Composition for preventing or treating inflammatory skin diseases comprising TAZ or agonist thereof as active ingredient

Abstract

The present invention relates to a composition for preventing or treating inflammatory skin diseases, comprising transcriptional coactivator with PDZ-binding motif (TAZ) or an agonist thereof as an active ingredient. The composition of the present invention can efficiently ameliorate skin inflammatory diseases, including psoriasis, by controlling the stability of TCTP, and thus can be widely used in related industrial fields.

Description

Composition for preventing or treating inflammatory skin diseases containing TAZ or its agent as an active ingredient

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2023-0148524, filed October 31, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a composition for preventing or treating inflammatory skin diseases, comprising TAZ or an agent thereof as an active ingredient.

[0003] Psoriasis and other inflammatory skin diseases are caused by genetic, environmental, and immunological factors. They are characterized by abnormalities in the stratum corneum, the outermost layer of the skin that acts as a protective barrier. These conditions are exacerbated in dry climates. Key symptoms include severe itching, dry skin, rashes, oozing, crusting, and scaly skin (ichthyosis), often accompanied by chronic skin inflammation. Scratching, triggered by itching, can disrupt the skin barrier and lead to secondary infections, further exacerbating the inflammation.

[0004] Psoriasis not only affects the skin, but also involves a widespread systemic inflammatory immune response, triggering inflammatory responses in various organs and systems, potentially leading to functional impairment. While numerous research and development efforts are underway to treat psoriasis (Korean Patent Publication No. 2021-0154983), most of this research focuses solely on the skin symptoms of psoriasis. Therefore, there is a pressing need to develop technologies that can control the widespread systemic inflammation of psoriasis.

[0005] One aspect is to provide a composition for preventing, treating or improving inflammatory skin diseases, comprising TAZ (Transcriptional coactivator with PDZ-binding motif) or an agonist thereof as an active ingredient.

[0006] Another aspect provides a method for screening a therapeutic agent for an inflammatory skin disease, comprising the steps of: (a) treating cells with a candidate substance in vitro; (b) measuring the expression level of TCTP after treating the candidate substance; and (c) determining a substance that reduces the expression level of TCTP compared to a group not treated with the candidate substance as a therapeutic agent for an inflammatory skin disease.

[0007] However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the relevant technical field from the description below.

[0008] One aspect is to provide a composition for preventing, treating or improving inflammatory skin diseases, comprising TAZ (Transcriptional coactivator with PDZ-binding motif) or an agonist thereof as an active ingredient.

[0009] The term "TAZ (Transcriptional coactivator with PDZ-binding motif)" used herein refers to a cellular protein identified as interacting with the 14-3-3 protein, and may also be referred to as WWTR1. TAZ may also be utilized in the regulation of glucose homeostasis by performing muscle cell-specific interactions with the insulin receptor substrate (IRS) within muscle cells. Therefore, the TAZ may be a protein encoded by the TAZ gene.

[0010] The sequence of the gene encoding the TAZ protein may be a sequence obtained from GeneBank Accession No. NM_000116.5, NM_001303465.1, NM_181311.3, NM_181312.3, or NM_181313.3 for humans, or from NM_001173547.2, NM_001242615.2, NM_001242616.2, NM_001290738.1, or NM_181516.6 for mice. The amino acid sequence of the TAZ protein may be a sequence obtained from GeneBank Accession No. NM_000116.5, NM_001303465.1, NM_181311.3, NM_181312.3, or NM_181313.3 for humans, or from NM_001173547.2, NM_001242615.2, NM_001242616.2, NM_001290738.1, or NM_181516.6 for mice. It may be an amino acid sequence obtained from NP_001161753, NP_001161753, NP_001335291.1, NP_000107.1, NP_001290394.1, NP_851828.1, NP_851829.1, or NP_851830.1. Even if some of the nucleic acid sequences or amino acid sequences do not match the nucleic acid sequences or amino acid sequences, a nucleic acid sequence or amino acid sequence having biologically equivalent activity may be considered as the mRNA or protein of TAZ.

