibit non-specific binding After the reaction was washed with perm wash buffer. Anti Foxp3-FITC and anti IL-17 PE were added thereto, reacted at 4 ° C. for 30 minutes, and washed with perm wash buffer. After staining the cells were washed with FACs buffer, flow cytometry (FACs, fluorescent-activated cell sorter) was used to analyze the expression of IL-17 cytokines and Foxp3 expression by DUSP5 overexpression.

<2-3> Confocal Microscopy Analysis

In addition, for the analysis of Th17 and Treg cell numbers according to DUSP5 overexpression, an optimal cutting temperature compound (OCT compound) was embedded using the spleen and lymph nodes of the mouse obtained by the above method, and then tissue was rapidly liquefied in liquefied nitrogen. Cool and attach to slides 7 μm thick using a frozen slicer. Sections were then fixed with acetone and 10% normal goat serum was applied to block nonspecific reactions for 30 minutes. In the Treg assay, the primary antibody FITC-labeled anti-mFoxp3 Ab, PE-labeled anti-mCD4 Ab, APC-labeled anti-CD25 Ab diluted 1: 100 in PBS (pH7.5) and FITC for Th17 cell analysis. Reaction with -labeled anti-mCD4 Ab, PE-labeled anti-mIL-17 at 4 ° C. overnight, washed with PBS solution the next day, and stained tissues were analyzed by confocal microscopy.

<2-4> RT-PCR Analysis

In order to analyze the simultaneous regulation of T h 17 and Tregs according to DUSP5 overexpression at the gene expression level, gene expression levels of factors related to TH17 cell differentiation and factors related to Treg cell differentiation were analyzed by RT-PCR. Each cell for analysis was using the cell group used for the flow cytometry, and after obtaining the total RNA from these cell groups, RT-PCR was performed using the primers in the table below.

Primer sequence Primer Name Primer sequence (5'-3 ') IL-17 forward CCT CAA AGC TCA GCG TGT CC IL-17 reverse GAG CTC ACT TTT GCG CCA AG IL-21 forward CCC TTG TCT GTC TGG TAG TCA TC IL-21 reverse ATC ACA GGA AGG GCA TTT AGC IRF4 forward GCA GCT CAC TTT GGA TGA CA IRF4 reverse CCA AAC GTC ACA GGA CAT TG AHR Forward AGC AGC TGT GTC AGA TGG TG AHR reverse CTG AGC AGT CCC CTG TAA GC RoRrt forward TGT CCT GGG CTA CCC TAC TG RoRrt reverse GTG CAG GAG TAG GCC ACA TT Foxp3 forward GGC CCT TCT CCA GGA CAG A Foxp3 reverse GCT GAT CAT GGC TGG GTT GT

<2-5> Analysis result

As a result of analysis using a flow cytometer, as shown in FIG. 3A, in the group in which DUSP5 was injected into the arthritis-induced mice, the number of TH17 cell groups in the spleen and lymph nodes was decreased in comparison with the control group without the DUSP5 injection. (Reduced from 4.83 to 3.10 for splenocytes and decreased from 3.30 to 1.21 for lymph nodes), whereas the number of Foxp3-expressing Treg cell groups increased in the DUSP5-injected group compared to the control group. Spleen cells increased from 7.76 to 11.06 and lymph nodes increased from 8.43 to 11.04.

As a result of confocal microscopy, as shown in FIG. 3B, the TH17 cell group of IL-17 + was more than doubled in the experimental group injected with DUSP5 compared to the control group, and Foxp3-expressing Treg cell group was 1.6 times. It was found to increase by a factor of 2.

These results were similarly confirmed by RT-PCR results. Gene expression of IL-17, IL-21, IRF4, AHR, and RORrt, which are factors related to TH17 cell differentiation, were all effectively suppressed by overexpression of DUSP5. On the other hand, the expression of Foxp3 was increased (see Figure 3c).

Therefore, the present inventors were able to confirm that DUSP5 of the present invention has an action of inhibiting the differentiation and activity of TH17 cells and at the same time promoting the differentiation and activity of Treg cells, and this action is TH17 / Treg cell differentiation. It was found that it is through the regulation of the gene associated with.

<Example 3>

Effect of Dusp5 on ERK, Th17 and Treg Regulation in Vitro

In Example 2 described above, the effect of THD / Treg cell differentiation in the mouse was injected by injecting the DUSP5 expression vector into the disease mouse model. In the following experiment, the activity was reproved through in vitro experiments.

