KR101302483B1 - The development of allergy therapeutics targeting histone deacetylase 3 - Google Patents

Abstract

The present invention relates to a method for providing information necessary for the diagnosis of allergic diseases and a method for developing an allergic agent, and specifically, to provide information necessary for the diagnosis of allergic diseases, histone deacetyl from a mammalian cell sample. Method for measuring the expression level of oxidase-3 and selecting a substance that inhibits the expression of histone deacetylase-3 or inhibits the crosslinking of histone deacetylase-3 and FcεRIβ. It is about.
According to the present invention, the expression levels of histone deacetylase-3, DNA methyltransferase-1 (DNMT-1) or monocyte chemotactic protein-1 (MCP-1) Measurement can provide information necessary for the diagnosis of effective allergic diseases, and targeting histone deacetylase-3 can effectively develop therapeutics for allergic diseases.

Description

The development of allergy therapeutics targeting histone deacetylase 3}

The present invention relates to a method for providing information necessary for the diagnosis of allergic diseases and a method for developing an allergic agent, and specifically, to provide information necessary for the diagnosis of allergic diseases, histone deacetyl from a mammalian cell sample. Method for measuring the expression level of oxidase-3 and selecting a substance that inhibits the expression of histone deacetylase-3 or inhibits the crosslinking of histone deacetylase-3 and FcεRIβ. It is about.

With the rapid development of modern industry, allergic diseases such as asthma, allergic rhinitis and atopic dermatitis continue to increase, and currently affect 10 to 40% of the industrialized country population. In Korea, allergic patients, which were less than 1% in the 1980s, surged to more than 5% by the 2000s, and it is estimated that more than 10% of the total population including potential patients was included. In the United States, where more than 22% of the population suffers from allergic rhinitis, allergy affects not only the quality of life but also the medical and economic activities, as the economic impact of allergic diseases is estimated at $ 6,400,000,000 in the early 1990s. The direct and indirect costs for this are enormous.

Allergy refers to a phenomenon in which foreign antigens, or allergens, generally cause harmful damage to the body through immunological mechanisms. Hypersensitivity reactions to allergens include diseases such as anaphylaxis, allergic rhinitis, asthma, atopic dermatitis, insect allergies, food allergies, drug allergies and urticaria ( urticaria), Wuthrich B., 1989, Int. Arch. Allergy Appl. Immunol., 90, 3-10].

Allergic reactions are mediated by mast cells, which are widely distributed in organs of the whole body such as the skin, respiratory tract, mucous membranes of the gastrointestinal tract, around lymphatic vessels, around blood vessels, and in the brain. Allergic reactions are caused by the action of IgE on mast cells to fight allergens such as house dust mites, pollen, animal dander, mold, egg albumin, milk proteins and peanuts [Wills-Karp M., et al., 1998, Science, 282, 2258-2261,; Grunig G., et al., 1998, Science, 282, 2261-2263.

IgE is a class of antibodies that normally exist in very low concentrations in serum. IgE production concentrations with binding specificities for specific antigens in serum of allergic patients sensitive to specific antigens are present at high levels when compared to serum of normal individuals. The mechanism of immune response to the antigen leading to IgE production is as follows.

Once our body is exposed to an antigen, the antigen is first recognized by Antigen presenting cells (APCs), which then differentiate Th0 into Th2 cells by presenting an allergen in the presence of IL-4. Let's do it. These differentiated Th2 cells secrete cytokines such as IL-4, IL-5 and IL-13, which promote the development of eosinophils in the bone marrow to inflamed tissue In addition to inducing eosinophilic leukocytes, they act on B cells to induce IgE production (Wills-Karp M., et al., 1998, Science, 282, 2258-2261).

The mechanism of action produced IgE binds strongly to apoptotic cells via the high affinity IgE receptor (FcεRI) via the Fc region [Ishisaka et al., 1970, Immunochemistry 7,687-702; Metzger et al., 1986, Ann. Rev. Immunol. 4, 419-470; Kinet, 1999, Ann. Rev. Immunol. 17, 931-972]. High affinity receptor (FcεRI) for IgE is a major mediator of immediate allergic symptoms. FcεRI is found in astigmatism cells as well as many other cell types, including basophils, platelets, and antigen presenting cells, such as monocytes and dendritic cells. For reference, low affinity receptors (FcεRII or CD23) for IgE are widely expressed in B lymphocytes, macrophages, platelets and many other cell types, such as airway smooth muscle. FcεRII may play a role in feedback regulation of IgE expression and consequently FcεRII surface expression.

When IgE binds to the plaque cells via FcεRI and then is exposed again to the same antigen, the antigen binds to the IgE bound with the FcεRI of the plaque, inducing cross-linking between these receptors, and a series of signaling responses activate the plaque. . Activated mast cells are free radicals such as histamine, proteoglycan, protease, leukotrien, prostaglandin, cytokine, and free radicals (ROS). By degranulation or newly synthesized secretion by degranulation, it mediates a series of allergic reactions such as vasodilation, intestinal and bronchial smooth muscle contraction, mucus secretion and local protein secretion. Since the allergic reaction is caused by activation of mast cells such as degranulation of mast cells, a substance that inhibits activation of mast cells can be used as a therapeutic agent for allergic diseases [J. Korean Soc. Appl. Biol. Chem., 2005, 48, 315-321.

