TWI767921B - Use of detection reagent of itac gene expression level in the manufacture of composition and kit for detecting vitiligo - Google Patents

Use of detection reagent of itac gene expression level in the manufacture of composition and kit for detecting vitiligo Download PDF

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TWI767921B
TWI767921B TW106123288A TW106123288A TWI767921B TW I767921 B TWI767921 B TW I767921B TW 106123288 A TW106123288 A TW 106123288A TW 106123288 A TW106123288 A TW 106123288A TW I767921 B TWI767921 B TW I767921B
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hdac5
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崔瑞娟
曺恩敬
賓範浩
李泰龍
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南韓商愛茉莉太平洋股份有限公司
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Abstract

Disclosed herein is a composition for the prevention or treatment of vitiligo or vitiligo spread comprising an interferon-inducible T-cell alpha chemoattractant (ITAC) inhibitor. The composition can suppress the action of ITAC to inhibit melanocyte migration and exhibit a hypopigmentation effect.In addition, the composition prevents or treats vitiligo or metastasis of vitiligo by suppressing the action of ITAC.

Description

以ITAC基因表現量之檢測試劑作為製備檢測白斑病組成 物及套組之用途 Using the detection reagent of ITAC gene expression as preparation to detect the composition of vitiligo Uses of objects and kits

在本文中揭示包含ITAC抑制劑之組成物。 Compositions comprising ITAC inhibitors are disclosed herein.

趨化激素(chemokines)的重要角色之一係藉由誘導細胞遷移(cell migration)以介導免疫反應及腫瘤發生。一些趨化激素參與小鼠白斑病(vitiligo)。干擾素誘導性T細胞α趨化因子(Interferon-inducible T-cell alpha chemoattractant，ITAC)對CXCR3具有比其他配體(ligands)更高的結合親和力，從免疫細胞激發強烈的趨化性反應。ITAC對黑色素細胞遷移(melanocyte migration)及色素沉著(pigmentation)的影響以及其相關病症的關聯性是未知的。第二類組蛋白去乙醯酶(Class II histone deacetylase，HDAC)，特別是HDAC5，將p53蛋白去乙醯化，該p53蛋白參與各種細胞過程其包括細胞遷移以及調控蛋白質穩定性及活性。 One of the important roles of chemokines is to mediate immune responses and tumorigenesis by inducing cell migration. Some chemokines are involved in vitiligo in mice. Interferon-inducible T-cell alpha chemoattractant (ITAC) has a higher binding affinity for CXCR3 than other ligands, eliciting strong chemotactic responses from immune cells. The effect of ITAC on melanocyte migration and pigmentation and its relevance to related disorders is unknown. Class II histone deacetylases (HDACs), particularly HDAC5, deacetylate the p53 protein, which is involved in various cellular processes including cell migration and regulation of protein stability and activity.

本發明係透過p53藉由HDAC5的去乙醯化(deacetylation)以提供ITAC對黑色素細胞的移動效應及色素過少效應(hypopigmentation effects)的活體外證據，並從轉譯上的觀點深入了解ITAC在病理狀況諸如白斑病中的角色。 The present invention provides in vitro evidence of the migratory effects and hypopigmentation effects of ITAC on melanocytes through the deacetylation of HDAC5 by p53, and provides insight into the role of ITAC in pathological conditions from a translational point of view Such as the role in vitiligo.

在一態樣中，本發明提供用於預防或治療白斑病或白斑病轉移的組成物，該組成物包含干擾素誘導型T細胞α趨化因子(ITAC)抑制劑。 In one aspect, the present invention provides a composition for preventing or treating vitiligo or metastases of vitiligo, the composition comprising an interferon-inducible T cell alpha chemokine (ITAC) inhibitor.

在另一態樣中，本發明提供一種用於檢測白斑病的組成物，其包含用於ITAC基因表現量(gene expression level)之檢測的試劑。 In another aspect, the present invention provides a composition for detecting vitiligo, comprising a reagent for detecting ITAC gene expression level.

