WO2018012889A1 - Composition including itac inhibitor - Google Patents

Abstract

The present specification describes and relates to a composition for preventing or treating vitiligo or vitiligo metastasis, wherein the composition includes an interferon-inducible T-cell alpha chemoattractant (ITAC) inhibitor. The composition may inhibit the movement of melanin-forming cells and exhibit a hypopigmentation effect by inhibiting the activity of ITAC. Moreover, the composition may exhibit a vitiligo or vitiligo metastasis prevention or treatment effect by inhibiting ITAC activity.

Description

Compositions Including Inhibitors of ITAC

The present specification describes a composition comprising an inhibitor of ITAC.

One important role of chemokines is to mediate immune responses and tumorigenesis through induction of cell migration. Some chemokines are involved in mouse vitiligo. Interferon-inducible T-cell alpha chemoattractant (ITAC) has a high binding affinity for CXCR3 compared to other ligands to elicit a strong chemotactic response from immune cells. The effect of ITAC on melanocyte migration and pigmentation and its relationship to related disorders is unknown. Class II histone deacetylases (HDACs), particularly HDAC5, deacetylate p53 proteins involved in various cellular processes, including cell migration, and regulate protein stability and activity.

The present study investigated the role of ITAC in pathological conditions such as vitiligo and in vitro evidence for the migratory and hypopigmentation effects of ITAC on melanocytes by deacetylation of p53 via HDAC5. Provides a review on the translation side.

In one aspect, the present invention provides a composition for the prevention or treatment of vitiligo or vitiligo metastasis comprising an interferon-inducible T-cell alpha chemoattractant (ITAC) inhibitor.

In another aspect, the present invention provides a composition for vitiligo or detection comprising a reagent for detecting the gene expression amount of ITAC.

In yet another aspect, the present invention provides a method for screening a prophylactic or therapeutic agent for vitiligo comprising identifying whether it inhibits the expression of one or more genes of ITAC, CXCR3 and CXCR7.

The present study investigated the role of ITAC in pathological conditions such as vitiligo and in vitro evidence for the migratory and hypopigmentation effects of ITAC on melanocytes by deacetylation of p53 via HDAC5. Provides a review on the translation side.

Figure 1a shows the results of comparing the migration of human epidermal melanocytes (NHEM) according to ITAC, MIG and IP-10 treatment.

Figure 1b shows the NHEM migration results according to the change in ITAC concentration.

1C shows Western blot results of CXCR3 and CXCR7 protein expression levels.

1D shows the results of migration analysis of NHEMs in the presence of ITAC after CXCR3 or CXCR7 neutralizing antibody treatment.

2A shows the results of analyzing biological processes that are significantly up- or down-regulated by ITAC treatment.

2B shows HDAC5 mRNA levels in NHEM following ITAC treatment.

2C shows HDAC9 mRNA levels following ITAC treatment.

Figure 3a shows the results confirmed the cell migration according to HDAC5 and HDAC9 knockdown in NHEM.

Figure 3b shows the results confirmed the cell migration following HDAC5 overexpression.

Figure 3c shows the results of confirming the HDAC5 protein level following ITAC treatment.

4A shows the results of confirming the levels of total p53 and acetylated p53 in NHEM following ITAC treatment.

Figure 4b shows the results of confirming the level of total p53 and acetylated p53 following HDAC5 overexpression.

Figure 4c shows the results of confirming the levels of total p53 and acetylated p53 according to HDAC5 knockdown.

FIG. 5A shows the results confirming the effects on cell migration in the absence or absence of ITAC when HDAC5 overexpression (HDAC5-myc), p53 overexpression (p53-Flag) or both are overexpressed (HDAC5-Myc + p53-Flag). Indicates.

Figure 5b shows the results confirming the effect on MMP1 expression following ITAC treatment.

Figure 6a shows the results of confirming the change in melanin amount according to the ITAC treatment.

Figure 6b shows the results of confirming the melanin amount, total p53 or acetylated p53 protein level after ITAC treatment after UV irradiation.

Figure 6c shows the result of confirming the amount of melanin according to HDAC5 overexpression.

7 shows the results of staining skin samples (# 1 and # 2) of normal (N) or hypopigmented lesions (L) with anti-ITAC antibodies (green) and anti-HMB45 (red). The nucleus is represented by DAPI (blue), arrow indicates melanin and triangle indicates melanin forming cells. DAPI, anti-ITAC antibody and anti-HMB45 stained portions in black and white drawings are represented in white.

8 is a result showing the mobility of NHEM to the skin cell-derived chemokine.

Figure 9a is a result confirming the cell migration effect of ITAC in human melanoma cell line (WM266-4).

Figure 9b is the result confirming the cell migration effect of ITAC in human melanoma cell line (SK-MEL-28).

Figure 9c is a result confirming the cell migration effect of ITAC in human melanoma cell line (MNT-1).

9D shows the results of detecting ITAC, CXCR3, and CXCR7 in melanocytes and melanoma cells.

Figure 10a is a mRNA expression result confirming the knockdown of HDAC5 by siRNA treatment.

10b is a mRNA expression result confirming the knockdown of HDAC9 by siRNA treatment.

Figure 11a is a result confirming that acetylation of alpha-tubulin is affected by ITAC treatment in NHEM.

11A shows the results confirming that acetylation of alpha-tubulin is affected by HDAC5 deletion in NHEM.

Figure 11c is a result confirming the changes in p53 and TYR expression according to ITAC treatment in melanoma cells.

Figure 12a is a result confirming the effect on TYR expression in NHEM following ITAC treatment.

Figure 12b is a result confirming the effect on MITF expression in NHEM following ITAC treatment.

Figure 12c is a result confirming the effect on TRP1 expression in NHEM following ITAC treatment.

Figure 12d is a result confirming the effect on the expression of DCT in NHEM by ITAC treatment.

Figure 13a is the result confirming the effect on TYR expression according to ITAC treatment in the case of p53 knockdown.

Figure 13b is the result confirming the effect on MITF expression according to ITAC treatment in the case of p53 knockdown.

Figure 13c is the result confirming the effect on TRP1 expression according to ITAC treatment in the case of p53 knockdown.

Figure 13d is the result confirming the effect on DCT expression according to ITAC treatment in the case of p53 knockdown.

Hereinafter, with reference to the accompanying drawings, it will be described embodiments of the present application in more detail. However, the technology disclosed in the present application is not limited to the embodiments described herein and may be embodied in other forms. It is merely to be understood that the embodiments introduced herein are provided so that the disclosure can be made thorough and complete, and that the spirit of the present application can be fully conveyed to those skilled in the art. In order to clearly express each component in the drawings, the size, such as the width or thickness of the component, is shown to be somewhat enlarged. In addition, although only a part of the components are shown for convenience of description, those skilled in the art will be able to easily understand the rest of the components. In addition, one of ordinary skill in the art may implement the spirit of the present application in various other forms without departing from the technical spirit of the present application.

As used herein, "neutralizing", "inhibiting" means reducing the activity in the presence of an inhibitor, as compared to the activity in the absence of the substance. The decrease in activity can be, for example, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

One embodiment of the present invention relates to a composition for the prevention or treatment of vitiligo or vitiligo metastasis comprising an interferon-inducible T-cell alpha chemoattractant (ITAC) inhibitor.

The composition according to the embodiments can effectively inhibit melanocyte-like cell migration, contribute to the prevention, treatment or improvement of their symptoms of vitiligo or vitiligo metastasis.

The ITAC inhibitor may be a substance that inhibits the expression or activity of the ITAC gene or the action or activity of the ITAC.

In one embodiment, the ITAC inhibitor may be an oligonucleotide that binds at least a portion of the ITAC mRNA or a neutralizing antibody of the ITAC.

The oligonucleotide may be any one or more of? SiRNA, shRNA, and miRNA. The gene expression or activity inhibitor may be any one or more of the siRNA, shRNA and miRNA that induces RNA interference (RNAi) phenomenon. In one aspect of the invention, RNAi phenomenon of inducing interference of the gene mRNA may be used to suppress the mRNA expression of the gene.

miRNA is a kind of endogenous small RNA existing in cells and is derived from hairpin-shaped transcripts derived from DNA which does not synthesize proteins. miRNA binds to the complementary sequence of the 3'-UTR of the target mRNA and induces translation inhibition or destabilization of the mRNA, ultimately acting as a repressor to inhibit protein synthesis of the target mRNA . One miRNA targets several mRNAs, and mRNAs are known to be regulated by multiple miRNAs. Other RNA inducing RNAi phenomenon is short interfering RNA (siRNA), which is short RNA of about 19 to 27 mer, and shRNA having a short hairpin structure.

In an embodiment, the oligonucleotide is double stranded and can be any one of siRNA, shRNA and miRNA.

In the case of neutralizing antibodies of the ITAC, the ITAC inhibitor may be an anti-ITAC antibody.

In one embodiment, the ITAC inhibitor may be a substance that inhibits the expression or activity of the ITAC receptor. In one embodiment, the ITAC receptor may be CXC-chemokine receptor type 3 (CXCR3), CXC- chemokine receptor type 7 (CXCR7) or a mixture thereof.

Substances that inhibit the expression or activity of the ITAC receptor may be oligonucleotides that bind to at least a portion of the ITAC receptor mRNA or neutralizing antibodies of the ITAC receptor.