IgE-mediated inflammatory reactions are immediate hypersensitivity reactions that cause degranulation of apoptotic cells within minutes of antigen binding to IgE antibodies, releasing a performance mediator such as histamine. The maximum intensity of this reaction occurs within 15 minutes after initial antigen exposure, accompanied by rash and pruritus (itch) erythema, focal edema, etc. Granule content induces local expression of Vascular Cell Adhesion Molecule (VCAM-1) to assist vascular permeation, and other chemokines with chemotactic activity of leukocytes and fibroblasts ultimately inflame the site Cause. The IgE-mediated immediate inflammatory response is specifically called type 1 hypersensitivity.

These IgE-mediated inflammatory responses include atopic allergies (eg fever, asthma and atopic dermatitis), systemic anaphylaxis reactions and allergic dimples (ticks), which cause rapid vasodilation and circulate solubility as described above. It can generally play a role as the first line of immune defense because it facilitates entry of factors and cells into antigenic contact sites. Approximately 6 to 24 hours after the initial mediated response of the apoptotic cells, acidic leukemia and infiltration of basic leukocytes and lymphocytes are issued, leading to chronic inflammation of tissues exposed to the antigen. This late phase immune response is specifically called type 4 hypersensitivity and is caused by a number of active factors released from sensitized T lymphocytes by the reaction of sensitized T lymphocytes with antigens. Clinical features include erythema, induration, warmth, pruritus, and burning sensation at the site of infection.

Regulation of expression of certain genes is regulated by the ability of transcription factors to access DNA, with stronger binding between DNA and histones restricting access by transcription factors and thus inhibiting gene transcription [Morrison et al, 2007, Cell Mol. Life Sci., 64, 22582269. Modification of histones or DNA by methyl group, phosphate group, or acetyl group covalent addition or the like changes their binding strength and consequently affects DNA transcription.

Methylation of DNA and acetylation of histones are the main forms of epigenetic regulation that inhibit gene expression through modification of chromatin structure [Herman JG., 2003, N. Engl. J. Med., 349, 2042-2054], studies have reported that these epigenetic regulatory mechanisms play a direct role in tumor development and genetic disease. In particular, it has been found that DNA methylation plays an important role in the development of rheumatoid arthritis, an autoimmune disease that causes systemic chronic inflammation.The genomic DNA of T cells isolated from the rheumatoid arthritis sample is hypomethylated and DNA methyltransferase ( DNMT) activity has been reported [Richardson B. et al., 1990, Arthritis Rheum., 33, 1665-1673], and rheumatoid arthritis has been reported to be associated with DNA methylation [ Kim YI. et al., J. Lab. Clin. Med., 1996,128, 165-172.

DNA methyltransferase (DNMT) is an enzyme that adds a methyl group by covalent bond to a specific nucleotide base of a DNA molecule. In mammals, four active DNA methyltransferases, DNMT1, DNMT2, DNMT3A and DNMT3B, have been identified.

DNMT1 is the most common DNA methyltransferase and is known as an important methyltransferase in mammals, mainly responsible for methylating hemimethylated CpG di-nucleotides in the mammalian genome and maintaining the methylation pattern established at the developmental stage. DNMT3 is a type of DNA methyltransferase capable of methylating the hemi-methylated CpG moiety and the unmethylated CpG moiety at the same rate and is structurally similar to DNMT1. Recently, the correlation between the polymorphism of the DNMT3B gene and the risk of rheumatoid arthritis and the rate of joint destruction in Koreans has been reported.Based on this, the DNMT3B polymorphism has been suggested as a genetic factor for diagnosing the prognosis of rheumatoid arthritis disease. [KR Patent Application 10-2007-0102741].

In addition, many proteins are known to interact with DNA methyltransferase, which is a constituent enzyme that forms large complexes with known proteins to induce transcriptional regulation and chromatin structural modifications rather than the DNMT enzyme alone. It can be seen that one. These facts will be of great help in understanding the various roles of DNA methylation in disease and in developing new therapies to prevent or repair these defects.

Reversible acetylation of histones is a major control factor for gene expression that changes the accessibility of transcriptional factors to DNA. In normal cells, histone deacetylases (HDACs) and histone acetyltransferases (HATs) together regulate the acetylation levels of histones to balance gene expression. While HATs are transcription activators, HDACs correspond to transcription inhibition and gene silencing [Kouzarides, 1999, Curr. Opin. Genet. Dev., 9, 40-48; Johnstone RW, 2002, Nat. Rev. Drug Discov., 1, 287-299].

Human HDACs are divided into classes I, II, and III based on homology. Class I (HDAC1, 2, 3 and 8), II (HDAC4, 5, 6, 7, 9 and 10), which are considered classical HDACs, are commonly inhibited by trichostatin A (TSA). Compared to the universally expressed class I, class II is tissue limited and has high expression levels, especially in the heart, skeleton and brain. Class III HDACs are a class of NAD + dependent proteins that are not affected by TSA.