而在另一態樣中，本發明提供一種用於篩選白斑病預防性或治療性試劑的方法，其包含鑑定至少一種選自ITAC、CXCR3、和CXCR7中的表現是否被抑制。 In yet another aspect, the present invention provides a method for screening a vitiligo prophylactic or therapeutic agent comprising identifying whether at least one expression selected from ITAC, CXCR3, and CXCR7 is inhibited.

本發明透過HDAC5對p53的去乙醯化，提供ITAC對黑色素細胞的移動效應及色素過少效應的活體外證據，並從轉譯上的觀點深入了解ITAC在病理狀況諸如白斑病中的角色。 The present invention provides in vitro evidence of the migratory and hypopigmented effects of ITAC on melanocytes through deacetylation of p53 by HDAC5, and provides insights into the role of ITAC in pathological conditions such as vitiligo from a translational perspective.

圖1A顯示根據用ITAC、MIG、或IP-10處理的正常人類表皮黑色素細胞(NHEM)遷移的比較結果。 Figure 1A shows comparative results according to the migration of normal human epidermal melanocytes (NHEM) treated with ITAC, MIG, or IP-10.

圖1B顯示隨著ITAC濃度變化，NHEM遷移的結果。 Figure 1B shows the results of NHEM migration with changes in ITAC concentration.

圖1C顯示CXCR3及CXCR7蛋白質表現量的西方墨點法(Western blot)結果。 Figure 1C shows the results of Western blotting of CXCR3 and CXCR7 protein expression levels.

圖1D顯示在ITAC存在下NHEM的遷移隨後用CXCR3或CXCR7中和抗體處理後的分析結果。 Figure ID shows the results of the analysis of migration of NHEM in the presence of ITAC followed by treatment with CXCR3 or CXCR7 neutralizing antibodies.

圖2A顯示藉由ITAC處理顯著地上調(up-regulated)或下調(down-regulated)生物過程的分析結果。 Figure 2A shows the results of analysis of biological processes that were significantly up-regulated or down-regulated by ITAC treatment.

圖2B顯示根據ITAC處理，在NHEM中的HDAC5 mRNA量。 Figure 2B shows the amount of HDAC5 mRNA in NHEM according to ITAC treatment.

圖2C顯示根據ITAC處理的HDAC9 mRNA量。 Figure 2C shows the amount of HDAC9 mRNA according to ITAC treatment.

圖3A顯示根據在NHEM中HDAC5及HDAC9的減弱(knockdown)，細胞遷移的結果。 Figure 3A shows the results of cell migration according to knockdown of HDAC5 and HDAC9 in NHEM.

圖3B顯示根據HDAC5過度表現(overexpression)，細胞遷移的結果。 Figure 3B shows the results of cell migration according to HDAC5 overexpression.

圖3C顯示根據ITAC處理的HDAC5蛋白量。 Figure 3C shows the amount of HDAC5 protein according to ITAC treatment.

圖4A顯示根據ITAC處理，在NHEM中總p53及乙醯化p53的量。 Figure 4A shows the amount of total p53 and acetylated p53 in NHEM according to ITAC treatment.

圖4B顯示根據HDAC5過度表現，總p53及乙醯化p53的量。 Figure 4B shows the amount of total p53 and acetylated p53 according to HDAC5 overexpression.

圖4C顯示根據HDAC5減弱，總p53及乙醯化p53的量。 Figure 4C shows the amount of total p53 and acetylated p53 according to HDAC5 attenuation.

圖5A顯示在ITAC存在或不存在的情況下，HDAC5(HDAC5-myc)的過度表現、p53(p53-Flag)的過度表現、或兩者(HDAC5-Myc+p53-Flag)的過度表現對細胞遷移的影響。 Figure 5A shows that overexpression of HDAC5 (HDAC5-myc), overexpression of p53 (p53-Flag), or both (HDAC5-Myc+p53-Flag) overexpression on cells in the presence or absence of ITAC Migration effects.

圖5B顯示根據ITAC處理，對MMP1表現的影響。 Figure 5B shows the effect on MMP1 expression according to ITAC treatment.