[0011] The above TAZ may refer to the TAZ gene, or may also refer to a polypeptide encoded by the TAZ gene.

[0012] The above pharmaceutical composition may contain, in addition to the TAZ gene, TAZ protein or TAZ agonist, other ingredients that may preferably have a synergistic effect on the main effect, within a range that does not impair the main effect intended by the pharmaceutical composition.

[0013] In addition, the pharmaceutical composition may further include pharmaceutically acceptable carriers, excipients, and diluents in addition to the above-described effective ingredients for administration.

[0014] For example, carriers, excipients and diluents that can be used include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc., and target organ-specific antibodies or other ligands can be combined with the carriers to specifically act on the target organ.

[0015] The above pharmaceutical composition can be prepared in various parenteral or oral dosage forms according to known methods. Representative parenteral dosage forms include aerosol dosage forms and injectable dosage forms.

[0016] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

[0017] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives.

[0018] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include withepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin. However, the present invention is not limited thereto, and pharmaceutical compositions according to one aspect may be formulated according to the intended purpose using any method known in the art without limitation.

[0019] The effective dosage of the above pharmaceutical composition may vary depending on the patient's age, sex, and weight, but may be administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, but is not limited thereto.

[0020] The above pharmaceutical composition may be administered in a pharmaceutically effective amount, and may be administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, but is not limited thereto. The dosage may vary depending on the patient's age, sex, weight, health condition, diet, administration period, administration method, disease severity, etc.

[0021] Another aspect provides a health functional food composition for preventing or improving inflammatory skin diseases, comprising TAZ (Transcriptional coactivator with PDZ-binding motif) or an agonist thereof as an active ingredient.

[0022] In health functional foods for the prevention or improvement of inflammatory skin diseases, the TAZ or its agent may be added directly or in combination with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of active ingredients mixed may be appropriately determined depending on the intended use, such as prevention, health, or treatment.

[0023] The formulation of health functional foods can be in the form of powders, granules, pills, tablets, capsules, or any other form of general food or beverage.

[0024] There is no particular limitation on the types of the above foods, and examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all foods in the conventional sense can be included.

[0025] Generally, when adding TAZ or its agent during the manufacture of food or beverages, it can be added in an amount appropriate for consumption. However, for long-term consumption for health and hygiene purposes or for health regulation, it can be added at a lower level. Furthermore, since the present invention utilizes a fraction from a natural product, there are no safety issues, and therefore, it can be used in amounts exceeding the above range.

[0026] Among the health functional foods according to the aspect, beverages may contain various flavoring agents or natural carbohydrates as additional ingredients, just like regular beverages. The natural carbohydrates mentioned above may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the beverage according to the present invention.

[0027] In addition to the above, health functional foods for preventing or improving inflammatory skin diseases according to one aspect may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. In addition, the composition for improving sleep of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in mixtures. The ratio of these additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health functional food of the present invention.

[0028] Another aspect provides a method for treating an inflammatory skin disease comprising administering the pharmaceutical composition to a subject.

[0029] The above entity may be a mammal other than a human, preferably a chimpanzee, gorilla, monkey, dog, cat, cow or rat.

[0030] Another aspect provides a use of the pharmaceutical composition for treating inflammatory skin diseases.

[0031] Another aspect provides a method for screening a therapeutic agent for an inflammatory skin disease, comprising the following steps.

[0032] (a) a step of treating cells with a candidate substance in vitro;

[0033] (b) a step of measuring the expression level of TCTP after treatment with the candidate substance; and

[0034] (c) A step of determining a substance that reduces the expression level of TCTP compared to the untreated group as a treatment agent for inflammatory skin diseases.

[0035] The expression level of the above step (b) is preferably performed by any one method selected from the group consisting of immunoprecipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunohistochemical analysis, RealTime-PCR, qRT-PCR, Western Blotting, and flow cytometry (FACS), but any method for measuring the level of protein expression known to those skilled in the art may be used without limitation.