The oligonucleotide may be any one or more of? SiRNA, shRNA, and miRNA. Since the siRNA, shRNA and miRNA are as described above, description thereof is omitted.

In the case of neutralizing antibodies of the ITAC receptor, the ITAC inhibitor may be an anti-CXCR3 antibody, an anti-CXCR7 antibody or a mixture thereof.

In one example, the ITAC inhibitor may be a substance that inhibits HDAC: histone deacetylase-5 (HDAC5), a downstream effector of ITAC. The substance that inhibits HDAC5 may be, for example, a substance that inhibits HDAC5 expression or activity.

The substance that inhibits the expression or activity of HDAC5 may be, for example, an oligonucleotide that binds to at least a portion of HDAC5 mRNA or a neutralizing antibody of HDAC5.

The oligonucleotide may be any one or more of? SiRNA, shRNA, and miRNA. Since the siRNA, shRNA and miRNA are as described above, description thereof is omitted.

The neutralizing antibody of HDAC5 may be an anti-HDAC5 antibody.

The mRNA sequence of the ITAC may be represented by NM_005409, NM_001302123, or SEQ ID NO: 1. The mRNA sequence of CXCR3 can be represented by NM_001142797, NM_001504 or SEQ ID NO: 2. The mRNA sequence of CXCR7 can be represented by NM_001047841, NM_020311, or SEQ ID NO: 3. The sequence of HDAC5 mRNA may be represented by NP_001015053, NM_005474, NM_139205, or SEQ ID NO: 4.

Compositions according to embodiments of the present invention may be provided in various forms of food additives or functional food. For example, it may be processed into fermented milk, cheese, yoghurt, juice, probiotic and health food, and may be used in the form of various other food additives.

The composition according to the embodiments of the present invention may be a composition for health food. The health food composition inhibits the action of ITAC, inhibits melanocyte-forming cells, and exhibits a low pigmentation effect. In addition, the health food composition may relieve or improve the symptoms or metastasis of vitiligo, vitiligo by inhibiting the action of ITAC.

In embodiments, the health food composition may be formulated as pills, capsules, tablets, granules, caramels or drinks. In other embodiments, it may be processed in the form of a liquid, powder, granules, tablets or tea bags and the like.

The composition may be administered by various methods, such as simple drinking, injection, spray or squeeze.

The composition may contain other ingredients and the like that can give a synergistic effect to the main effect within a range that does not impair the main effect of the present invention. For example, it may further include additives such as perfumes, pigments, fungicides, antioxidants, preservatives, moisturizers, thickeners, inorganic salts, emulsifiers and synthetic polymer materials to improve physical properties. In addition, supplementary ingredients such as water soluble vitamins, oil soluble vitamins, polymer peptides, polymer polysaccharides and seaweed extract may be further included. The above ingredients may be suitably selected and formulated by those skilled in the art according to the dosage form or purpose of use, and the amount thereof may be selected within a range that does not impair the object and effect of the present invention. For example, the addition amount of the components may be 0.01% to 5% by weight, for example 0.01% to 3% by weight based on the total weight of the composition.

The composition according to the embodiments of the present invention may be a pharmaceutical composition. The pharmaceutical composition may inhibit the action of ITAC to inhibit melanocyte-like cell migration, exhibit a hypopigmentation effect, and prevent, treat or alleviate the symptoms or metastasis of vitiligo or vitiligo.

In embodiments, the pharmaceutical composition may be formulated in oral or parenteral dosage forms in various forms, such as solid, semi-solid or liquid, further comprising a commercially available inorganic or inorganic carrier in accordance with conventional methods.

The oral dosage form can be, for example, tablets, pills, granules, soft / light capsules, powders, granules, powders, emulsions, syrups, pellets, and the like. Parenteral dosage forms are, for example, injections, drops, and the like. Agents, ointments, lotions, sprays, suspensions, emulsions, suppositories, and the like. The pharmaceutical composition may further contain preservatives, stabilizers, hydrating or emulsifying accelerators, pharmaceutical auxiliaries such as salts or buffers for controlling osmotic pressure and other therapeutically useful substances.

The pharmaceutical composition may be administered orally, parenteral, topical, transdermal, subcutaneous, rectal, intravenous, intramuscular, intraperitoneal, and the like.

It is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the severity of the symptom, the route of administration chosen, the age, sex, weight and health of the subject. Generally, the dosage of the active ingredient is 0.001 mg / kg / day to 2000 mg / kg / day, for example 0.5 mg / kg / day to 1500 mg / kg / day.

In another aspect, an embodiment of the present invention may provide a composition for detecting vitiligo including a reagent for detecting an ITAC gene expression amount.

The reagent for detecting the expression of the ITAC gene may be one capable of measuring the level of a transcript of the ITAC gene. In one example, the detection reagent may be a primer pair or probe that specifically binds to an ITAC transcript.

When the amount of ITAC expression is increased by the composition compared to the normal group, it can be determined that there is a vitiligo or vitiligo metastasis.

The primer is complementary to the gene mRNA and may include, but is not limited to, a primer pair capable of amplifying the mRNA.

The probe comprises a polynucleotide consisting of the sequence of the gene mRNA; And polynucleotides including 10 or more consecutive nucleotides as fragments of the polynucleotides, but are not limited thereto.

In the above embodiments, the expression level assay of the gene was polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), northern blot analysis, western blot analysis, and dot blot. (dot blot assay), enzyme-linked immunosorbent assay (ELISA), and microarrays such as the TaqManTM probe probe using allele-specific hybridization, allele specific hybridization through polymerization Probe method using -specific hybridization, restriction fragment length polymorphism (RFLP) method using restriction enzyme, dynamic allele-specific hybridization using melting temperature (Tm, melting temperature) Hybridization, pyrosequencing using polymerization, microarray using allele-specific extension, etc. But it is not limited to this.

The sample may be epithelial cells, keratinocytes, melanocytes, mast cells, or fibroblasts isolated from humans, for example For example, it may be a keratinocyte.

Obtaining a sample from a subject may be performed according to a conventional method in the art, and may be performed by a non-invasive method without causing irritation to the skin, such as tape stripping to the skin, but is not limited thereto. In one example, the tape stripping may be performed, for example, 1-20 times, for example 5 times, and a sample may be obtained without causing skin irritation within the above range.

Assaying the expression level of the gene may comprise measuring the level of a transcript of the gene or a protein expressed therefrom in the sample. When using the primer pair or probe, the level of transcript of a gene can be measured by measuring the amount of transcript hybridized with at least one of the primer pair and probe in the sample. When using the antibody, the expressed protein level can be measured by measuring the amount of protein hybridized with the antibody in the sample.

According to another embodiment of the present invention, one or more of a primer pair or probe specifically binding to a transcript of one or more genes of ITAC, CXCR3 and CXCR7, an antibody specifically binding to a protein expressed from the one or more genes It may provide a kit for diagnosing vitiligo.

In another aspect, an embodiment of the present invention may provide a method of diagnosing vitiligo or vitiligo through ITAC expression.

The method detects the amount of ITAC expression in the sample and can be diagnosed as vitiligo when the ITAC is overexpressed compared to normal. The sample may be an epidermal tissue to be diagnosed. In one embodiment, the sample may be one or more of immune cells, such as epithelial cells, keratinocytes, melanocytes, mast cells, etc., isolated from a diagnosis subject.

The overexpression is more than 1.1 times, more than 1.2 times, more than 1.3 times, more than 1.4 times, more than 1.5 times, more than 1.6 times, more than 1.7 times, more than 1.8 times, more than 1.9 times the amount of gene expression compared to normal expression. More than 2.0 times, More than 2.1 times, More than 2.2 times More than 2.3 times, More than 2.4 times, More than 2.5 times, More than 2.6 times, More than 2.7 times, More than 2.8 times, More than 2.9 times, More than 3.0 times, More than 3.1 times, 3.2 3.3 times or more, 3.3 times or more, 3.4 times or more, 3.5 times or more, 3.6 times or more, 3.7 times or more, 3.8 times or more, 3.9 times or more, 4.0 times or more, 4.1 times or more, 4.2 times or more, 4.3 times or more, 4.4 times or more , 4.5 times or more, 4.6 times or more, 4.7 times or more, 4.8 times or more, 4.9 times or more, or 5.0 times or more, 20.0 times or less, 19.5 times or less, 19.0 times or less, 18.5 times or less, 18.0 times or less, 17.5 times or less , 17.0 times or less, 16.5 times or less, 16.0 times or less, 15.5 times or less, 15.0 times or less, 14.5 times or less, 14.0 times or less, 13.5 times or less, 13.0 times or less, 12.5 times or less, 12.0 times or less, 11.5 times or less, 11.0 times or less, 10.5 times or less, 10.0 times or less, 9.5 times or less, 9.0 times or less, 9.0 times or less, 8.5 times or less, 8.0 times or less, 7.5 times or less, 7.0 times or less, 6.5 times or less, 6.0 times or less, 5.5 times Or less than 5.0 times, for example 1.1 to 20.0 times, for example 1.5 to 10.0 times, for example 2 to 8 times can be diagnosed as vitiligo.

The method may further comprise measuring the expression of the ITAC receptor CXCR3 or CXCR7, or both, in addition to ITAC expression. As with ITAC expression, overexpression of CXCR3 or CXCR7 can diagnose vitiligo or vitiligo metastases.