圖6A顯示根據ITAC處理，黑色素含量中的變化。 Figure 6A shows changes in melanin content according to ITAC treatment.

圖6B顯示根據UV照射，ITAC處理後的黑色素含量及總p53或乙醯化p53蛋白的量。 Figure 6B shows the melanin content and the amount of total p53 or acetylated p53 protein after ITAC treatment according to UV irradiation.

圖6C顯示根據HDAC5過度表現的黑色素含量。 Figure 6C shows melanin content according to HDAC5 overexpression.

圖7顯示用抗ITAC抗體(綠色)及抗HMB45(紅色)染色正常皮膚(N)或有色素過少病灶的皮膚(L)樣本(#1及#2)的結果。核用DAPI(藍色)染色，箭頭表示黑色素，而三角形表示黑色素細胞。 Figure 7 shows the results of staining normal skin (N) or skin with hypopigmented lesions (L) samples (#1 and #2) with anti-ITAC antibody (green) and anti-HMB45 (red). Nuclei are stained with DAPI (blue), arrows indicate melanin and triangles indicate melanocytes.

圖8顯示皮膚細胞衍生的趨化激素誘導NHEM遷移的能力。 Figure 8 shows the ability of skin cell-derived chemokines to induce NHEM migration.

圖9A顯示ITAC在人類黑色素瘤(melanoma)細胞系(cell line)(WM266-4)中的細胞遷移效應。 Figure 9A shows the cell migration effect of ITAC in a human melanoma cell line (WM266-4).

圖9B顯示ITAC在人類黑色素瘤細胞系(SK-MEL-28)中的細胞遷移效應。 Figure 9B shows the cell migration effect of ITAC in a human melanoma cell line (SK-MEL-28).

圖9C顯示ITAC在人類黑色素瘤細胞系(MNT-1)中的細胞遷移效應。 Figure 9C shows the cell migration effect of ITAC in a human melanoma cell line (MNT-1).

圖9D顯示在黑色素細胞及黑色素瘤細胞中ITAC、CXCR3、及CXCR7的檢測結果。 Figure 9D shows the detection results of ITAC, CXCR3, and CXCR7 in melanocytes and melanoma cells.

圖10A顯示mRNA表現的結果，確認HDAC5是否因siRNA處理而減弱。 Figure 10A shows the results of mRNA expression, confirming whether HDAC5 is attenuated by siRNA treatment.

圖10B顯示mRNA表現的結果，確認HDAC9是否因siRNA處理而減弱。 Figure 10B shows the results of mRNA expression, confirming whether HDAC9 is attenuated by siRNA treatment.

圖11A顯示在NHEM中，鑑定α-微管蛋白(alpha-tubulin)的乙醯化是否因ITAC處理而受影響的結果。 Figure 11A shows the results of identifying whether acetylation of alpha-tubulin is affected by ITAC treatment in NHEM.

圖11B顯示在NHEM中，鑑定α-微管蛋白的乙醯化是否因HDAC5缺失(deletion)而受影響的結果。 Figure 11B shows the results of identifying whether acetylation of alpha-tubulin is affected by deletion of HDAC5 in NHEM.

圖11C顯示在黑色素瘤細胞中根據ITAC處理，p53及TYR之表現的變化的結果。 Figure 11C shows the results of changes in the expression of p53 and TYR in melanoma cells upon ITAC treatment.

圖12A顯示在NHEM中，ITAC處理對TYR表現的影響。 Figure 12A shows the effect of ITAC treatment on TYR performance in NHEM.

圖12B顯示在NHEM中，ITAC處理對MITF表現的影響。 Figure 12B shows the effect of ITAC treatment on MITF performance in NHEM.

圖12C顯示在NHEM中，ITAC處理對TRP1表現的影響。 Figure 12C shows the effect of ITAC treatment on TRP1 expression in NHEM.

圖12D顯示在NHEM中，ITAC處理對DCT表現的影響。 Figure 12D shows the effect of ITAC treatment on DCT performance in NHEM.

圖13A顯示在p53減弱的情況下，ITAC處理對TYR表現的影響。 Figure 13A shows the effect of ITAC treatment on TYR expression in the context of p53 attenuation.