[0036] In addition, the step of measuring the expression level of TCTP after the candidate substance treatment (b) above is not limited to the target organ to be measured, but may be measuring the expression level of TCTP in the spleen.

[0037] The present invention relates to a composition for preventing or treating inflammatory skin diseases, comprising TAZ (Transcriptional coactivator with PDZ-binding motif) or an agonist thereof as an active ingredient. The composition of the present invention can effectively improve inflammatory skin diseases, including psoriasis, by controlling the stability of TCTP.

[0038] Figure 1 is a diagram showing the structure of the TAZ protein.

[0039] Figure 2 is a diagram showing the pathology of skin affected by psoriasis.

[0040] Figure 3 is a diagram showing a strategy for inducing psoriasis in mice and the change in body weight of mice induced with psoriasis accordingly.

[0041] Figure 4 is a diagram showing the inflammatory state of the skin on the ears and back of the mouse group used in the psoriasis induction experiment.

[0042] Figure 5 is a photograph showing the state of the back and ears of the mouse group used in the psoriasis induction experiment.

[0043] Figure 6 shows the results of observing the back and ear skin tissues of the mouse group used in the psoriasis induction experiment after staining with H&E (hematoxylin and eosin), Trichrome (collagen), and Sirius red (collagen).

[0044] Figure 7 is a diagram showing the normal differentiation and maturation characteristics of T cells confirmed by photographing, weight analysis, and flow cytometry of thymus collected from a group of mice used in a psoriasis-inducing experiment.

[0045] Figure 8 is a photograph of the spleen taken from a group of mice used in a psoriasis induction experiment, confirming an increase in the total number of cells in the spleen and lymph nodes.

[0046] Figure 9 is a diagram showing the results of collecting spleens and lymph nodes from a group of mice used in a psoriasis-inducing experiment, staining them with CD3, and performing flow cytometry analysis to determine changes in T cells.

[0047] Figure 10 is a diagram showing the expression of TCTP protein by collecting spleen proteins and blood proteins from a group of mice used in a psoriasis induction experiment.

[0048] Figure 11 shows changes in TCTP protein in the back and ear skin tissues of the mouse group used in the psoriasis induction experiment.

[0049] Figure 12 is a diagram showing the results of flow cytometry analysis of TCTP-expressing cells in spleen cells of the control (WT) and TAZ KO (TAZ KO) mouse groups following LPS stimulation.

[0050] Figure 13 shows the change in TCTP expression confirmed by performing immunoblotting on spleen cells from the control (WT) and TAZ KO (TAZ KO) mouse groups treated with LPS, CHX, and MG132.

[0051] Figure 14 is a diagram showing the results of confirming changes in TCTP expression by performing immunofluorescence experiments, immunoblotting, and flow cytometry on control (WT), TAZ KO group (TAZ KO), and TAZ reintroduced KO (KO / T) MEF cells.

[0052] Figure 15a is a photograph showing changes in TCTP protein after CHX treatment in the above MEF cells.

[0053] Figure 15b is a diagram showing the quantification of the level of TCTP protein change after CHX treatment in the above MEF cells.

[0054] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0055] As used herein, the singular forms include the plural forms unless the context clearly dictates otherwise. Furthermore, as used herein, the words “comprise” and “include” and / or “comprising” and “including” specify the presence of stated features, numbers, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, numbers, operations, elements, components, and / or groups thereof.

[0056]

[0057] Example

[0058] Example 1. Experiment and material preparation

[0059] 1-1. Antibodies

[0060] Antibodies used in the experiments were purchased from Abcam (Cambridge, MA, USA), Santa Cruz Biotecnology (Dallase, TX, USA), Cell Signaling, Sigma Aldrich (St Louis, MO, USA), BioLegend (San Diego, CA, USA), and BD Biosciences (NJ, USA). A list of specific antibodies used in the experiments is shown in Table 1 below.