The overexpression is more than 1.1 times, more than 1.2 times, more than 1.3 times, more than 1.4 times, more than 1.5 times, more than 1.6 times, more than 1.7 times, more than 1.8 times, more than 1.9 times the amount of gene expression compared to normal expression. More than 2.0 times, More than 2.1 times, More than 2.2 times More than 2.3 times, More than 2.4 times, More than 2.5 times, More than 2.6 times, More than 2.7 times, More than 2.8 times, More than 2.9 times, More than 3.0 times, More than 3.1 times, 3.2 3.3 times or more, 3.3 times or more, 3.4 times or more, 3.5 times or more, 3.6 times or more, 3.7 times or more, 3.8 times or more, 3.9 times or more, 4.0 times or more, 4.1 times or more, 4.2 times or more, 4.3 times or more, 4.4 times or more , 4.5 times or more, 4.6 times or more, 4.7 times or more, 4.8 times or more, 4.9 times or more, or 5.0 times or more, 20.0 times or less, 19.5 times or less, 19.0 times or less, 18.5 times or less, 18.0 times or less, 17.5 times or less , 17.0 times or less, 16.5 times or less, 16.0 times or less, 15.5 times or less, 15.0 times or less, 14.5 times or less, 14.0 times or less, 13.5 times or less, 13.0 times or less, 12.5 times or less, 12.0 times or less, 11.5 times or less, 11.0 times or less, 10.5 times or less, 10.0 times or less, 9.5 times or less, 9.0 times or less, 9.0 times or less, 8.5 times or less, 8.0 times or less, 7.5 times or less, 7.0 times or less, 6.5 times or less, 6.0 times or less, 5.5 times Or less than 5.0 times, for example 1.1 to 20.0 times, for example 1.5 to 10.0 times, for example 2 to 8 times can be diagnosed as vitiligo.

According to another embodiment of the present invention, a method for screening a prophylactic or therapeutic substance for vitiligo may be provided.

In one embodiment, the method for screening for or preventing vitiligo is to treat a test substance in human epidermal tissue and determine whether the test substance inhibits the expression of one or more genes of ITAC, CXCR3 and CXCR7 in human epidermal tissue. It may include.

The epidermal tissue may be one or more of immune cells, such as epithelial cells, keratinocytes, melanocytes, mast cells, etc., isolated from a diagnosis subject.

In one example, the determination as the prophylactic or therapeutic substance may be determined as a prophylactic or therapeutic substance when the expression of the expression of the untreated group is significantly suppressed. For example, inhibition of expression may be a P <0.05 reduction in expression for untreated groups.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to experimental examples, examples and comparative examples. However, the following Experimental Examples, Examples and Comparative Examples are provided only for the purpose of illustration in order to facilitate understanding of the present invention, but the scope and scope of the present invention is not limited thereto.

ITAC HDAC5  Through p53 Deacetylation  Through melanocyte-forming and Low pigmentation  Induces: Possible Role of ITAC in Pigment-Related Disorders

One important role of chemokines is to mediate immune responses and tumorigenesis through induction of cell migration.

Some chemokines are involved in mouse vitiligo.

Interferon-inducible T-cell alpha chemoattractant (ITAC) has a high binding affinity for CXCR3 compared to other ligands to elicit a strong chemotactic response from immune cells.

The effects of ITAC on melanocyte migration and pigmentation and its association with related disorders are unknown.

Class II histone deacetylases (HDACs), particularly HDAC5, deacetylate p53 proteins involved in various cellular processes, including cell migration and regulate protein stability and activity.

Only ITAC in CXCR3-targeting chemokines efficiently induces migration and hypopigmentation of melanocytes and melanoma cells.

ITAC expression is increased in the epidermis of vitiligo skin.

HDAC5 deacetylates and destabilizes its substrate, p53, to participate in ITAC-mediated cellular processes.

This study is based on in vitro evidence of the migratory and hypopigmentation effects of ITAC on melanocytes by deacetylation of p53 via HDAC5 and on pathological conditions such as melanoma and vitiligo. Provide a review of the role of the ITAC in terms of translation.

Abbreviation

CXCR3: CXC-chemokine receptor type 3; HDAC: histone deacetylase; IP-10: interferon-gamma-induced protein 10; ITAC: interferon-inducible T-cell alpha chemoattractant (ITAC); MIG: monookine induced by interferon-gamma; MMP1: matrix metallopeptidase 1; NHEM: normal human epidermal melanocytes; TYR: tyrosinase; MITF: microphthalmia-associated transcription factor; TRP1: tyrosinase-related protein-1; DCT: dopachrome tautomerase; HMB45: human melanoma black-45

Cell migration regulation and skin disease management by inhibition of ITAC and its receptor

ITAC (chemokine): induces migration of melanocytes, melanoma and immune cells

Signal transduction through ITAC receptors CXCR3, CXCR7

-Cell signaling is induced through the lower signaling factor HDAC5 activity, p53 inhibition

Neutralizing antibody (anti-CXCR3, anti-CXCR7) treatment inhibits ITAC-induced cell migration

Cell migration-related skin diseases: melanoma, vitiligo

-Increased ITAC and receptor expression in metastatic melanoma tissue

-Increased ITAC expression in vitiligo lesions

Thus, inhibiting ITAC and its receptors contributes to the improvement of melanoma and vitiligo.

In this connection, compositions for the management / treatment of certain skin diseases using neutralizing antibodies of ITAC and ITAC receptors.

-Effect of cell migration of various chemokines / cytokines

Use of chemokines to promote the migration of melanocytes

summary

background

Cell migration plays a major role in immune response and tumorigenesis. Interferon-inducible T-cell alpha chemoattractant (ITAC) elicits a strong chemotactic response from immune cells.

purpose

To examine the effects of ITAC on melanocyte migration and pigmentation and its related disorders, and to investigate potential key elements of these processes.

Way

Human melanocytes or melanoma cells were treated with ITAC and subjected to a migration assay. Global gene expression analysis was performed to find genes regulated by ITAC treatment. Knockdown or overexpression experiments with ITAC treatment were used to determine the role of key factors involved in ITAC-induced cellular processes. ITAC expression in inflammation-associated hypopigmentation vitiligo was tested.

result

Only ITAC of CXCR3 ligand induces melanogenesis. ITAC treatment up-regulated histone deacetylase 5 (HDAC5) and down-regulated p53, a known target of HDAC5. Knockdown or overexpression of HDAC5 and p53 confirmed that HDAC5 mediates ITAC-induced migration by decreasing p53 levels through deacetylation. ITAC treatment can also reduce pigmentation with p53- and HDAC5-dependency. Finally, increased migration of human melanoma cells by ITAC treatment and increased ITAC expression in the epidermis of vitiligo skin were identified.

conclusion

This study provides in vitro evidence for the migratory and hypopigmentation effects of ITAC on melanocytes by deacetylation of p53 via HDAC5 and ITAC in pathological conditions such as melanoma and vitiligo. It provides a review on the role of translation and suggests HDAC5 and its substrate, p53, as a potential target for regulating ITAC-induced cellular processes.

Keyword: hdac5, p53, itac, melanocytes, migration, melanoma, vitiligo

Introduction

Lysine (lysine) acetylated suggest that grows a key regulator of the deformed ^first, acetylation / deacetylation upset physiologically active protein after transfer that can affect more than 1750 proteins in human cells. Histoneacetyltransferases transfer acetyl moieties from acetyl coenzyme A to ε-amines of lysine residues to catalyze lysine acetylation on histone proteins, histone deacetylases. (HDAC) catalyzes the opposite process. Unlike histone acetyltransferases with opposite structure and function, HDAC is classified into four distinct classes based on sequence homology to the yeast counterpart: Class I (HDAC1, 2, 3) , And 8), Class II (IIa: HDAC4, 5, 7, 9, and MITR; IIb: HDAC6 and 10), Class III (SIRT1-7), and Class IV (HDAC11). ² Class I HDACs are essentially expressed in the nucleus and found in all tissues, and Class II HDACs exhibit tissue specific expression patterns and involve nucleocytoplasmic shuttling, which is responsible for deacetylation of non-histone proteins. It suggests that you are involved. Among non-histone proteins, p53, a transcription factor that functions in cell growth arrest, apoptosis, and senescence, is a representative protein that is stably regulated by lysine deacetylation via HDAC. ^3-4 It has also recently been reported that HDAC5 acts p53dp by deacetylating lysine 120 (K120). ⁵

Although HDAC and its targets have been studied relatively well, the factors that activate particular HDACs are less understood. Because HDAC participates in various cellular processes, its dysregulation is associated with the development of cancer and inflammatory diseases. Although HDAC inhibitors have been studied in clinical trials for the treatment of these diseases, the molecular basis for HDAC inhibition in patients is not fully known. ⁶ HDAC has been shown to be important for the motility of various cells and tumors through deacetylation of motility related proteins such as alpha-tubulin and cortactin. ^7-8 The discovery and potential role in HDAC metastasis and inflammation suggest a relationship between HDAC and disease processes involved in cell migration.