HDACs have been known to modulate the activity of non-histone proteins by modifying their acetalization levels. These include steroid receptors such as estrogen and androgen receptors [Wang et al, 2001, J. Biol. Chem., 276, 18375-18383; Gaughan et al, 2002, J. Biol. Chem., 277, 25904-25913], p53, transcription factors such as E2F [Luo et al, 2000, Nature, 408, 377-381; Ito et al, 2001, EMBO J., 19, 1176-1179; Sartorelli et al, 1999, Mol. Cell, 4, 725-734] and cytoplasmic proteins such as α-tubulin (Hubbert et al, 2002, Nature, 417, 455-458).

Such variations in epigenetic regulation are signs of many diseases, including cancer. Therefore, the fundamental mechanisms of epigenetic control in the development of drugs provided as therapeutic agents for these diseases are intensive areas of interest, and the search for epigenetic regulators has become a very promising field for drug discovery.

Chemokines are low molecular weight (8-10 kDa) and are inducible, secretory, proinflammatory, chemoattractant cytokines that play a pivotal role in the perivascular migration and accumulation of certain subpopulations of white blood cells at the site of tissue damage. Seems to do.

The monocyte chemotactic protein MCP1 (called Monocyte Chemotactic Protein-1, or CCL2), belongs to the β subfamily of chemokines, which shows the highest specificity for monocytes and macrophages, among all known chemokines, T lymphocytes, mast cells and neutrophils. Has a strong chemotactic effect on basic leukocytes [Rollins BJ, 1997, Blood 90, 909-928; M. Baggiolini M., 1998, Nature 392, 565-568].

MCP1 binds and signals through the chemokine receptor CCR2. CCR2 is expressed in many cells, including monocytes, T-cells, B-cells and basophils, resulting in the production of several inflammatory factors (active oxygen, arachidonic acid and derivatives, cytokines / chemokines) and increased phagocytosis .

MCP1 plays an important role during the inflammatory response and has been demonstrated as a new target in various lesions. Evidence of significant physiological contributions of MCP1 has been reported in patients with joint and kidney inflammatory diseases (rheumatic arthritis, lupus nephritis, diabetic nephropathy and post-transplant rejection).

Recently, MCP1 has been treated with atopic dermatosis, colitis, interstitial lung disorders, restenosis, atherosclerosis, surgical treatment (with or without inflammatory abnormalities and obvious inflammatory components of the CNS (Multiple Sclerosis, Alzheimer's Disease, HIV-related dementia)). For example, they are involved in pain and other abnormalities and pains, including angiogenesis, arterectomy, transplantation, organ and / or tissue replacement, prosthetic implants, cancer (adenoma, carcinoma and metastasis) and metabolic diseases such as insulin resistance and obesity Among the arguments.

Representative allergic agents currently commonly used include antihistamines, steroidal or nonsteroidal anti-inflammatory drugs, which are primarily for the treatment of symptoms, and are fundamental to allergies that alleviate excessive humoral immunity or inhibit IgE production. There has been no effect on treating the cause. On the other hand, clinical trials of IgE antibody (CGP-51901) and recombinant humanized monoclonal antibody demonstrated that suppression of IgE production improved the symptoms of allergy [Fahy JV. et al., Am. J. Respir. Crit. Care. Med., 1997, 155, 1828-1834], diacyl benzimidazole homologues (US Pat. No. 6,369,091) and bacterial polysaccharides (US Patent Application Publication No. 20020041885) have been identified as inhibitors of IgE synthesis and secretion.

As mentioned above, conventional methods for the treatment of allergies include systemic treatment with anti-histamines and methods for alleviating patient sensitivity, and these studies have demonstrated basic IgE-mast / Basic cell interactions or FcRI interactions. It is not based on mechanism.

The present inventors studied a protein complex that binds to the IgE high affinity receptor FcεRI based on the known mechanisms of IgE-mediated allergy and cutaneous anaphylaxis induction mechanisms. It was confirmed that the inflammatory response is mediated through a series of signals and deeply involved in allergic diseases such as negatively regulating the response of DNMT1. Accordingly, it is possible to provide information for relatively easy and accurate diagnosis of allergic diseases by confirming the expression level of related factors, including the HDAC3, and affect the expression or activity of these factors, Using methods of screening for affected substances can effectively develop new allergy medications.

Accordingly, a main object of the present invention is to provide a method for providing information for efficiently diagnosing an allergic disease.

Another object of the present invention is to provide a method for developing a therapeutic agent for an allergic disease, that is, a new target for the development of a therapeutic agent for an allergic disease.

According to one aspect of the present invention, the present invention relates to histone deacetylase-3 (HDAC3), DNA methyl transferase- from mammalian cellular samples to provide information necessary for the diagnosis of allergic diseases. A method for measuring the expression level of 1 (DNA methyltransferase-1, DNMT-1) or monocyte chemotactic protein-1 (MCP-1) is provided.