[0061] Antibodycatalogcompanyapplicationdilution factor / concTAZ560235BD PharmingenIF1:100TCTPab37506AbcamIB, IF,IHC, FACS1:500 ~ 1:3000b-actinsc-47778Santa CruzIB1:1000Alexa Fluor TM 488,Goat anti-Rat IgG (H+L)A11006InvitrogenIHC1:500Alexa Fluor TM 488,Goat anti-rabbit IgG (H+L)A11008InvitrogenIHC, FACS1:500Alexa Fluor TM647,Goat anti-mouse IgG (H+L)A21235InvitrogenIHC1:500APC Rat IgG1, κ Isotype Ctrl400412BiolegendFACS1:500APC anti-mouse CD25 Antibody102012BiolegendFACS1:500FITC Mouse IgG1, κIsotype Control 550616BD PharmingenFACS1:500FITC anti-mouse / human CD45R / B220103206BiolegenFACS0.5PE Hamster Anti-Mouse CD3e553064BD PharmingenFACS1:500

[0062] 1-2. Primer

[0063] The primer sequences used in the experiment are as shown in Table 2 below.

[0064] PrimerSequenceIL-17a FGCT CCA GAA GGC CCT CAG AIL-17a RAGC TTT CCC TCC GCA TTG AIL-22 FTTG AGG TGT CCA ACT TCC AGC AIL-22 RAGC CGG ACA TCT GTG TTG TTAFoxp3 FGGC CCT TCT CCA GGA CAG A Foxp3 RGCT GAT CAT GGC TGG GTT GTIFNg FAGC AAC AGC AAG GCG AAA AIFNg RCTG GAC CTG TGG GTT GTT GARORgt FACC TCC ACT GCC AGC TGT GTG CTG TCRORgt RTGA CGC GTG CAG GAG TAG GTCTP FACC GAA AGC ACA GTA GTC ACCTCTP RAGT CAC ACC ATC TTC ACG Gb-actin FAGA GGG AAA TCG TGC GTG ACb-actin RCAA TAG TGA TGA CCT GGC CG

[0065] 1-3. Laboratory animals

[0066] WT C57BL / 6 mice and TAZ-deficient (KO) mice were purchased from Jackson Laboratory (Minneapolis, MN, US) and were prepared as previously described. All mice were bred and maintained in a specific pathogen-free animal facility at Ewha Womans University. All animals were housed under controlled conditions (temperature 22 ± 2 °C, humidity 60 ± 5%, 12-h light / 12-h dark), and all animal experiments were approved by the Institutional Animal Care and Use Committee of Ewha Womans University and performed in accordance with their guidelines (IACUC 19-031, 21-072).

[0067]

[0068] 1-4. Production of psoriasis-induced mice treated with imiquimod

[0069] Mouse experiments were conducted using wild-type (WT) mice (male, 8–12 weeks old, 25–30 g) and TAZ-deficient (KO) mice (male, 8–12 weeks old, 20–25 g). The mouse groups were divided into WT, TAZ KO, imiquimod (IMQ)-treated WT (WT + IMQ), and imiquimod-treated TAZ KO (TAZ KO + IMQ). In the imiquimod-treated group, 62.5 mg of 5% IMQ (Aldara cream) was applied to the depilated back skin and left ear. In the imiquimod-free group, the same amount of petrolatum was applied.

[0070] The above mouse groups were evaluated for skin inflammation levels for 5 days using the PASI (Psoriasis Area and Severity Index) scoring system, and the evaluation criteria included skin thickness and weight loss. Skin thickness before treatment was measured using a dial thickness gauge (Peacock G1-A, Ozaki MFG. Co., Ltd., Tokyo, Japan).