In the skin, immune cells and skin cells secrete a variety of cytokines and chemokines in response to steady state and environmental stresses. In particular, exposure to UVB wavelengths causes dysregulation of melanocyte-microenvironment through the secreted factors, and causes hyper-proliferation and migration of melanocytes to abnormal regions. ⁹ However, little is reported about the effects of chemokines on mature melanogenesis. We demonstrated that SDF1, a skin-derived chemokine, induces chemotaxis in mature melanocytes. ¹⁰ other chemokines, in particular CXC-chemokine receptor type 3 (CXCR3) -targeting chemokines, monocaine (MIG) induced by interferon-g, interferon-g inducing protein (IP-10), and interferon-induced T-cells Alpha chemotactic factor (ITAC) plays an important role in the recruitment of immune cells of other subtypes to the site of inflammation. ¹¹ Interestingly, the expression patterns and binding affinity of these chemokines differ: IP-10 and ITAC are expressed in basal keratinocytes, but MIG is expressed in dermal infiltrate, and ITAC is expressed in IPX in CXCR3. Binds with higher affinity than 10 or MIG. ¹³ MIG and IP-10 are involved in delayed pigmentation under chronic UVB exposure and ¹⁴ induce pigmentation in co-culture of keratinocytes and melanocytes, but ¹⁵ melanocyte migration and pigmentation The role of ITAC in is unknown. We predicted that ITAC would play a distinct role in these processes based on the fact that ITAC exhibited different expression patterns and receptor-binding activity compared to other CXCR3 ligands.

In this study, we established the effects of ITAC on human melanocytes and melanoma migration. We identified HDAC5, a Class IIa HDAC, as a mediator of ITAC-induced melanogenesis cell migration and p53 as a deacetylation target of HDAC5. We also demonstrated that ITAC treatment reduced pigmentation in a p53- and HDAC5-dependent manner and increased ITAC expression in the epidermis of vitiligo skin.

Materials and methods

Cell culture

NHEM derived from intermediate pigmented skin (Cascade Biologics, Portland, OR) and containing 5% CO ₂ in medium 254 supplemented with human melanocyte growth aid (HMGS; Life Technologies, Carlsbad, Calif.) Incubation was in a wet incubator. NHEMs were treated with the indicated chemokines of varying concentrations (Table S1; R & D R & D system, Minneapolis, MN).

Cell migration assay

Transfer assays were performed in a Boyden chamber after treatment indicated on the cells according to published method ¹⁰ . In selected experiments, NHEMs were pretreated with 50 μg / ml of neutralizing antibody against CXCR3 (R & D system), CXCR7 (Biolegend, San Diego, Calif.), Or each isotype-matched control antibody. Specific protocols for the Boyden chamber assay are provided in the supplementary information.

Western blot analysis and transient transfection

Western blot was performed using 25 mg of cell eluate. Specific protocols and antibody information are described in the supplementary information. 2 mg samples of plasmid DNA for siRNA 50nM or HDAC5-Myc and p53-Flag (GeneCopoeia, Rockville, MD) for HDAC5, HDAC9 and p53 (Dharmacon, Lafayette, Co.), respectively, were prepared using Lipofectamine RNAiMAX or Lipofectamine 2000 (Invitrogen, Washington, Cells) were transfected for 48 hours according to manufacturer's instructions.

Microarray analysis

NHEM was treated with 50 ng / ml of ITAC for 48 hours and total RNA was isolated using TRIzol reagent (Gibco-BRL, Gaithersburg, MD). 15 ng of total RNA was used for analysis. Microarray analysis methods are described in detail in Supplementary Information.

Quantitative real-time PCR analysis

2 μg of total RNA was used for cDNA synthesis using random hexamers and reverse transcriptase (Promega, Madison, Wis.) According to the manufacturer's instructions. Specific protocols are given in the supplementary information.

Melanin Assay

Cells were treated with ITAC 50 or 200 ng / ml for 4 days or transfected with various concentrations of HDAC5-Myc expressing plasmids for 2 days. Specific methods of cell elution and melanin assays are provided in the supplemental information.

Immunohistochemical Analysis of Human Vitiligo Skin Immunohistochemical  analysis of human vitiligo skins)

Two patients diagnosed with vitiligo were included in the study after written cooperation. Three pairs of hypopigmented and normal pigmented skin samples were biopsied with a 3 mm-diameter punch and immunohistochemistry using anti-ITAC (R & D system) and anti-HMB45 (melanocyte marker) antibodies (GeneTex, Irvine, CA) Analyzed. The study was approved by the Institutional Review Board of Dongguk University Ilsan Hospital and was in accordance with the Declaration of Helsinki.

Statistical analysis

All data are expressed as mean ± SD. The Student's t-test was used to analyze the differences between the two groups.

result

ITAC Induces Human Epidermal Melanocytes

Dermal cells such as macrophages, Langerhans cells, gamma delta-T cells, mast cells, keratinocytes and melanocytes produce various chemokines (Table 1, Supplemental Information). Reference). One important role of chemokines is to mediate cell migration. We confirmed the ability of skin-derived chemokines to induce migration of normal human epidermal melanocytes (NHEM) using a chamber assay. NHEMs migrate to FK506, a known inducer of melanogenesis cell migration, and SDF1, which appears to induce NHEM migration (see FIG. 8, Supplemental Information). Many other chemokines, such as ¹⁰ CTACK, PARC, ITAC, RANTES, TARC and Lptn, also increased significantly in untreated groups at the same concentration of SDF1 (FIG. 8). We noted the ITAC as a CXCR3-targeting chemokine involved in recruiting immune subtypes of other subtypes to the site of inflammation, since only ITAC, not MIG and IP-10, induced NHEM migration (Figure 8, Figures 1a, b). ). In addition, the migration effect of ITAC was confirmed in three different human melanoma cell lines, and the migration of these cells was significantly increased (FIG. 9). Given that MIG and IP-10 are involved in conventional pigmentation, while ITAC is unknown, ITAC will play a distinct role in melanogenesis, i.e. by regulating melanocyte-like cell migration. . To elucidate the ITAC-induced NHEM mechanism, we first assessed the levels of the ITAC receptors CXCR3 and CXCR7 ¹⁶ , which initiate ITAC-related signals using specific antibodies, and confirmed that both proteins were expressed in NHEM. (FIG. 1C). To determine the respective involvement in ITAC-induced migration, we performed transfer assays after removal of endogenous CXCR3 or CXCR7 using specific neutralizing antibodies. ITAC-induced migration was significantly reduced after treatment of each of the two neutralizing antibodies as compared to each isotype control (FIG. 1D; lane 4 vs. 5 and lane 6 vs. 7), indicating that both CXCR3 and CXCR7 were ITAC-induced melanin Suggests that it is involved in constituent cell migration. To investigate whether the expression of ITAC and both receptors changes in pathological conditions such as melanoma, we integrate the analysis of the publicly available Microarray Gene Expression Omnibus (GEO) dataset and the expression of ITAC and both receptors is melanocytes. In contrast to (GSE29377), it was confirmed that the melanoma tissue tends to increase. Although there is a variation between patients, these statistical results suggest that ITAC and its receptors are closely related and involved in some melanoma pathologies.

ITAC up-regulates mRNA expression of migration-related genes in NHEMs.

Global gene expression analysis was performed using control or ITAC-treated NHEMs to understand the underlying signaling pathways of ITAC-induced NHEM migration. ITAC treatment up-regulated 391 genes and down-regulated 350 genes with more than two-fold changes compared to untreated groups (Table 2). Based on functional classification analysis, we observed that many cell motility-related genes were upregulated, and several melanogenesis-related genes such as tyrosinase (TYR) and dopachrometomerase (DCT) were downregulated ( Figure 2a; Table 2). Of the motility-related genes (23), we selected two genes, HDAC5 and HDAC9, which, as Class IIa members, were able to regulate protein activity by deacetylating non-histone proteins and suggested to be involved in cell-motility. We found that the mRNA levels of these two genes were significantly increased by ITAC treatment in RT-qPCT analysis (Figure 2b, c).

HDAC5 mediates ITAC-induced migration of NHEMs.

To confirm that these genes are involved in ITAC-induced migration, siRNAs specific for HDAC5 and HDAC9 were knocked down HDAC5 and HDAC9 and chamber assays performed in the presence or absence of ITAC were performed. Each gene knockdown was confirmed by measuring mRNA expression levels by RT-qPCT (FIG. 10). NHEMs responded to FK506 with about a 2-fold increase in migration as expected (FIG. 3A, lane 2). Compared with scrambled siRNA control that induced NHEM migration without ITAC (lane 3), HDAC5 knockdown reduced migration but HDAC9 knockdown had no effect (lanes 4 and 5). In the presence of ITAC, only HDAC5 significantly inhibited migration compared to the scrambled control (lane 6 vs. 7), and similar levels of migration were observed for HDAC5 knockdown without ITAC (lane 4 vs. 7), indicating that ITAC-induced migration was Mostly mediated by HDAC5. We therefore noted the role of HDAC5 in melanocyte migration. HDAC5 overexpression using HDAC5-Myc expression plasmids significantly increased NHEM migration (FIG. 3B, first two lanes). By ITAC treatment, migration of control cells was markedly increased to a level comparable to that of HDAC5 overexpression, and HDAC5 overexpressing cells showed an additive migration effect (FIG. 3b, the last two lanes). Upregulation via HDAC5 overexpression or ITAC treatment effectively mediates NHEM migration. We confirmed that HDAC5 protein levels increased with upregulation of HDAC5 mRNA after ITAC treatment (FIG. 3C).