Histone deacetylase-3 may consist of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. SEQ ID NO: 1 is the amino acid sequence of histone deacetylase-3 of mouse (Mus musculus), SEQ ID NO: 3 is the amino acid sequence of histone deacetylase-3 of human (Homo sapiens). In addition, the gene encoding histone deacetylase-3 may consist of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. SEQ ID NO: 2 is a nucleotide sequence of histone deacetylase-3 gene of mouse (Mus musculus), SEQ ID NO: 4 is a nucleotide sequence of histone deacetylase-3 gene of human (Homo sapiens).

DNA methyl transferase-1 may consist of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7. SEQ ID NO: 5 is the amino acid sequence of DNA methyl transferase-1 of mouse (Mus musculus), SEQ ID NO: 7 is the amino acid sequence of DNA methyl transferase-1 of human (Homo sapiens). In addition, the gene encoding DNA methyl transferase-1 may be composed of the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 8. SEQ ID NO: 6 is a nucleotide sequence of a DNA methyl transferase-1 gene of a mouse (Mus musculus), SEQ ID NO: 8 is a nucleotide sequence of a DNA methyl transferase-1 gene of a human (Homo sapiens).

Monocyte chemotactic protein-1 may consist of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11. SEQ ID NO: 9 is the amino acid sequence of the monocyte chemotactic protein-1 of mouse (Mus musculus), SEQ ID NO: 11 is the amino acid sequence of the monocyte chemotactic protein-1 of human (Homo sapiens). In addition, the gene encoding the monocyte chemotactic protein-1 may be composed of the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 12. SEQ ID NO: 10 is the nucleotide sequence of the monocyte chemotactic protein-1 gene of mouse (Mus musculus), SEQ ID NO: 12 is the nucleotide sequence of the monocyte chemotactic protein-1 gene of human (Homo sapiens).

In the method of the present invention, the histone deacetylase-3 (HDAC3), DNA methyl transferase-1 (DNA methyltransferase) as a target for measuring the expression level for providing information necessary for the diagnosis of allergic disease -1, DNMT-1) or monocyte chemotactic protein-1 (MCP-1), one of three enzymes (protein) can be selected, and more than one to provide more accurate information It is preferable to select all three, most preferably.

Since the present invention is based on a study of the skin anaphylaxis model, the method of the present invention is most preferably used to provide information necessary for prognostic diagnosis of skin anaphylaxis among allergic diseases. It can also be used to provide anaphylactic shock and similar information for the prognosis of allergic diseases such as bronchial asthma, urticaria and hay fever.

In the method of the present invention, the cell sample is preferably a cell sample selected from tissue, serum, plasma and sugar.

In the method of the present invention, in order to measure the expression level can be used to directly determine whether the expression of the enzyme, or a method of confirming the expression of mRNA for the gene of the enzyme.

In order to directly confirm the expression of the enzyme, an antibody that specifically binds to the enzyme may be used. In the method of the present invention, the antibody specifically binds to histone deacetylase-3, DNA methyl transferase-1. Antibodies that bind specifically, and antibodies that specifically bind to monocyte chemotactic protein-1 can be used.

In order to confirm the expression of mRNA for the gene of the enzyme, a primer set that specifically binds to the gene sequence of the enzyme can be used. In the method of the present invention, specific to the base sequence of the histone deacetylase-3 gene. Primer set specifically binding to the nucleotide sequence of the DNA methyl transferase-1 gene, and primer set specifically binding to the nucleotide sequence of the monocyte chemotactic protein-1 gene.

Western blot, ELISA, radioimmunoassay, radioimmunoassay, electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS or protein chip assay using each antibody may be used to measure expression levels.

In addition, in order to measure the expression level of the histone deacetylase-3, an aptamer coated with HDAC3 may be used on the surface, and as the support, a membrane, a slide glass, or a metal body may be used. Can be.

According to another aspect of the present invention, the present invention provides a method for screening an allergic agent by targeting histone deacetylase 3.

Attempts have been made to develop a cure for a number of diseases or conditions, but many of these attempts cannot be made because the cause is unknown. Therefore, in order to develop a therapeutic agent, it is necessary to first analyze the cause of the disease or disease, and research on the factors involved therein is required. When finding a related factor, various attempts can be made to develop a new therapeutic agent for this, and therefore, it is very meaningful to provide a new target factor in developing a therapeutic agent for a disease or disorder.

The present invention provides a new factor involved in allergy not known so far, and an attempt has been made to select a substance that targets histone deacetylase 3 to affect its expression, activity or binding to other factors. If so, new allergy medications can be developed effectively.

Since there are a variety of known methods for screening for substances that inhibit or increase the expression of specific proteins, substances that inhibit or activate the activity of enzymes, or substances that interfere with the binding of proteins to DNA, RNA, etc. These may be selected and applied to the development of therapeutic agents.