[0071]

[0072] 1-5. Cell culture

[0073] ① Spleen cell culture

[0074] Spleens obtained from mice were placed in a 30-mm culture dish containing RPMI 1640 medium (Hyclone, Logan, VA, USA). The tissue was ground using a translucent slide glass for spleen, and the suspension was transferred to a 50-ml conical tube. The suspension was spun at 1,000 rpm for 3 min, and red blood cells were lysed with RBC lysis buffer. The lysed suspension was filtered through a 70-μm strainer and transferred to a new 50-ml conical tube. The suspension was re-neutralized with sufficient RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS; Gendepot, Katy, TX, USA), and then resuspended. The total spleen cells obtained were plated (5 × 10 6 cells / ml).

[0075]

[0076] ② Fibroblast culture

[0077] Mouse embryonic fibroblasts were isolated from wild-type and TAZ-deficient mouse embryos and cultured in Dulbecco's Modified Eagle's Medium (DMEM, Hyclone, Logan, VA, USA) containing 10% FBS.

[0078]

[0079] 1-6. Histological analysis

[0080] Tissues from the back and ears of mice were fixed in Bouin's solution for 3–4 hours and then fixed overnight in PBS containing 4% formaldehyde. They were then embedded in paraffin. The embedded tissues were sectioned into 4-μm-thick slices on glass slides using an automatic rotary microtome. The skin sections were deparaffinized with Neo-Clear (Merck Millipore, MA, USA), rehydrated with absolute alcohol, and then with 70% alcohol, and then washed with distilled water. The washed skin sections were stained with hematoxylin and eosin (Sigma-Aldrich, St Louis, MO, USA). For collagen staining, a trichrome staining kit (DAKO) and a Sirius red staining kit (Abcam) were used.

[0081]

[0082] 1-7. Immunofluorescence staining

[0083] ① Tissue staining

[0084] Paraffin-embedded skin tissues were cut into 4-μm-thick slices on coated glass slides (Matsunami, Japan). The skin sections were deparaffinized and rehydrated. Antigen retrieval was performed with 10 mM sodium citrate buffer (pH 6.0), permeabilized with 1% Triton X-100 in PBS, blocked with 3% bovine serum albumin (BSA) in PBST, and stained with primary antibodies specific for TAZ and TCTP, each diluted to 1%. After washing three times with BSA in PBST overnight at 4°C, the slides were incubated with Alexa 647, 488 goat anti-mouse and goat anti-rabbit (Invitrogen) for 40 min at room temperature in a darkroom. Nuclei were stained with DAPI (1 μg / ml). Images were analyzed using a confocal microscope Zeiss LSM880 using Aryscan and then processed with ZEN5 software (Zeiss, Oberkochen, Germany).

[0085]

[0086] ② MEF cell staining

[0087] MEF cells were cultured in 24-well plates containing coverslips coated with 0.01% poly-L-lysine (Sigma-Aldrich). Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes, permeabilized with 0.1% Triton X-100 in PBS for 10 minutes, and blocked with 3% BSA in PBST for 30 minutes. Cells were incubated overnight at 4°C with primary antibodies against YAP, TAZ, and TCTP. Cells were washed three times with PBST and incubated with secondary antibodies against Alexa 647, 488 for 40 minutes. Nuclei were then stained with DAPI for 5 minutes and mounted with fluorescent mounting medium. Images were analyzed using an Aryscan confocal microscope Zeiss LSM880 and processed with ZEN5 software (Zeiss, Oberkochen, Germany).

[0088]

[0089] 1-8. Immunoblot analysis

[0090] Proteins were extracted from mouse tissues or cultured cells and resuspended in 10% glycerol, 25 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 1 mM PMSF, 1 mM DTT, 1 mM Na₃VO₄, protease inhibitor cocktail (Roche, BS, SUI) and centrifuged at 12,000 rpm for 10 min at 4°C.

[0091] Lysates were subjected to SDS-PAGE under reducing or non-reducing conditions and transferred to nitrocellulose membranes. After blocking with 3% BSA and 0.1% Tween-20 in Tris-buffered saline (TBS), the membranes were incubated overnight at 4°C with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies and ECL Western blotting reagent. The results were quantitatively analyzed using Image J software.