HDAC5 affects the p53 acetylation status in ITAC-treated NHEMs.

The downstream target of HDAC5 was identified in ITAC-induced migration. We first tested alpha-tubulin, which is deacetylated by HDAC5 in neurons as a migration-related protein. ⁷ However, the acetylation status of alpha-tubulin was not affected by ITAC treatment (FIG. 11A) or HDAC5 deletion in NHEM (FIG. 11B), indicating that alpha-tubulin induced HDAC5 in ITAC-induced melanogenesis cell migration. Imply that it is not a target. Of the non-histone proteins regulated by acetylation and deacetylation, p53 plays a role in cell migration. ¹⁷ p53 is an HDAC5-interacting protein that has been shown to overexpress HDAC5 in cells by immunoaffinity isolation, ¹⁸ HDAC5 directly deacetylates p120 K120, resulting in a ⁵ MDM2-dependent analog Leads to quitylation and degradation pathways. Therefore, it was examined whether acetylated p53 or total p53 levels were affected when HDAC5 mRNA and protein levels were increased by ITAC treatment (FIGS. 2B, 3C). In ITAC treated NHEMs, the amount of acetylated p53 revealed by the antibody recognizing at least two acetylated lysine residues 373 and 382 of p53 showed a slight decrease in total p53 (FIG. 4A). In the human melanoma cell line SK-MEL-28, which showed increased migration by ITAC treatment (FIG. 9B), total p53 protein was significantly reduced by ITAC treatment (FIG. 11C). HDAC5 was overexpressed or knocked down to examine the effect of HDAC5 on p53 expression levels. HDAC5 overexpression corresponded to decreases in acetylated p53 and total p53 levels (FIG. 4B) and both p53 increased when HDAC5 knocked down to siRNA (FIG. 4C). This data suggests that the acetylation status of p53 is regulated by HDAC5 and that downregulation of p53 is involved in ITAC-induced cell activity.

p53 overexpression inhibits ITAC-induced and HDAC5-mediated NHEM migration.

After the ITAC treatment, HDAC5 dependently overexpressed HDAC5, p53, or both, and performed Bodenchamber assay to determine whether p53 affects NHEM migration. HDAC5 overexpression in the absence of ITAC significantly increased cell migration (FIG. 5a, lane 2), but this effect was lost by p53 overexpression (lane 4), suggesting an involvement of p53 in HDAC5-induced cell migration ( Lane 5 vs. 7). HDAC5 overexpression in ITAC treated cells increased cell migration more compared to mock-treated controls (lane 5 vs. 6) and this effect was reduced by p53 overexpression (lane 6 vs. 8). ). These data suggest that p53 is a downstream effector of HDAC5 and is involved in ITAC- and HDAC5-induced cell migration. P53 transcription targets also known to be negatively regulated by p53, migration-related gene matrix metallopeptidase 1 (MMP1) ²⁰ and collagen ²¹ Reversed upregulation by ITAC treatment as measured by microarray analysis of MMP1 (Table 2) and RT-qPCR (FIG. 5B), indicating that the transcriptional activity of p53 is reduced in ITAC-treated cells.

ITAC treatment results in low pigmentation of NHEM through p53 downregulation.

Some CXCR3 ligands, such as IP-10 and MIG, are upregulated at delayed pigmented sites. ^{In 14} ITAC-treated SK-MEL28 cells, the amount of TYR protein decreased markedly with decreasing p53 expression level (FIG. 11C). To determine whether these CXCR3 ligands, including ITACs, were effective in melanogenesis, each NHEM was treated with 200 ng / ml. Consistent with the results of global gene expression analysis with down-regulation of TYR and DCT in ITAC treated cells (FIG. 2A, Table 2), ITAC is the melanogenesis-related gene TYR, tyrosinase-related protein-1. ) And DCT mRNA expression levels were reduced (FIG. 12). However, MIG and IP-10 did not alter the mRNA levels of these genes (FIG. 12), indicating a different effect on melanogenesis in the CXCR3 ligand, similar to cell migration results (FIG. 1A). Furthermore, after treatment with 200ng / ml of ITAC for 4 days in NHEM, it was measured that the melanin amount was markedly dose-dependently (FIG. 6A). Conventional p53 has been reported to play a role in the skin pigmentation after UV irradiation by upregulation of pigmentation-related genes such as TYR and TRP1. ^22-23 present inventors that the test can be ITAC reduces the p53- mediated pigmentation after UV irradiation. The amount of melanin in UV-treated NHEMs increased, and the total p53 protein or acetylated p53 protein levels increased with this (Figure 6b, lane 1 vs 2). The effect after these UV irradiations was attenuated by ITAC treatment, resulting in a decrease in melanin amount and p53 protein levels (FIG. 6B, lane 2 vs 3). The effect of p53 in ITAC-induced hypopigmentation was also confirmed by analyzing melanogenesis-related gene expression after siRNA treatment in the presence of p53 ITAC. As a result, these gene expressions were markedly decreased by the p53 siRNA treatment compared to the scrambled control (FIG. 13, lanes 1 and 2) and by the ITAC treated groups (TYR and TRP1; FIG. 13, lane 3), but with the p53 knockdown effect. Were similar before or after ITAC (FIG. 13, lane 2 vs. 4). In addition, HDAC5 overexpression in NHEM was found to induce hypopigmentation (FIG. 6C). Overall, these results suggest that ITAC-induced hypopigmentation is mediated in part by p53 deacetylation by HDAC5 and thus destabilization in melanocytes.

Although ITAC causes an anti-melanogenic effect in p53-associated pigmentation, ITAC overproduction may be related to hypopigmentation disorders because of its migration to immune and melanogenic cells. Vitiligo is a common type of immune-associated hypopigmentation disorder. We investigated whether ITAC expression changes in vitiligo lesions through immunohistochemical analysis using anti-ITAC antibodies. As expected, melanin (arrows) and melanocytes (triangles) were observed along the epidermal basement membrane only in normal skin (FIG. 7, N # 1). Interestingly, ITAC expression in vitiligo lesion epidermis was significantly upregulated and abundant compared to normal skin where little ITAC was found (Figure 7, L # 1, L # 2, vs N # 1). Consistent with ITAC expression analysis data in human melanoma tissue (GSE29377), these results indicate that ITAC can be upregulated in pathologies including vitiligo and melanoma and affect their cell migration and melanogenesis. It suggests that they are involved in the invention.

discussion

In this study, it was shown that ITAC, a chemokine produced by skin cells, upregulates HDAC5 expression and consequently downregulates p53 levels through deacetylation, leading to melanogenesis. ITAC treatment increased melanocyte migration as efficiently as FK506 treatment, which was mediated by known receptors CXCR3 and CXCR7. Comparing melanocytes with melanoma tissue based on global genetic analysis of the GEO database, ITAC and both receptors CXCR3 and CXCR7 tended to increase in melanoma tissue. The finding that a reduction in CXCR3 or CXCR7 completely blocks NHEM migration suggests that CXCR3 and CXCR7 form a heterodimer to mediate melanogenesis. Heterodimerization between CXCRs (eg CXCR4 and CXCR7) has been previously studied. ²⁴ However, the heterodimerization that occurs between CXCR3 and CXCR7 in melanocytes should be explained further.

It was found that HDAC5 and HDAC9 levels increased in ITAC treated cells compared to untreated controls. Only HDAC5 of these genes was involved in ITAC-induced NHEM migration (FIG. 3A). Based on previous reports, ⁷ we first tested alpha-tubulin as a target of HDAC5 to account for the shifting effects of ITAC. ITAC treatment and siRNA knockdown of HDAC5 were not associated with changes in acetylated alpha-tubulin levels (FIG. 11A, B), suggesting that other targets are involved in ITAC-induced melanogenesis. p53 is an important regulatory protein in a variety of signal pathways, including DNA damage response ²⁵ and migration of various cell types, including tumor cells, ²⁶ fibroblasts, ²⁷ and keratinocytes ²⁸ . The activity and stability of p53 is highly dependent on its acetylation state, which is antagonized by certain HDAC protein complexes, including HDAC1 or SIRT1. HDAC1 is primarily present in the nucleus, while other HDACs can regulate p53 acetylation in the cytoplasm. ^{4 It} has recently been reported that HDAC5 regulates p53 acetylation at K120. Based on the hypothesis that ⁵ p53 is deacetylated by HDAC5 in ITAC-induced melanogenesis cell migration, HDAC5 reduces acetylated p53 protein and total p53 protein (FIG. 4b, c), thereby reducing melanogenesis cell migration. Mediation (FIG. 5A). Although specific lysine residues that were deacetylated targets by HDAC5 were not identified in this study, it was expected that not only 120 of p53 but also lysine residues of 373/382 could be the target position. To find the ⁵ acetylated p53 protein, an anti-acetylated p53 antibody that was thought to recognize at least two acetylated lysine residues at positions 373 and 382 was used, and the acetylated p53 on these residues was subject to HDAC5 overexpression or knockdown. By down- or up-regulation, respectively (FIG. 4B, c), suggesting that these residues are possible target sites for deacetylation by HDAC5.