圖13B顯示在p53減弱的情況下，ITAC處理對MITF表現的影響。 Figure 13B shows the effect of ITAC treatment on MITF performance in the context of p53 attenuation.

圖13C顯示在p53減弱的情況下，ITAC處理對TRP1表現的影響。 Figure 13C shows the effect of ITAC treatment on TRP1 expression in the context of p53 attenuation.

圖13D顯示在p53減弱的情況下，ITAC處理對DCT表現的影響。 Figure 13D shows the effect of ITAC treatment on DCT performance in the context of p53 attenuation.

現在將參照附圖更詳細地描述本申請的實施例。然而，本申請所揭示的技術不限於在本文中描述的實施例，而是可以其他形式實施。應當理解，提供在本文中揭示的實施例使得本揭露將是徹底且完整的，以及將向所屬技術領域中具有通常知識者充分地傳達本發明的範疇。在圖中，放大組分的寬度及厚度，以清楚地闡述每個組分。進一步，雖然為了便於說明，僅示出了一部分組分，但是所屬技術領域中具有通常知識者將容易理解組分的其餘部分。對於所屬技術領域中具有通常知識者顯而易見的是，在不脫離本發明的精神和範疇下，可以在本發明中進行各種修改及變化。 Embodiments of the present application will now be described in more detail with reference to the accompanying drawings. However, the techniques disclosed in this application are not limited to the embodiments described herein, but may be implemented in other forms. It should be understood that the embodiments disclosed herein are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In the figures, the width and thickness of the components are exaggerated to clearly illustrate each component. Further, although only a portion of the components are shown for ease of explanation, the rest of the components will be readily understood by those of ordinary skill in the art. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the inventions.

如本文所用，用語「中和(neutralize)」及「抑制(inhibit)」意指活性因抑制劑的存在而降低，此是與沒有物質存在的活性相比。活性可降低例如，約10%、20%、30%、40%、50%、60%、70%、80%、或90%、或更多。 As used herein, the terms "neutralize" and "inhibit" mean that the activity is reduced by the presence of the inhibitor as compared to the activity in the absence of the substance. The activity can be reduced, for example, by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or more.

本發明之一實施例關於用於預防或治療白斑病或白斑病轉移的組成物，該組成物包含干擾素誘導性T細胞α趨化因子(ITAC)抑制劑。 One embodiment of the present invention relates to a composition for preventing or treating vitiligo or metastases of vitiligo, the composition comprising an interferon-inducible T cell alpha chemokine (ITAC) inhibitor.

根據上述實施例之組成物可有效抑制黑色素細胞遷移，以及有助於預防或治療白斑病或白斑病轉移、或改善其症狀。 The compositions according to the above embodiments can effectively inhibit the migration of melanocytes, and help prevent or treat vitiligo or metastases of vitiligo, or improve symptoms thereof.

ITAC抑制劑可係抑制ITAC基因表現或活性、或ITAC的作用或活性的物質。 An ITAC inhibitor may be a substance that inhibits the expression or activity of the ITAC gene, or the action or activity of ITAC.

在一實例中，ITAC抑制劑可係與至少一部分的ITAC mRNA結合的寡核苷酸(oligonucleotide)、或對抗ITAC的中和抗體。 In one example, the ITAC inhibitor can be an oligonucleotide that binds to at least a portion of ITAC mRNA, or a neutralizing antibody against ITAC.

寡核苷酸可係siRNA、shRNA、及miRNA中的一或多者。抑制基因表現或活性的物質可係siRNA、shRNA、及miRNA中的一或多者，其包括RNA干擾(RNAi)現象。在本發明一態樣中，誘導基因的mRNA干擾的RNAi現象可用以抑制基因的mRNA的表現。 Oligonucleotides can be one or more of siRNA, shRNA, and miRNA. Substances that inhibit gene expression or activity can be one or more of siRNA, shRNA, and miRNA, including the phenomenon of RNA interference (RNAi). In one aspect of the present invention, the phenomenon of RNAi, which induces mRNA interference of a gene, can be used to inhibit the expression of mRNA of a gene.