Since the present invention is based on the results of the study of the skin anaphylaxis model, it is most preferable to use the screening agent for screening anaphylaxis among allergic diseases, but is not limited thereto. It can be used to screen for therapeutic agents for allergic diseases such as anaphylaxis shock and similar bronchial asthma, urticaria and hay fever, and can also be used for screening for other allergic diseases.

It is desirable to target compounds or biological molecules for the selection of therapeutic substances. The biological molecule may be a nucleic acid or a protein, the nucleic acid may be an aptamer or an interfering RNA, and the protein may be a transcription factor that binds to the promoter sequence of HDAC3 to reduce the expression level of HDAC3.

As an example for selecting therapeutic substances, a method of selecting a substance that reduces the expression level of HDAC3 among compounds or biological molecules can be used.

According to another aspect of the present invention, the present invention provides a composition for diagnosing allergic disease, comprising a marker for measuring the expression level of histone deacetylase-3 (HDAC3).

The diagnostic composition of the present invention comprises a marker for measuring the expression level of DNA methyltransferase-1 (DNA methyltransferase-1, DNMT-1); And markers for measuring expression levels of monocyte chemotactic protein-1 (MCP-1); It is preferable to further include a marker selected from among.

The marker for measuring the expression level of each enzyme may be an antibody specifically binding to each enzyme or a primer set specifically binding to the nucleotide sequence of each enzyme gene.

In the diagnostic composition of the present invention, the marker for measuring the expression level of histone deacetylase-3 is preferably an antibody specifically binding to histone deacetylase-3, and the DNA methyl transferase-1 The marker for measuring the expression level of is preferably an antibody that specifically binds to DNA methyl transferase-1, and the marker for measuring the expression level of the monocyte chemotactic protein-1 is specific for monocyte chemotactic protein-1. It is preferable that it is an antibody couple | bonded with.

According to another aspect of the invention, the present invention provides a kit for diagnosing allergic diseases comprising the composition.

The diagnostic kit of the present invention may include a buffer for reacting the marker with an enzyme or mRNA, or a tool for measuring the expression level of each enzyme or mRNA.

As described above, measuring the expression level of histone deacetylase-3, DNA methyl transferase-1 or monocyte chemotactic protein-1 according to the present invention can provide information necessary for the diagnosis of effective allergic diseases. Targeting histone deacetylase-3 can effectively develop therapeutics for allergic diseases.

A of FIG. 1 shows that after IgE sensitization, a TpCR (Triphasic passive cutaneous reaction) is induced by antigen (DNFB) stimulation, resulting in an increase in ear thickness over time.
FIG. 1B shows changes in expression of HDAC3 and DNMT1 at different times in the ear tissue of FIG. 1A.
1C shows that HDAC3 is a negative regulator in the IgE-DNFB mediated anaphylaxis reaction.
1D shows that a decrease in DNMT1 expression occurs due to HDAC3 expression in the ear tissue, and HDAC3 complexes with FcεRIβ.
FIG. 2 shows that HDAC3 expression and DNMT1 expression are induced by antigen stimulation in the ear tissue of FIG. 1C, and expression of c-Kit and Tryptase, which are marker proteins of apoptotic cells, occurs.
3A shows that HDAC3 complexes with FcεRIβ by antigen (DNP-HSA) stimulation in RBL2H3 and BMMCs cell lines.
FIG. 3B shows the results of FcεRIβ-HDAC3 complex formation by IF (immunofluorescence) by antigen stimulation in RBL2H3.
3C shows the same result as above in IgE-DNFB mediated ear tissue.
4A to 4 show that HDAC3-Rac1 complex causes nitration of DNMT1 tyrosine residues by antigen stimulation, thereby inhibiting proteasome-dependent expression of DNMT1.
4F shows that expression of HDAC3 is cross-regulated by DNMT1.
5A shows that chemokine MCP1 secretion in IgE-DNFB mediated anaphylaxis is regulated by HDAC3.
5B shows that protein expression of MCP1 is regulated by HDAC3 in the ear tissue of FIG. 5A.
5C shows that IgE-DNFB mediated TpCR is regulated by MCP1.
5D shows that degranulation by antigen stimulation in control cell lines is regulated by MCP1.
6A to 6C show that MCP1 is regulated by DNMT1.
6, D and E show that apoptotic cells are degranulated and activated by recombinant MCP1 protein.
A and B of Figure 7 shows that the expression of HDAC3, DNMT1, MCP1 by antigen stimulation in apoptotic cell lines is regulated by aspirin.
7C to E show the same results as above in the IgE-DNFB mediated TpCR reaction.
8A shows that inhibition of DNMT1 expression increases ear thickness.
8B shows that expression of HDAC2, HDAC3 and angiogenesis-related proteins is regulated by DNMT1 in the ear tissue.
8, C and D show that the inflammatory immune response is induced by DNMT1, and the inflammatory cells migrate and infiltrate into the ear tissues. In this case, the expression of DNMT1 and HDAC2 is suppressed and the expression of HDAC3 is increased.
8E shows that HDAC3 and FcεRIβ are complexed in the same rat lung tissue.
8 F shows that the expression of HDAC3, MCP1, tryptase, c-Kit is regulated by DNMT1 in the same lung tissue.
9A shows that inhibition of expression on DNMT1 in the same ear tissue increases vascular permeability.
9B shows that vascular permeability by antigen stimulation is regulated by MCP1.
9C shows that expression of PECAM-1 in ear tissues is increased by antigen stimulation.
9D shows that inhibition of expression of DNMT1 induces expression of PECAM-1 in ear tissue.
10A to 10C show that RAECs increase angiogenesis when treated with the adaptation medium of the apoptotic cell line in which the expression of DNMT1 is suppressed.
10, D and E show that the angiogenic factor VEGF is regulated by DNMT1.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Role and Regulatory Mechanisms of HDAC3 in Skin Anaphylaxis