In addition to migration, ITAC treatment was observed to induce hypopigmentation of normal and UV-treated melanocytes (Figures 6a, b). ITAC treatment down-regulated melanogenesis-related gene expression and p53 protein levels (FIG. 2; FIG. 4A, FIG. 6B). p53 was up-regulated by UV exposure and induced melanogenesis by increasing TYR and TRP1 expression. ^22-23 p53 was found to be involved in ITAC-induced hypopigmentation (FIG. 6B). Since HDAC5 is the primary regulator of p53 stability through deacetylation in ITAC-induced melanogenesis, and HDAC5 overexpression is associated with depigmentation (FIG. 6C), HDAC5 is p53 deacetylated, resulting in melanogenesis- Relevant genes may be directly involved in ITAC-induced hypopigmentation through down-regulation. Genes affected by p53 down-regulation may be the cause of melanogenesis cell migration, hypopigmentation or both after ITAC treatment. Collagens and MMP1, cell migration-related genes negatively regulated by p53, were found to be up-regulated in ITAC-treated cells (FIG. 5B; Table 2). These up-regulated genes may also be involved in ITAC-induced hypopigmentation. These data also suggest that destabilization of p53 via deacetylation via Class IIa HDAC, especially HDAC5, plays an important role in ITAC-induced cellular processes such as migration and hypopigmentation.

Acute and chronic dermatitis can cause hypopigmentation or hyperpigmentation, depending on the interaction between environmental and genetic factors. Among the ²⁹ CXCR3 ligands, MIG and IP-10 are up-regulated at delayed pigmented sites in which inflammatory cells are depressed, resulting in melanocyte activity and chronic melanin synthesis. ¹⁴ In contrast, ITAC is upregulated in the epidermis of low pigmented disease vitiligo skin (FIG. 7), and IP-10 is not deregulated when compared to healthy controls, ³⁰ which is a different function between CXCR3 ligands and humans of ITAC. It suggests potential involvement in the development of vitiligo.

Previously, it has been claimed that MIG and IP-10 are important in mouse vitiligo and that ITAC does not play a major role. ³¹ These differences are due to epidermal structural differences between mice and humans, for example: 1) the human epidermis is made up of multiple layers, creating a different environment than the thin epidermis of the mouse; Although present in all, mouse melanocytes are present only in the vesicle area. ITAC secreted from keratinocytes on the basement membrane will directly affect melanocytes in the intervesicle region where no melanocytes are present in the mouse.

In the co-culture system of human keratinocytes and melanocytes, low concentrations (10 ng / ml) of MIG, IP-10 and ITAC increased the amount of melanin. ^15, however, three kinds of chemokine thereof Jeodong furnace melanin forming cells only produce melanin in the culture system that had no effect on related gene expression (data not yet displayed), only ITAC them down from the high concentration (200ng / ml) - was controlled (Figure 12 ). The observation of this difference may be due to soluble factors secreted by keratinocytes in the co-culture system after ITAC treatment, and soluble factors may indirectly affect melanocyte activation. MIG and IP-10 had no effect on pigmentation or melanogenesis at high concentrations, suggesting that they would not directly affect melanocytes and would play a different role than ITAC. Although MIG, IP-10, and ITAC are closely related interferon-g-reactive chemokines and mediate signals for attracting T lymphocytes through CXCR3, their effects on melanocyte migration and melanogenesis are influenced by cellular context. Depends on This finding is explained in part by their different binding affinity for CXCR3, different ITAC co-receptors on melanocytes such as CXCR7, and their different expression levels in pathological conditions. Given that ITAC is primarily secreted by keratinocytes on the basement membrane around lesion spots and by skin cells in various types of inflammatory skin diseases, ¹² its downstream effectors, HDAC5 and p53, act on mature melanocytes. This may be important to control. Several pigmentation-related disorders such as solar lentigo, melasma, and melanoma are represented by and exacerbated by melanocyte condensation. This study highlights the possibility that p53 down-regulation via HDAC5 stimulation is one of the strategies to mitigate these disorders by inducing migration of condensed melanocytes and simultaneously inducing hypopigmentation.

references

Choudhary C, Kumar C, Gnad F et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 2009; 325 : 834-40.

Yang XJ, Gregoire S. Class II histone deacetylases: from sequence to function, regulation, and clinical implication. Mol Cell Biol 2005; 25 : 2873-84.

3. Luo J, Su F, Chen D et al . Deacetylation of p53 modulates its effect on cell growth and apoptosis. Nature 2000; 408 : 377-81.

4. Bode AM, Dong Z. Post-translational modification of p53 in tumorigenesis. Nat Rev Cancer 2004; 4 : 793-805.

5. Sen N, Kumari R, Singh MI et al. HDAC5, a key component in temporal regulation of p53-mediated transactivation in response to genotoxic stress. Mol Cell 2013; 52 : 406-20.

6.Huang L. Targeting histone deacetylases for the treatment of cancer and inflammatory diseases. J Cell Physiol 2006; 209 : 611-6.

7. Marcus AI, Zhou J, O'Brate A et al. The synergistic combination of the farnesyl transferase inhibitor lonafarnib and paclitaxel enhances tubulin acetylation and requires a functional tubulin deacetylase. Cancer Res 2005; 65 : 3883-93.

8. Janke C, Bulinski JC. Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions. Nat Rev Mol Cell Biol 2011; 12 : 773-86.

9. Zaidi MR, Davis S, Noonan FP et al. Interferon-γ links ultraviolet radiation to melanomagenesis in mice. Nature 2011; 469 : 548-53.

10. Lee E, Han J, Kim K et al. CXCR7 mediates SDF1-induced melanocyte migration. Pigment Cell Melanoma Res 2013; 26 : 58-66.

11. Wenzel J, Bekisch B, Uerlich M et al. Type I interferon-associated recruitment of cytotoxic lymphocytes: a common mechanism in regressive melanocytic lesions. Am J Clin Pathol 2005; 124 : 37-48.

12. Flier J, Boorsma DM, van Beek PJ et al. Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation. J Pathol 2001; 194 : 398-405.

13. Booth V, Clark-Lewis I, Sykes BD. NMR structure of CXCR3 binding chemokine CXCL11 (ITAC). Protein Sci 2004; 13 : 2022-8.

14.Aoki H, Moro O. Upregulation of the IFN-gamma-stimulated genes in the development of delayed pigmented spots on the dorsal skin of F1 mice of HR-1xHR / De. J Invest Dermatol 2005; 124 : 1053-61.

15. Kurata R, Fujita F, Oonishi K et al. Inhibition of the CXCR3-mediated pathway suppresses ultraviolet B-induced pigmentation and erythema in skin. Br J Dermatol 2010; 163 : 593-602.

16. Lasagni L, Francalanci M, Annunziato F et al. An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig, and I-TAC, and acts as functional receptor for platelet factor 4.J Exp Med 2003; 197 : 1537-49.

17. Alexandrova A, Ivanov A, Chumakov P et al. Changes in p53 expression in mouse fibroblasts can modify motility and extracellular matrix organization. Oncogene 2000; 19 : 5826-30.

18. Greco ™, Yu F, Guise AJ et al. Nuclear import of histone deacetylase 5 by requisite nuclear localization signal phosphorylation. Mol Cell Proteomics 2011; 10 : M110.004317.

19.Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett 1997; 420 : 25-7.

20. Sun Y, Sun YI, Wenger L et al. p53 down-regulates human matrix metalloproteinase-1 (collagenase-1) gene expression. J Biol Chem 1999; 274 : 11535-40.

21. Muller PA, Vousden KH, Norman JC. p53 and its mutants in tumor cell migration and invasion. J Cell Biol 2011; 192 : 209-18.

22. Khlgatian MK, Hadshiew IM, Asawanonda P et al. Tyrosinase gene expression is regulated by p53. J Invest Dermatol 2002; 118 : 126-32.

23. Nylander K, Bourdon JC, Bray SE et al. Transcriptional activation of tyrosinase and TRP-1 by p53 links UV irradiation to the protective tanning response. J Pathol 2000; 190 : 39-46.

24. Levoye A, Balabanian K, Baleux F et al. CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling. Blood 2009; 113 : 6085-93.

25. Gadea G, Lapasset L, Gauthier-Rouviere C et al. Regulation of Cdc42-mediated morphological effects: a novel function for p53. EMBO J 2002; 21 : 2373-82.

26. Muller PA, Vousden KH. p53 mutations in cancer. Nat Cell Biol 2013; 15 : 2-8.

27. Guo F, Zheng Y. Rho family GTPases cooperate with p53 deletion to promote primary mouse embryonic fibroblast cell invasion. Oncogene 2004; 23 : 5577-85.

28. Sablina AA, Chumakovm PM, Kopnin MP. Tumor suppressor p53 and its homologue p73alpha affect cell migration. J Biol Chem 2003; 278 : 27362-71.

29. Slomanoki A, Paus R. Melanogenesis is coupled to murine anagen: toward new concepts for the role of melanocytes and the regulation of melanogenesis in hair growth. J Invest Dermatol 1993; 101 : 905-75.

30. Regazzetti C, Joly F, Marty C et al. Transcriptional analysis of vitiligo skin reveals the alteration of WNT Pathway: A promising target for repigmenting vitiligo patients. J Invest Dermatol 2015; 135 : 3105-14.