[0092]

[0093] 1-9. Flow cytometry

[0094] Single-cell suspensions from lymph nodes and spleens were stained with antibodies conjugated to PE, FITC, or APC. Fixed cells were stained with anti-TCTP and Alexa 488-anti-rabbit IgG or Alexa 633-anti-rabbit. Stained cells were analyzed using flow cytometry and Cell Quest Pro analysis. All analyses were performed by gating on 10,000 live cells. FITC, PE, or APC isotype Ctrl was used as a negative control for all analyses.

[0095]

[0096] 1-10. Protein stability analysis

[0097] Single-cell suspensions obtained from the spleen were plated in six wells at a density of 5 × 10 cells / ml. All wells were stimulated with lipopolysaccharide (LPS, 1 μg / ml) for 24 h, and then treated with MG132 (20 μM) and cycloheximide (CHX, 20 μg / ml) 6 h and 4 h before harvest. The harvested cells were lysed using RIPA lysis buffer, and proteins were detected by SDS-PAGE. The intensity of each band was analyzed by normalizing it to b-actin using Image J software. MEF cells were treated only with MG132 and CHX without LPS stimulation. MEF cells were seeded in 24 wells containing poly-L-lysine-coated cover slides at a density of 1 × 10 6 Cells were seeded at a density of 10 cells / ml and proteins were identified by immunofluorescence staining.

[0098]

[0099] Example 2. Confirmation of psoriasis symptoms according to the presence or absence of the TAZ gene.

[0100] The experiment was conducted by dividing the mice into four groups. The wild-type (WT) and TAZ KO groups were treated with or without IMQ, and a total of four experimental groups were prepared. Each group was repeated three times with n=5 per group. IMQ was continuously treated for 5 days, and the control group was treated with Vaseline in the same way. Body weight was measured every day before treatment with IMQ and Vaseline, and the value on Day 0 was set as 100% to check the change in body weight (Fig. 3).

[0101] The Psoriasis Area Severity Index (PASI) was used to evaluate body weight, changes in back skin thickness, and scaling (4 points each), giving a total of 12 points. In the IMQ-untreated group, no significant difference was observed between WT and TAZ-deficient mice (Fig. 4). However, in the IMQ-treated mouse group, a significant difference was observed between WT and TAZ-deficient mice. Specifically, when IMQ was treated in TAZ KO mice, increased sensitivity to weight changes and more severe psoriasis was observed. In addition, the dorsal skin visually thickened from day 1, and dramatic changes in the ears were also observed from day 3. When the mice were observed on day 5, partial redness and keratinization were observed in the red areas of the dorsal skin in the IMQ-treated WT mice (Fig. 5), whereas more keratin was found and erythema increased overall in the IMQ-treated TAZ KO mice compared to the WT mice. In ear analysis, greater erythema and more pronounced skin keratinization were observed in TAZ KO mice.

[0102] Even when skin thickness was checked, no significant difference was confirmed in the group not treated with IMQ (Fig. 6), and no significant difference was confirmed in the dermal layer thickness in the IMQ-treated groups. However, in the IMQ-treated group, the epidermal layer of TAZ KO mice was confirmed to be thicker overall than that of WT mice.

[0103] The above results demonstrated that psoriasis induced in TAZ-deficient mice was significantly increased compared to the control group. Therefore, the TAZ gene appears to influence psoriasis susceptibility. Therefore, we conducted an experiment to determine its effect on the immune-inflammatory response observed during the psoriasis pathogenesis.

[0104]

[0105] Example 3. Confirmation of inflammatory response according to the presence or absence of the TAZ gene.