For this purpose, CD4 ⁺ T cell separation from spleen and lymph node cells was first performed by spleen and lymph node cells with CD4 coated microbeads at 4 ° C. for 15 minutes. After the reaction, washing with MACs buffer, CD4 ⁺ T cells were isolated. Isolated CD4 ⁺ T cells were washed with PBS and cell cultures containing 10% fetal calf serum, penicillin (100 U / mL) and streptomycin (100 g / mL) inactivated at 55 ° C. for 30 minutes (RPMI1640 medium). , Gibco BRL, USA). Then, for differentiation from isolated CD4 ⁺ T cells to Th 17 cells, the cells were seeded in a 24-well plate coated with 1 μg / mL of anti-CD3 antibody to 1 × 10 ⁶ cells, and Th17 cells were seeded. After treatment with anti-CD28 antibody 1μg / mL, TGF-β 2ng / ml, IL-6 20ng / ml, anti-IL-4 10ng / ml, anti-IFNr 10ng / ml Cells were cultured for 3 days to induce differentiation into Th17 cells.

<3-1> Analysis method using RT-PCR

Cells for in vitro experiments were isolated from CD4 T cells from spleens of 35-day-old mice after the first immunization with the CII, and the cells were transduced with pcDNA-DUSP5 or MOCK vector and then 4 hours later. The cells were treated with 1 μg / mL of anti-CD28 antibody, 2 μg of TGF-β, 20 ng / ml of IL-6, 10 μg / ml of anti-IL-4, and 10 μg / ml of anti-IFNr. After incubation, cells were collected and subjected to the RT-PCR method described above to analyze mRNA levels of IL-17, Foxp3 and ERK2 following DUSP5 overexpression under Th17 differentiation conditions.

As a result, as shown in FIG. 4A, the expression of IL-17 and ERK2 was decreased when DUSP5 was overexpressed, whereas the expression of Foxp3 was significantly increased.

<3-2> Analysis method using ELISA

In addition, the present inventors collected the culture supernatant of the cells used in the above <3-1> and confirmed that the supernatant was monoclonal anti-IL-17, anti-inhibition to determine whether the production of inflammatory cytokines is inhibited by DUSP5 in vitro -TNF-a and anti-IL-21 were reacted at 4 ° C. overnight at 2 μg / mL, respectively, followed by blocking nonspecific binding with blocking solution (1% BSA / PBST). Thereafter, the biotinylated anti-IL-17, anti-TNF-a and anti-IL-21 were reacted at room temperature for 2 hours, washed four times, and then diluted with an ExtraAvidin-Alkaline Phosphatase conjugate and added for 2 hours at room temperature. Reacted. Thereafter, PNPP / DEA solution was added, followed by color development, and absorbance at 405 nm was measured.

As a result, as shown in Figure 4b, all of the inflammatory cytokines IL-17, TNF-a and IL-21 appeared to be inhibited by DUSP5, especially IL-17 was found to have excellent inhibitory effect.

<3-3> Analysis of IL-17, ERK2 and Foxp3 Expression Change by siRNA Treatment

In <3-1>, we analyzed the changes of IL-17, ERK2 and Foxp3 expression according to overexpression of DUSP5. This time, siRNA (Dharmacon, DUSP5) for DUSP5 was detected in CD4 T cells isolated from spleen in <3-1>. siGENOME SMARTpool (M-057231-01-0010), 19 and siGENOME non-targeting siRNA pool (D-001810-01-20) were treated to inhibit the expression of DUSP5 in cells. After 4 hours of incubation with siRNA, the cells were incubated for 20 hours under the Th17 differentiation condition of <3-1>, and the cells were collected and subjected to the RT-PCR method described above to perform IL-17, Foxp3 and MRNA levels of ERK2 were analyzed.

As shown in FIG. 4C, Foxp3 expression was significantly suppressed in Th17-differentiated cells while DUSP5 expression was suppressed, and mRNA levels of IL-17 and ERK2 were increased.

<3-4> Analysis of expression changes of inflammatory cytokines by siRNA treatment

Collect the cell culture solution of each experimental cell group used in the above <3-3> and IL-17, TNF-a and IL- in the state where the expression of DUSP5 is suppressed using the ELISA assay described in <3-2>. The degree of production of 21 was analyzed.

As a result, as shown in Figure 4d, when the expression of DUSP5 is suppressed, the production of inflammatory cytokines IL-17, TNF-a and IL-21 all increased in Th17 differentiated cells.

<Example 4>

Analysis of the effect of Dusp5 on STAT3 and STAT5

The inventors performed the following experiment to determine whether DUSP5 of the present invention also affects the activity of STAT3 and STAT5. First, in Example 1, the group was first immunized with CII and the mice injected with the DUSP5 expression vector and the mice injected with the MOCK vector were killed, and the spleen was separated, and then the degree of phosphorylation of STAT3 and STAT5 by DUSP5 in the tissues was determined. Observation was made through confocal microscopy and Western blot.