31. Miekus K, Jarocha D, Trzyna E et al. Role of I-TAC-binding receptors CXCR3 and CXCR7 in proliferation, activation of intracellular signaling pathways and migration of various tumor cell lines. Folia Histochem Cytobiol 2010; 48 : 104-11.

Figure 1. ITAC induces migration of human epidermal melanocytes

Migration of normal human neonatal epidermal melanocytes (NHEM) was measured using a Boyden chamber assay after treatment with 200 ng / ml MIG, IP-10 or ITAC (a) or various concentrations of ITAC (b). Cells were allowed to migrate for 24 hours. The data represent three independent experiments (* P <0.05, ** P <0.01). (c) Protein expression levels of CXCR3 and CXCR7 were measured by Western blot. Western blot analysis was performed repeatedly and representative images are shown. (d) NHEM was pretreated with neutralizing antibody of CXCR3 or CXCR7 for 30 minutes, and migration analysis was performed in the presence of ITAC. Anti-IgG (CXCR3) and anti-IgG (CXCR7) antibodies were used as isotype control antibodies of anti-CXCR3 and anti-CXCR7 neutralizing antibodies, respectively. The data represent three independent experiments (*** P <0.001).

Figure 2. ITAC up-regulates mRNA expression of migration-related genes in NHEMs.

NHEMs were treated for 48 hours with 50 ng / ml ITAC and microarray analysis was performed using 50ng total RNA. (a) Biological processes significantly up- or down-regulated by ITAC treatment were grouped using gene ontology analysis (GO) analysis via DAVID. BH P value: Benjamini-Hochberg multiple test-corrected p value. (bc) HDAC5 (b) and HDAC9 (c) mRNA levels in NHEMs measured by RT-qPCR. The data represent three independent experiments (* P <0.05).

3. HDAC5 mediates ITAC-induction in NHEMs.

(a) siRNA was transfected in NHEM for 48 hours. Cells were allowed to migrate for 24 hours in the absence or presence of 50 ng / ml ITAC after transfection and fixed for counting. Scrambled siRNA and FK506 were used as negative and positive controls, respectively. The data represent three independent experiments (* P <0.05, *** P <0.001). (b) HDAC5-Myc plasmid was introduced into NHEM for 48 hours. Cells were allowed to migrate for 24 hours in the absence or presence of 50ng / ml ITAC. The migrated cells were counted. The data represent three independent experiments (* P <0.05, *** P <0.001). (c) HDAC5 protein expression levels were measured by Western blot after 48 hours of 50 ng / ml ITAC treatment. Western blot analysis was performed repeatedly and representative images are shown. Relative band intensity.

4. HDAC5 affects the p53 acetylation status in ITAC-treated NHEMs.

(a) NHEMs were treated with 50 ng / ml ITAC for 48 hours. The levels of acetylated p53 protein and total p53 protein were measured by Western blot with the indicated antibodies. HDAC5-Myc overexpression construct (b) or siRNA for HDAC5 (c) were transfected into NHEM for 48 hours. Mock siRNA and scrambled siRNA were used as controls for HDAC5-Myc and HDAC5-specific siRNA, respectively. The levels of acetylated p53 protein and total p53 protein were measured by Western blot with the indicated antibodies. GAPDH was used as loading control. Western blot analysis was performed repeatedly and representative images are shown. Relative band intensity. a-Ac-p53, anti-acetylated p53 antibody

5. p53 overexpression inhibits ITAC-induced and HDAC5-mediated NHEM migration.

(a) NHEMs were transfected with mock-transfection (lanes 1, 5), or HDAC5-Myc expressing plasmids (lanes 2, 6), p53-flag (lanes 3, 7) or both (lanes 4, 8). Specified. 48 hours after transfection were allowed to migrate for 24 hours in the absence or presence of 50ng / ml ITAC and fixed for counting. Data is representative of three independent experiments (* P <0.05). (b) mRNA levels of MMP1 in NHEM were measured before and after ITAC treatment with RT-qPCT. The value was normalized to GAPDH. The data represent three independent experiments (* P <0.05).

Figure 6. ITAC treatment leads to low pigmentation of NHEM through p53 downregulation.

(a) NHEMs were treated with various concentrations of ITAC for 4 days. Each pellet was visualized to determine the amount of melanin. Data is representative of three independent experiments (* P <0.05). (b) exposed and maintained twice with UV (20 mJ / cm ² ) in NHEM for 2 weeks. ITAC (50 ng / ml) was treated every 3 days after exposure. Data is representative of three independent experiments (* P <0.05). The levels of acetylated p53 protein and total p53 protein were measured by Western blot with the indicated antibodies. GAPDH was used as loading control. Western blot analysis was performed repeatedly and representative images are shown. Relative band intensity. a-Ac-p53, anti-acetylated p53 antibody. (c) NHEMs were transfected for 48 hours with mock-transfection or various concentrations of HDAC5-Myc expression plasmids. Each pellet was visualized after centrifugation and the melanin amount was measured. The data represent three independent experiments (* P <0.05).

ITAC is abundant in the epidermis of vitiligo skin.

Skin samples (# 1 and # 2) of normal (N) or hypopigmented lesions (L) from two patients with vitiligo were stained with anti-ITAC antibody (green) and anti-HMB45 (red) and the nucleus was DAPI. Stained (blue). Bright field images were taken to visualize melanin in the tissues (arrows). Triangles represent melanocytes. HMB45, human melanoma black-45. Scale bar 50 μm.

<Supplement information>

ITAC On HDAC5  By p53 Destabilization  Through melanocyte-forming and Low pigmentation  Induces: Possible Roles in Pigment-Related Abnormalities of ITAC

Supplementary Materials and Methods:

Cell culture

Human melanoma MNT-1 cell line was provided by Dr. Lee Ae-young of Dongguk University who received the cell line from Dr. Vincent J. Hearing of National Institutes of Health, Bethesda, Maryland, USA. Cells were incubated at 37 ° C. in DMEM (Lonza, Basel, Switzerland) containing 20% FBS and antibodies. WM266-4 human melanoma cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). SK-Mel-28 human melanoma cell line was obtained from Korean Cell Line Bank (KCLB, Seoul, Korea). These cells were maintained at 37 ° C. in EMEM medium (ATCC) containing 10% FBS and antibodies.

Western blot analysis

Cells (2 × 10 ⁶ ) were removed in elution buffer (20 mM Tris [pH 7.5], 0.1% Triton X-100, 0.5% deoxycholate, 1 mM PMSF, 10 μg / ml aprotinin, and 10 μg / ml leupeptin). Incubated for 10 minutes at ℃, and centrifuged for 5 minutes at 13,000 rpm. Protein concentrations were measured using a BCA assay kit (BCA assay kit; Sigma, St. Louis, Mo.) according to the manufacturer's instructions. 25 mg of total protein was separated by SDS / PAGE and transferred to PVDF membrane (Invitrogen, Washington, DC). After blocking 0.5 hours at room temperature with 0.3% casein containing PBS, the membranes were incubated overnight at 4 ° C. in PBS containing 0.1% Tween 20 with 3-5 μg / ml primary antibody. Signal strength was quantified using ImagePro Plus software version 4.0 and normalized to GAPDH. Each antibody information is as follows: anti-CXCR3, anti-CXCR7 (Abcam, Cambridge, UK), anti-HDAC5, anti-p53 (Cell Signaling, Danvers, MA), anti-acetylated p53 (Millipore, MA), Anti-alpha-tubulin, anti-acetylated alpha-tubulin (Sigma-Aldrich, St. Louis, MO), anti-ITAC (R & D system, Minneapolis, MN), anti-HMB45 (GeneTex, Irvine, CA,) , Anti-GAPDH antibody (Santa Cruz Biotechnology, Dollas, TX). All data were obtained from at least two independent experiments in three runs.

Cell migration assays of primary melanocytes and melanoma cell lines

Filters in a Boyden chamber (pore size, 8 μg; Corning Inc., Corning, NY) were coated with type IV collagen for 3 hours at room temperature. 5 × 10 ⁴ cells were placed in the upper wells of the chamber and the lower wells filled with designated Chemokine containing EpiLife medium (Life Technologies, Carlsbad, CA, USA) containing 0.5% bovine serum albumin and different concentrations of ITAC. . After 24 hours of incubation at 37 ° C., the filter inserts were fixed with methanol for staining with hematoxylin and eosin (Merck, Darmstadt, Germany). Images were taken under phase-contrast microscopy and the cells were counted for quantitation. All data were obtained from at least two independent experiments in three runs.

Microarray Analysis

NHEMs were treated for 48 hours with 50 ng / ml ITAC and total RNA was isolated using TRIZOL reagent (GIbco-BRL, Gaithersburg, MD). 50 ng of total RNA was converted to Cy3 or Cy5-labeled cRNA according to the manufacturer's protocol using the Quick Amp Kit (Agilent Technologies, Palo Alto, Calif.). Absorbance ratios and concentrations at 260 nm and 280 nm were measured using an Agilent Bioanalyzer for qualitative evaluation. The same amount of Cy3- or Cy5-labeled cRNA from another sample was hybridized to Agilent Human Whole Genome 4 × 44k Microarrays. Data was extracted from the scanned image using Feature Extraction version 10.2 (Agilent Technologies). The hydration reaction was performed twice for each sample.