In this example, the role and regulation mechanism of HDAC3 was confirmed using IgE-DNFB dermal anaphylaxis model of Balb / C species mice.

Dermal anaphylaxis reaction was performed by injecting anti-DNP IgE (10 ㎍) into the tail vein of Balb / C mice, sensitizing the skin, and containing DNFB antigen in 0.15% concentration in acetone-olive oil mixture (3: 1) 24 hours later. 25 µl of the prepared DNFB antigen solution was applied to both ears of the rat. 30 minutes after application, ear thicknesses were measured at time intervals shown in FIG.

As can be seen in Figures A and B, after 60 minutes of anaphylaxis induction, the ear thickness increased as a result of the initial IgE-mediated immediate hypersensitivity reaction, and chronic inflammation progressed. It was confirmed that expression of DNMT1 was suppressed while expression increased. This indicates that in skin anaphylaxis, DNMT1 and HDAC3 act as negative regulators of each other.

In order to confirm the role of HDAC3 under these conditions, HDAC3-specific siRNA was administered to the tail vein one day before IgE sensitization in the same model, and ear thickness measurements and western blots were performed for 24 hours after administration. As shown in C and D of FIG. 1, it was confirmed that siHDAC3 was inhibited by an increase in ear thickness and a decrease in expression of DNMT1 by immediate hypersensitivity after 1 hour of antigen stimulation. It was also confirmed that FcεRIβ activity by antigen stimulation is regulated by HDAC3 and complexed with each other.

After embedding the paraffin in the ear tissues, sections were prepared to a thickness of 4 μm, and tissue immunostaining was performed using antibodies to HDAC3, DNMT1, c-Kit, and Tryptase. As shown in FIG. 2, the results of the same pattern as in FIG. 1 D were confirmed, and it was confirmed that the expression of Tryptase, which is a marker of the non-blast cells, was controlled by HDAC3.

As described above, it was confirmed that HDAC3 is an essential regulator in the IgE-mediated apoptotic cell dependent anaphylaxis reaction.

Example 2 HDAC3 Complexes with FcεRIβ to Mediate Skin Anaphylaxis

Based on the results of complex formation of HDAC3 and FcεRIβ through FIG. 1, the complex formation by antigen stimulation in RBL2H3 and BMMCs apoptotic cell lines was confirmed by immunofluorescence staining and Western blot. Antigen stimulation was performed for 1 hour after IgE sensitization to the apoptotic cell lines, followed by Western blot (see FIG. 3A) and immunofluorescence (see FIG. 3B) to confirm HDAC3-FcεRIβ complex formation. The same results were confirmed in cutaneous anaphylaxis model ear tissue (see FIG. 3C).

As described above, it was confirmed that HDAC3 complexed with FcεRIβ to mediate IgE-mediated allergy and skin anaphylaxis.

Example 3 HDAC3 Induces Expression of MCP1 as a Cross-Negative Regulator with DNMT1

In order to confirm the regulation mechanism of DNMT1 expression by antigen stimulation, Western blot was performed for 1 hour after antigen stimulation to a control cell line pretreated with 1 μM solution of MG132, a proteasome inhibitor, for 12 hours before antigen stimulation, to perform HDAC3, DNMT1, HDAC2. Expression was confirmed. As shown in FIG. 4A, DNMT1 and HDAC2 are proteasome-dependently degraded by antigen stimulation, thereby regulating HDAC3 expression.

As a result of confirming GTP-Rac1 activity after antigen stimulation to confirm the regulation mechanism of DNMT1 expression by HDCA3, binding of HDAC3-Rac1 was achieved by antigen stimulation (B, C in FIG. 4), and Rac1 was a dominant negative plasmid of Rac1. As a result of antigen stimulation on the apoptotic cell lines which inhibited the activity of, it was confirmed that HDAC3 expression was suppressed and DNMT1 expression inhibition was inhibited (FIG. 4D). In addition, inhibition of expression of DNMT1 by antigen stimulation was confirmed that Rac1 activation by HDAC3-Rac1 conjugates was induced by proteasome dependent degradation by ubiquitation, inducing epigenetic modifications such as nitration of DNMT1 Tyr residues. 4D). Expression of HDCA3 by DNMT1 inhibition was induced in apoptotic cell lines treated with 5-aza-2-deoxycytidine, a DNMT1 inhibitor, by concentration for 24 hours (FIG. 4F), and inhibition by si DNMT also showed the same pattern. (G in Fig. 4). In the expression of HDAC3 by antigen stimulation, it was confirmed once again by ChIP assay that DNMT1 is a negative regulator (FIG. 4F).