First, as in the method used in the above <2-3> for confocal microscopy analysis, the mouse joints are separated and prepared as an Optimal Cutting Temperature compound (OCT compound), and then embedded in the tissue, and then the tissue is removed from the liquid nitrogen. Rapid cooling and attached to slides 7 μm thick using a frozen slicer. Sections were then fixed with acetone and 10% normal goat serum was applied to block nonspecific reactions for 30 minutes. And PE-labeled anti-STAT3 Tyr 705 (pSTAT3 ^{Tyr 705} ), PE-labeled anti-STAT3 Ser 727 (pSTAT3 ^{Ser 727} ) and anti-STAT5 Tyr 694 (pSTAT3 ^Tyr ) to detect phosphorylated STAT3 and STAT5. ⁶⁹⁴ ) overnight at 4 ° C., washed with PBS solution the next day, and stained tissues were analyzed by confocal microscopy. In addition, the same experiment was performed using PE-labeled anti-ERK antibody to analyze the degree of activation of ERK in the same manner.

In addition, these results were confirmed by Western blot, which was analyzed using a conventional Western blot method, wherein each antibody had a pSTAT3 ^{Tyr. 705} (1: 200), pSTAT3 ^{Ser 727} (1: 200) and pSTAT5 ^{Tyr 694} (1: 200) were purchased from Cell signaling.

As shown in FIG. 5, the number of CD4 + pSTAT3 (Tyr705) + T cells and CD4 + pSTAT3 (Ser727) + T cells was decreased in the group injected with DUSP5, compared to the control group without injection. + pSTAT5 (Tyr694) + T cells were found to increase (FIG. 5A). These results also showed the same trend in Western blot results, DUSP5 showed a decrease in the phosphorylation of STAT3, STAT3 phosphorylation was shown to increase (see Figure 5b).

In addition, it can be seen from the results of FIG. 5c that DUSP5 of the present invention plays a role of reducing CD4 + ERK + cell number and also phosphorylation of ERK.

Therefore, based on these results, the present inventors found that DUSP5 also influences the activity of STAT3 and STAT5, and that DUSP5 affects the differentiation of TH17 and Treg through the regulation of STAT3 and STAT5 activity, in particular DUSP5. It was found that the activity of STAT3 and STAT5 by the opposite results.

As a result, the present inventors have confirmed that DUSP5 can simultaneously regulate TH17 / Treg by regulating the activity of ERK and transcription factors that regulate TH17 / Treg cells.

Example 5

Inhibitory Effect of Dusp5 on Osteoclast Differentiation

<5-1> RANKL, RANK, NFATc1 and TRAP Analysis

In Example 1, primary immunization with CII and the injection of DUSP5 expression vector were performed 56 days later, and mice were killed to obtain bone tissue and bone marrow cells of the mice. In this case, a group of MOCK vector injected instead of DUSP5 expression vector was used. The obtained joint tissues were measured by immunohistochemical staining to confirm the expression level of RANKL, RANK, and NFATc1, which induce osteoclasts. For this purpose, the Vectastain ABC kit was used, and primary antibodies (antibodies against RANKL, RANK, and NFATc1) were reacted at 4 ° C. for at least 12 hours, washed with PBS, and reacted with a biotin-bound secondary antibody (antirabbit IgG antibody) for 20 minutes. After washing with PBS, the solution of streptavidin bound to peroxidase was further reacted for 20 minutes, washed again, and then developed using DAB chromogen.

As a result, as shown in FIG. 6A, the joints of the collagen-induced arthritis disease animal model showed increased expression of RANKL, RANK, and NFATc1 markers of osteoclasts, whereas in the joints of mice injected with DUSP5 The expression of cell markers was shown to be markedly reduced.

In addition, the present inventors obtained bone marrow cells from the mouse group used in the experiment, and obtained monocytes from the bone marrow cells, and then, bone marrow of the animal model injected with the DUSP5 vector was treated with RANKL (50ng / ml) and M-CSF (10ng / ml) After stimulation, TRAP-positive cells, osteoclast factors, were observed by TRAP staining using a commercial kit.

As a result of the analysis, as shown in Figure 6b, the DUSP5-treated group showed a decrease in the number of TRAP positive marker cells and the expression level of TRAP compared to the non-treated group.