[0106] First, to determine whether there were differences in the intrinsic immune system, the thymus was collected and its size and weight were determined. As a result, no significant difference was observed between the IMQ-untreated group and the two groups. Therefore, it was confirmed that TAZ deficiency does not affect the intrinsic immune system. Even when IMQ was treated, thymic atrophy due to inflammation was confirmed, but no difference was observed between the wild-type and TAZ KO groups. In addition, there was no difference in the generation of CD4 / CD8 double-positive cells and CD4 T and CD8 T single-positive cells generated during T cell maturation between the wild-type and TAZ KO groups (Figure 7).

[0107] To confirm the changes in the immune response at the extremities, the spleen and lymph nodes were examined, and splenomegaly was observed by IMQ treatment, and it was confirmed that the total number of immune cells constituting the spleen and lymph nodes increased. In addition, the spleen of the TAZ KO group was somewhat enlarged, but the increase was more marked by IMQ treatment, and the number of cells constituting the spleen and lymph nodes also increased significantly (Fig. 8). As a result of staining T cells present in the lymph nodes and spleen, the number of T cells in the lymph nodes increased in both the IMQ-treated control group and the TAZ KO group, and the composition ratio of T cells in the spleen decreased by IMQ treatment, and no significant change was confirmed between the control group and the TAZ KO group (Fig. 9).

[0108]

[0109] Example 4. Confirmation of TCTP protein levels in TAZ-deficient mice.

[0110] To understand the mechanism of inflammation exacerbation due to increased susceptibility to psoriasis in the TAZ KO group, we analyzed the expression of inflammatory cytokines, particularly TCTP, a key factor in the development of psoriasis. The protein expression level of TCTP in the spleen of each group was compared with that of b-actin. TCTP plays an essential role in various physiological processes, including cell proliferation, apoptosis, the cell cycle, the cytoskeleton, protein synthesis, immune responses, malignant transformation, and nuclear reprogramming. Furthermore, TCTP influences the migration of inflammatory cells to inflammatory sites and interacts with signaling pathways, including NF-κB, to participate in the development of allergic responses. Therefore, we confirmed the interaction between the TAZ gene and TCTP protein. As a result, as shown in Figure 11, we confirmed that TCTP protein levels increased in the IMQ-treated group, and among them, the expression level of TCTP was significantly higher in the TAZ KO group. In addition, we further confirmed that endogenous TCTP expression increased in the TAZ KO group that was not treated with IMQ compared to the control group (Fig. 10A). Furthermore, when we obtained serum and confirmed the level of TCTP expression in the serum, TCTP expression increased in the serum of the TAZ KO group, and the increase in TCTP serum concentration in both the control group and TAZ KO group due to IMQ treatment was confirmed by Western blot and ELISA methods (Fig. 10B).

[0111] To confirm TCTP expression in the skin, immunohistochemistry (IHC) was performed on back and ear skin to determine TCTP protein levels (Fig. 11). Fluorescence was observed, indicating significantly higher levels of TCTP expression in TAZ-deficient mice. Furthermore, Western blot analysis confirmed a relative increase in TCTP protein in TAZ KO mice.

[0112]

[0113] Example 5. Confirmation of TAZ's ability to regulate TCTP protein stability.

[0114] As confirmed in Example 4 above, it was confirmed that the protein expression of TCTP increased in TAZ KO mice, but when the mRNA level was checked separately, no significant increase in expression was confirmed. Therefore, it was judged that the TAZ gene would function to regulate the stability, not the expression, of TCTP, and so additional experiments using cycloheximide (CHX) and MG132 were performed. Specifically, CHX was applied for 4 hours under LPS stimulation conditions, and MG132 was administered for 6 hours. As a result, it was confirmed that when WT mice were treated with CHX, TCTP expression was reduced by more than 50%, and this was restored to the original state when MG132 was treated (Figs. 12 and 13). However, in the case of TAZ KO mice, it was confirmed that TCTP was reduced to a minimal level when CHX was treated. That is, it is understood that in the absence of TAZ, TCTP is sufficiently stabilized and therefore the protein amount is not reduced by CHX treatment, and therefore is not significantly affected by MG132.