This example confirmed that HDAC3 and DNMT1 regulate the expression of MCP1 through the role of negative regulators.

Example 4 Expression Mechanism and Role of MCP1 by HDAC3 and DNMT1

In the present example, to determine the cytokines and chemokines regulated by HDAC3 / DNMT1 in IgE-mediated allergy and cutaneous anaphylaxis, the cytokine array (Profiler Mouse Cytokine Array Kit, R & D) of serum of the skin anaphylaxis model rats of Example 1 was used. Was carried out. Serum reacted for 1 hour with the cytokine antibody mixture was incubated for 12 hours at 4 ° C. in a membrane in which 40 kinds of mouse cytokine antibodies were integrated, and then reacted with HRP-conjugated streptavidin secondary antibody for 1 hour. And chemokine expression was confirmed. As shown in A and B of FIG. 5, the expression of chemokine MCP1 and angiogenesis related protein sICAM was induced by antigen stimulation, and they were respectively regulated by HDAC3. To determine whether chemokine MCP1 regulates skin anaphylaxis, anti-MCP1 neutralizing antibody nMCP1, which neutralizes MCP1, was administered to the tail vein with IgE and stimulated with DNFB 24 hours later to measure the increase in ear thickness in the same manner as in Example 1. Western blot was performed. It was confirmed that MCP1 is an important regulator in skin anaphylaxis as shown in C of FIG. 5, and the same pattern of results was obtained through degranulation reaction in the apoptotic cell line (see FIG. 5D).

In order to confirm the expression mechanism of MCP1, the cytokine arrays were performed with the serum of mice that induced dermal anaphylaxis under the same conditions as the adaptation medium of the apoptotic cell line in which DNMT1 was suppressed by siRNA. As shown in A and B of FIG. 6, ChIP assay was performed with the MCP1 promoter based on the expression inhibition of DNMT1 inducing the expression of MCP1. DNA was isolated from the antigen-stimulated plaque cell line, followed by IP with anti-HDAC3 antibody, anti-DNMT1 antibody, anti-HDAC2 antibody, anti-Ac-H3, -H4 antibody, respectively, and RT-PCR was performed with primers containing MCP1 promoter. . It was confirmed that DNMT1 and HDAC2, which were bound to the MCP1 promoter as transcription repression regulators, were released from the MCP1 promoter by antigen stimulation, thereby inducing histone acetylation by acting as a transcriptional activator. Epigenetic modifications of histones, such as acetylation, are known to modulate the activity of transcription factors by altering the binding force between histones and DNA. As shown in C of Figure 6 it can be seen that epigenetic modification plays an important role in regulating the expression of MCP1 by HDCA3-DNMT1. In order to confirm the effect of MCP1 on the activation of apoptotic cells, the treatment of recombinant MCP1 on apoptotic cell lines induced degranulation, which is a marker of apoptotic cell activation, as shown in D of FIG. 6, and confirmed the activity of CCR2, a receptor of MCP1, by IP. It was.

Thus, in this example, the expression and role of MCP1 mediating the IgE-mediated allergic reaction by HDAC3 and DNMT1, which are epigenetic genetic regulators, were confirmed.

Example 5 Validity of HDCA3, DNMT1, MCP1 in Anti-allergic Drug Development

Aspirin is used as an anti-rheumatic agent as an inhibitor of the prostaglandin synthase PGES, an inflammatory mediator. In addition, since prostaglandin secretion has been reported in response to increased expression of PGES in antigen-stimulated apoptotic cell lines, this example confirms the regulation of expression of HDAC3, DNMT1, and MCP1 by aspirin, thereby confirming the feasibility of targeting three proteins in the development of anti-allergic agents. Was intended.

For this purpose, it was confirmed in A and B of FIG. 7 that degranulation by antigen stimulation and expression of three proteins are regulated by aspirin in apoptotic cell lines pretreated with 0.1 mM aspirin for 30 minutes. In the animal model of skin anaphylaxis under the same conditions, an increase in ear thickness due to antigen stimulation was reduced, and Western blot and tissue immunity staining resulted in the same pattern.

This confirms that the three proteins may be important targets in the development of allergy symptomatics and therapeutics.

Example 6 DNMT1 Regulates Blood Vessel Formation by Anaphylaxis

Based on previous studies showing that granules such as IgE-mediated degranulated histamine increase the expression of VCAM-1, a cell adhesion protein, and vascular permeability, resulting in inflammation by chemotactic chemokines such as MCP1, DNMT1 We investigated the function of vascular permeability and vascular formation. In order to confirm this, the ear thickness of the rats that were periodically tail vein administered with 100nM siDNMT was measured for a predetermined time, and the expression and complex formation of proteins were confirmed through tissue staining, immunostaining, and Western blot in the same ear tissue.