Quantitative Real-Time PCR Analysis

CDNA was synthesized using the Superscript II Reverse Transcriptase Kit (Life Technologies, Grand Island, NY) using 2 μg total RNA from each sample. TaqMan® probe (Themo Fisher Scientific, Waltham, Mass.) Was purchased and qPCR was performed using an ABI Fast Real-Time PCR System (Life Technologies). Relative expression levels of each gene in the sample were normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a housekeeping gene, and the difference in gene expression between samples was determined using formula 2 ^-ΔΔCt . Calculated by

Melanin assay

Normal or UV-treated NHEMs in 0.1M Tris-HCl (pH 7.2) buffer containing 1% Nonidet P-40, 0.01% SDS and protease inhibitor cocktail; Roche Molecular Biochemical, Indianapolis, IN. The cell eluate was prepared by sonication at. Melanin was isolated from the cell eluate by centrifugation and dissolved in 1N NaOH. The melanin amount was measured by absorbance at 490 nm using a Synergy H2 microplate reader (BioTek, Winooski, VT, USA) and normalized to the injected protein. Three independent experiments were performed.

Supplementary References

Avery-Kiejda, K.A., Bowden, N.A., Croft, A.J., Scurr, L.L. et al. (2011). P53 in human melanoma fails to regulate target genes associated with apoptosis and the cell cycle and may contribute to proliferation. BMC Cancer 11, 203

Camp, RD, Bacon, KB, and Fincham, NJ, (1990). Lymphocyte chemoattractants in psoriasis and normal skin. J. Invest. Dermatol. 95, 22S-23S

Gunther, C., Zimmermann, N., Berndt, N., Groβer, M., Stein, A., Koch, A., and Meurer, M. (2011). Up-regulation of the chemokine CCL18 by macrophages is a potential immunomodulatory pathway in cutaneous T-cell lymphoma. Am. J. Pathol. 179, 1434-1442

Horikawa, T., Nakayama, T., Hikita, I., Yamada, H., Fujisawa, R., Bito, T., Harada, S., Fukunaga, A., Chantry, D., Gray, P.W. et al. (2002). IFN-gamma-inducible expression of thymus and activation-regulated chemokine / CCL17 and macrophage-derived chemokine / CCL22 in epidermal keratinocytes and their roles in atopic dermatitis. Int. Immunol. 14, 767-773

Hub, E., and Rot, A. (1998). Binding of RANTES, MCP-1, MCP-3, and MIP-1alpha to cells in human skin. Am. J. Pathol. 152, 749-757

Kanda, N., Mitsui, H., and Watanabe, S. (2004). Prostaglandin E (2) suppresses CCL27 production through EP2 and EP3 receptors in human keratinocytes. J. Allergy Clin. Immunol. 114, 1403-1409

Menezes-Souza, D., Guerra-Sa, R., Carneiro, CM, Vitoriano-Souza, J., Giunchetti, RC, Teixeira-Carvalho, A., Silveira-Lemos, D., Oliveira, GC, Correa-Oliveira , R., and Reis, AB (2012). Higher expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 chemokines in the skin associated with parasite density in canine visceral leishmaniasis. PLoS Negl. Trop. Dis. 6, e1566

Papadopoulos, E.J., Sassetti, C., Saeki, H., Yamada, N., Kawamura, T., Fitzhugh, D.J., Saraf, M.A., Schall, T., Blauvelt, A., Rosen, S.D. et al. (1999). Fractalkine, a CX3C chemokine, is expressed by dendritic cells and is up-regulated upon dendritic cell maturation. Eur. J. Immunol. 29, 2551-2559

Uchi, H., Terao, H., Koga, T., and Furue, M. (2000). Cytokines and chemokines in the epidermis . J. Dermatol. Sci. 24 Suppl 1, S29-38

Vestergaard, C., Bang, K., Gesser, B., Yoneyama, H., Matsushima, K., and Larsen, C. G. (2000). A Th2 chemokine, TARC, produced by keratinocytes may recruit CLA + CCR4 + lymphocytes into lesional atopic dermatitis skin. J. Invest. Dermatol. 115, 640-646

Yaszay, B., Trindade, M. C., Lind, M., Goodman, S. B., and Smith, R. L. (2001). Fibroblast expression of C-C chemokines in response to orthopaedic biomaterial particle challenge in vitro. J. Orthop. Res. 19, 970-976

Figure 8. Migration of NHEMs to skin cell derived chemokines

Cell migration was analyzed by a Boyden Chamber Assay as described in Supplementary Materials and Methods. Cells were added to the upper wells for the Boyden Chamber Assay, and designated chemokines (200 ng / ml) were added to the lower wells. Cells were allowed to migrate for 24 hours, fixed, stained, photographed and quantified. The results shown are reproducible in two independent experiments (*** P <0.001).

9. Effect of ITAC migration in human melanoma cell line

Cell migration was analyzed by the Boyden Chamber Assay. Human melanoma cell lines WM266-4 (a), SK-MEL-28 (b), and MNT-1 (c) are without I-TAC (control) or with 200ng / ml I-TAC (I-TAC) Moved 24 hours in the medium. Cells were fixed, stained and quantified. The data represent three independent experiments (* P <0.05, ** P <0.01).

10. Efficiency of HDAC5- and HDAC9-specific siRNA in NHEMs

Each siRNA against HDAC5, HDAC9, JUB, or THBS was transfected into NHEM for 48 hours and total RNA extracted. Scrambled siRNA was used as a negative control. MRNA levels of HDAC5 (a) and HDAC9 (b) in NHEMs were measured by RT-qPCR. Each value was normalized by GAPDH. The data represent 3 independent experiments (*** P <0.001).

Figure 11. NHEM  Within or within human melanoma cells ITAC  Effect of Treatment on Protein Expression

NHEMs were (a) treated with 50ng / ml ITAC for 48 hours or (b) transfected with siRNA against HDAC5. Acetylation levels of alpha-tubulin were measured by Western blot with the indicated antibodies. Scrambled siRNA was used as a control for HDAC5 specific siRNA. (c) Human melanoma cell line SK-MEL-38 was harvested for Western blot using anti-p53 and anti-tyrosinase (TYR) antibodies after 48 hours treatment with ITAC (50 ng / ml). Western block analysis was performed repeatedly and representative images are shown. GAPDH was used as loading control. a-Ac-p53, anti-acetylated p53 antibody. α-Ac-alpha-tubulin, anti-acetylated alpha-tubulin antibody.

12. Effect on melanogenesis of MIG, IP-10 or ITAC in NHEM

NHEMs were treated with 200 ng / ml of MIG, IP-10 or ITAC for 48 hours and total RNA was extracted. MRNA levels of TYR (a), MITF (b), TRP1 (c) and DCT (d) in NHEMs were measured using RT-qPCR. Each value was normalized by GAPDH. The data represent two independent experiments. (* P <0.05)

Figure 13. ITAC -Judo From low pigmentation  Effect of p53 knockdown on melanogenesis-related gene expression

(ad) NHEMs were transfected 48 hours with scrambled siRNA or siRNA for p53 and further treated with 200 ng / ml ITAC for 48 hours. Each value was normalized by GAPDH. The data represent three independent experiments ( ^** P <0.01, ^*** P <0.001).

[Correction under Rule 91] 15.09.2017

Claims (11)

1. A composition for the prevention or treatment of vitiligo or vitiligo metastasis comprising an interferon-inducible T-cell alpha chemoattractant (ITAC) inhibitor.
2. The method of claim 1,

   Wherein the ITAC inhibitor is an oligonucleotide that binds to at least a portion of the neutralizing antibody or ITAC mRNA of the ITAC.
3. The method of claim 1,

   The ITAC inhibitor is a neutralizing antibody of the ITAC receptor or an oligonucleotide that binds to at least a portion of the ITAC receptor mRNA.
4. The method of claim 3,

   Wherein the ITAC receptor is one or more of CXCR3 and CXCR7
5. The method of claim 1,

   The ITAC inhibitor is a neutralizing antibody of HDAC5 or an oligonucleotide that binds to at least a portion of the mRNA of HDAC5.
6. The method according to any one of claims 1 to 5,

   Wherein said oligonucleotide is any one or more of siRNA, shRNA and miRNA.
7. Composition for detecting vitiligo comprising a reagent for detecting the gene expression amount of the ITAC.
8. The method of claim 7, wherein

   The detection reagent comprises one or more of a primer pair or probe specifically binding to a transcript of the gene, and an antibody specifically binding to a protein expressed from the gene.
9. The method of claim 8,

   The primer pair is complementary to the gene mRNA and comprises a primer pair capable of amplifying the mRNA;

   The probe comprises a polynucleotide consisting of the sequence of the gene mRNA; And a polynucleotide comprising at least 10 contiguous nucleotides as a fragment of the polynucleotide.
10. A diagnostic kit for vitiligo comprising a composition for detecting vitiligo of any one of claims 7 to 9.
11. A method of screening for a prophylactic or therapeutic agent for vitiligo,

    A method for screening a prophylactic or therapeutic agent for vitiligo comprising treating a test substance to human epidermal tissue and determining whether the test substance inhibits the expression of one or more genes of ITAC, CXCR3 and CXCR7 in human epidermal tissue. .

Images