[0115] To confirm whether the results showing that TAZ regulates TCTP protein stability as described above are observed in the same way not only in spleen cells but also in other types of cells expressing TCTP, experiments were conducted using MEFs. Three types of MEF cells were prepared for the experiment: wild-type (WT), TAZ KO, and cells in which TAZ was reintroduced into TAZ KO cells to overexpress TAZ (KO / T). First, when immunofluorescence staining was performed on the above cells, TCTP expression increased in TAZ KO compared to WT, and decreased in KO / T MEF cells into which TAZ was reintroduced. This tendency was confirmed to be the same in WB and FACS staining experiments. That is, it was demonstrated that TCTP expression increased in the absence of TAZ, and decreased when TAZ expression was reintroduced (Fig. 14).

[0116] Furthermore, while TCTP expression in control MEF cells was drastically reduced by CHX treatment, it was barely affected by CHX in MEF cells lacking TAZ, and MEF cells in which TAZ expression was reintroduced showed a decrease in TCTP by CHX treatment, similar to the control cells (Fig. 15a and Fig. 15b). Therefore, it was confirmed that TAZ is an important factor in regulating TCTP protein stability.

Claims

1. A pharmaceutical composition for preventing or treating inflammatory skin diseases, comprising TAZ (Transcriptional coactivator with PDZ-binding motif) or an agonist thereof as an active ingredient.

2. In claim 1, A pharmaceutical composition, wherein the inflammatory skin disease is selected from the group consisting of psoriasis, hyperkeratosis, ichthyosis, atopic dermatitis, keratosis pilaris, actinic keratoses, seborrheic keratoses, pemphigus, corns, warts, and lichen planus.

3. In claim 1, The pharmaceutical composition above is a pharmaceutical composition that reduces the expression level of TCTP (Translationally controlled tumor protein).

4. In claim 3, A pharmaceutical composition wherein the reduction in the above TCTP expression level is due to a reduction in the stability of TCTP.

5. In claim 3, A pharmaceutical composition wherein the reduction in the above TCTP expression level is a reduction in the expression level of TCTP in the spleen.

6. In claim 1, A pharmaceutical composition wherein the above inflammatory skin disease is induced by imiquimod.

7. In claim 1, A pharmaceutical composition wherein the above inflammatory skin disease is caused by lipopolysaccharide.

8. A health functional food composition for preventing or improving inflammatory skin diseases, comprising TAZ (Transcriptional coactivator with PDZ-binding motif) or an agonist thereof as an active ingredient.

9. In claim 8, A health functional food composition, wherein the inflammatory skin disease is selected from the group consisting of psoriasis, hyperkeratosis, ichthyosis, atopic dermatitis, keratosis pilaris, actinic keratoses, seborrheic keratoses, pemphigus, corns, warts, and lichen planus.

10. A method for screening an inflammatory skin disease treatment agent comprising the following steps: (a) a step of treating cells with a candidate substance in vitro; (b) a step of measuring the expression level of TCTP after treatment with the candidate substance; and (c) A step of determining a substance that reduces the expression level of TCTP compared to the untreated group as a treatment agent for inflammatory skin diseases.

11. In claim 10, A method for screening an inflammatory skin disease treatment agent, wherein the expression level of the above step (b) is measured by any one method selected from the group consisting of immunoprecipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunohistochemical analysis, RealTime-PCR, qRT-PCR, Western Blotting, and flow cytometry (FACS).

12. In claim 10, A method for screening an inflammatory skin disease treatment agent, wherein the measurement of the expression level of step (b) above is performed in the spleen.

13. In claim 10, A method for screening an inflammatory skin disease treatment agent, wherein the inflammatory skin disease is selected from the group consisting of psoriasis, hyperkeratosis, ichthyosis, atopic dermatitis, keratosis pilaris, actinic keratoses, seborrheic keratoses, pemphigus, corns, warts, and lichen planus.

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