<5-2> Analysis of osteoclast differentiation inhibitory effect by DUSP5 in vitro

In addition, the present inventors were able to confirm that DUSP5 of the present invention has an activity for inhibiting osteoclast differentiation through the experiment of <5-1>. In vitro experiments confirmed the inhibitory activity of osteoclast differentiation as follows, ie for this purpose, the cells were stimulated with RANKL (50ng / ml) in RAW264.7 cell line, and then pcDNA- DUSP5 vector and MOCK vector (control) were injected, and the stimulated cells were treated with siRNA and scrambled siRNA (control) for DUSP5, respectively, and then cultured these cell lines for 72 hours. After that, gene expression levels of DUSP5, Cathepsin K, integrin beta3, RANK, and TRAP were analyzed by RT-PCR.

Primer sequence Primer Name Primer sequence (5'-3 ') Cathepsin K Forward CAG CAG AGG TGT GTA CTA TG Cathepsin K Reverse GCG TTG TTC TTA CGA GC Integrin-β forward CTG TGG GCT TTA AGG ACA GC Integrin-β reverse GAG GGT CGG TAA TCC TCC TC RANK forward CGA GGA AGA TTC CCA CAG AG RANK reverse CAG TGA AGT CAC AGC CCT CA TRAP forward TCC TGG CTC AAA AAG CAG TT TRAP reverse ACA TAG CCC ACA CCG TTC TC

As shown in FIG. 6C, the gene expression of the osteoclast factors Cathepsin K, integrin beta3, RANK, and TRAP was increased when the siRNA for DUSP5 was inhibited in the cells. In the case of overexpression of DUSP5 in RAW264.7 cells, all of these osteoclast factors were significantly inhibited in expression.

Therefore, the present inventors have found that DUSP5 of the present invention can effectively inhibit the differentiation of osteoclasts and thus can be usefully used for the treatment of bone diseases caused by osteoclast differentiation.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (15)

Pharmaceutical composition for the prevention or treatment of bone metabolic diseases, including both DUSP5 as an active ingredient. The method of claim 1, The DUSP5 is a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, characterized in that consisting of the peptide of SEQ ID NO: 1. The method of claim 2, Peptide of DUSP5 is a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, characterized in that encoded by the nucleotide sequence of SEQ ID NO: 2. The method of claim 1, The DUSP5 inhibits osteoclast differentiation by inhibiting the expression or activity of RANKL, RANK, NFATc1 and TRAP. The method of claim 1, The bone metabolic diseases include osteoprosis, paget disease, metastatic cancer or rheumatoid arthiritis, bone metastatic cancer, solid cancer bone metastasis, solid cancer bone metastasis Musculoskeletal complications, hypercalcemia due to malignant tumors, multiple myeloma, primary bone tumor, degenerative arthritis, periodontal disease, inflammatory alveolar bone disease and inflammatory bone resorption disease Pharmaceutical composition for the prophylaxis or treatment of. The method of claim 1, The DUSP5 inhibits or reduces the activity of Th17 and promotes or increases the activity of Regulatory T cells (Treg). The method of claim 1, The DUSP5 is a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, characterized in that it has an activity of inhibiting the phosphorylation of STAT3. The method of claim 1, The DUSP5 is a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, characterized in that it is contained in the composition in the form of an expression vector comprising a promoter and the DUSP5 base sequence of SEQ ID NO: 2 operably linked thereto. Pharmaceutical composition for the prevention or treatment of STAT3-mediated diseases, including both DUSP5 as an active ingredient. The method of claim 9, The STAT3-mediated disease is selected from the group consisting of arthritis, peritonitis, multiple sclerosis, psoriasis, asthma, edema, bronchial spasms, allergies, inflammatory diseases, autoimmune diseases, infectious diseases, mitochondrial related syndromes and necrotic diseases Pharmaceutical compositions for the prevention or treatment of STAT3-mediated diseases. The method of claim 9, The DUSP5 is a pharmaceutical composition for preventing or treating STAT3-mediated disease, characterized in that it has an activity of inhibiting the phosphorylation of STAT3. The method of claim 9, The DUSP5 is a pharmaceutical composition for the prevention or treatment of STAT3-mediated disease, characterized in that consisting of the peptide of SEQ ID NO: 1. The method of claim 9, The peptide of DUSP5 is a pharmaceutical composition for the prevention or treatment of STAT3-mediated disease, characterized in that encoded by the nucleotide sequence of SEQ ID NO: 2. The method of claim 9, The DUSP5 inhibits or reduces the activity of Th17, and promotes or increases the activity of Regulatory T cells (Treg), wherein the pharmaceutical composition for preventing or treating STAT3-mediated disease. Transforming the cell with an expression vector comprising a promoter and a DUSP5 nucleotide sequence of SEQ ID NO: 2 operably linked thereto to express the DUSP5 gene in vitro, thereby inducing the activity or expression of STAT3 in vitro. How to reduce.

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