In A of FIG. 8, the increase in ear thickness according to DNMT1 inhibition was confirmed, and expression of proteins related to cell adhesion and angiogenesis was regulated along with HDAC3 and HDAC2 expression control (see FIGS. 8B and D). It was confirmed in FIG. 8C that the increase in ear thickness due to the inhibition of DNMT1 was caused by the migration and invasion of inflammatory cells, and the same pattern was obtained in the lung tissue (see E and F of FIG. 8).

In this example, it was confirmed that the inhibition of expression of DNMT1 causes chronic inflammation due to migration and invasion of inflammatory cells to other organs such as the lungs as well as the initial immediate hypersensitivity reaction.

It was confirmed that the vascular permeability by DNMT1 was controlled by administering 2% Evans blue solution to the tail vein, and the increase in vascular permeability by antigen stimulation was controlled by MCP1 in A and B of FIG. 9. The expression of vascular endothelial cell factor PECAM-1 by antigen stimulation was confirmed by whole-mounted staining (whole-mount) in antigen-stimulated ear tissues, and the same pattern of results was obtained by inhibition of expression of DNMT1 (Fig. 9C and See D).

Endothelilal cell tube formation and aortic ring spouting technique were performed by treating the vascular endothelial cell RAEC obtained from the rat aorta with the adaptation medium of the RBL2H3 squamous cell line which suppressed the expression of DNMT1. (Aortic ring sprouting assay) was performed. The invasion technique using transwell confirmed the interactions of apoptotic cell lines with vascular endothelial cell lines by antigen stimulation. Translocation of 10 nM siDNMT to RBL2H3, and 48 h later, trypsinized 2 x 10 ⁴ cell lines in the upper chamber of the transwell, RAEC cells in the lower chamber with M199 medium containing 20% FBS And the degree of infiltration was confirmed. After 14 hours, the infiltrated cells were fixed with methanol and H & E stained to check the degree of infiltration. As shown in Figs. 10A to 10C, angiogenesis of rat vascular endothelial cells was increased, and the degree of infiltration was also increased by the adaptation medium of the apoptotic cell line in which the expression of DNMT1 was suppressed. This may be due to chemokines or cytokines secreted by DNMT1 expression inhibition and MCP1 may play a role. It was confirmed by Western blot and RT-PCR in D and E of FIG. 10 that angiogenesis by inhibition of DNMT1 was due to VEGF production and activation of its receptor.

In this example, it was confirmed that DNMT1 is a negative regulator of allergy and anaphylaxis.

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Claims (22)

A method of measuring the expression level of histone deacetylase-3 (HDAC3) from mammalian cell samples to provide information necessary for the diagnosis of allergic diseases. The method of claim 1,
DNA methyltransferase-1 (DNMT-1) or monocyte chemotactic protein-1 (MCP-) from mammalian cell samples to provide information necessary for the diagnosis of allergic diseases. The method of further measuring the expression level of 1). The method of claim 1, wherein the allergic disease
Anaphylactic shock, bronchial asthma, urticaria and hay fever. The method of claim 1, wherein the cell sample
And a cell sample selected from tissues, serum, plasma and sugar. The method of claim 1,
To measure the expression level of the histone deacetylase-3
A method characterized by using an antibody that specifically binds to histone deacetylase-3 (HDAC3). The method of claim 2,
To measure the expression level of the DNA methyl transferase-1
Using an antibody that specifically binds to DNA methyltransferase-1 (DNMT-1),
To measure the expression level of the monocyte chemotactic protein-1
Method for using an antibody that specifically binds to monocyte chemotactic protein-1 (MCP-1). The method according to claim 5 or 6,
The measurement is characterized in that consisting of an analysis method selected from Western blot, ELISA, radioimmunoassay, radioimmunoassay, electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS and protein chip analysis using the antibody . The method of claim 1,
To measure the expression level of the histone deacetylase-3 (HDAC3),
Using a support coated with an aptamer for HDAC3 on the surface. The method of claim 8,
And the support is selected from a membrane, a slide glass, and a metal body. delete delete delete delete delete delete delete A composition for diagnosing allergic diseases, comprising a marker measuring the expression level of histone deacetylase-3 (HDAC3). 18. The composition of claim 17, wherein the composition is
Markers for measuring expression levels of DNA methyltransferase-1 (DNMT-1); And
Markers for measuring expression levels of monocyte chemotactic protein-1 (MCP-1); The composition characterized in that it further comprises a marker selected from. 18. The composition of claim 17, wherein the marker measuring the expression level of histone deacetylase-3 is an antibody that specifically binds to histone deacetylase-3. The composition of claim 18, wherein the marker measuring the expression level of DNA methyl transferase-1 is an antibody that specifically binds to DNA methyl transferase-1. 19. The composition of claim 18, wherein the marker measuring the expression level of monocyte chemotactic protein-1 is an antibody that specifically binds to monocyte chemotactic protein-1. 22. An allergic disease diagnostic kit comprising the composition of any one of claims 17-21.

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