miRNA是一種內源性(endogenous)小RNA。它衍生自不合成蛋白質的DNA，且產生自髮夾形轉錄本(hairpin-shaped transcript)。miRNA與標靶mRNA的3'-UTR的互補序列(complementary sequence)結合，以誘導mRNA轉譯(translation)或去穩定化(destabilization)的抑制作用，最終作為抑制標靶mRNA的蛋白質合成的抑制子(repressor)。已知一種miRNA靶向數種mRNA，而mRNA也可被數種miRNA調控。誘導RNAi現象的其他RNA包括短干擾RNA(short interfering RNA，siRNA)，其是約19至27個單體單元(mer)的短RNA，以及具有短髮夾結構的shRNA。 miRNA is an endogenous small RNA. It is derived from DNA that does not synthesize proteins, and produces spontaneous hairpin-shaped transcripts. miRNA binds to the complementary sequence (complementary sequence) of the 3'-UTR of the target mRNA to induce the inhibition of mRNA translation or destabilization, and finally acts as an inhibitor of protein synthesis that inhibits the target mRNA ( repressor). One miRNA is known to target several mRNAs, and mRNAs can also be regulated by several miRNAs. Other RNAs that induce the phenomenon of RNAi include short interfering RNAs (siRNAs), which are short RNAs of about 19 to 27 monomer units (mers), and shRNAs with short hairpin structures.

在一具體實施例中，寡核苷酸可係雙股(double-stranded)，且係siRNA、shRNA、及miRNA中的任一種。 In a specific embodiment, the oligonucleotide can be double-stranded, and is any of siRNA, shRNA, and miRNA.

當ITAC抑制劑是對抗ITAC的中和抗體時，ITAC抑制劑可係抗ITAC抗體。 When the ITAC inhibitor is a neutralizing antibody against ITAC, the ITAC inhibitor can be an anti-ITAC antibody.

在一實例中，ITAC抑制劑可係抑制ITAC受體(receptor)的表現或活性的物質。在一實例中，ITAC受體可係第3型CXC-趨化激素受體(CXC-chemokine receptor type 3，CXCR3)、第7型CXC-趨化激素受體(CXC-chemokine receptor type 7，CXCR7)、或其混合物。 In one example, an ITAC inhibitor can be a substance that inhibits the expression or activity of an ITAC receptor. In one example, the ITAC receptor can be a CXC-chemokine receptor type 3 (CXCR3), a CXC-chemokine receptor type 7 (CXCR7) ), or a mixture thereof.

抑制ITAC受體的表現或活性的物質可係與至少一部分ITAC受體mRNA結合的寡核苷酸或對抗ITAC受體的中和抗體。 The substance that inhibits the expression or activity of the ITAC receptor can be an oligonucleotide that binds to at least a portion of the ITAC receptor mRNA or a neutralizing antibody against the ITAC receptor.

寡核苷酸可係siRNA、shRNA、及miRNA中的一或多者。siRNA、shRNA、及miRNA係如上所述，因此將省略其描述。 Oligonucleotides can be one or more of siRNA, shRNA, and miRNA. siRNA, shRNA, and miRNA are as described above, so their description will be omitted.

當ITAC抑制劑是對抗ITAC受體的中和抗體時，ITAC抑制劑可係抗CXCR3抗體、抗CXCR7抗體、或其混合物。 When the ITAC inhibitor is a neutralizing antibody against the ITAC receptor, the ITAC inhibitor can be an anti-CXCR3 antibody, an anti-CXCR7 antibody, or a mixture thereof.

在一實例中，ITAC抑制劑可係抑制組蛋白去乙醯酶-5(histone deacetylase-5，HDAC5)(一種ITAC的下游效應子(effector))的物質。抑制HDAC5的物質可係，例如，抑制HDAC5的表現或活性的物質。 In one example, an ITAC inhibitor can be a substance that inhibits histone deacetylase-5 (HDAC5), a downstream effector of ITAC. The substance that inhibits HDAC5 can be, for example, a substance that inhibits the expression or activity of HDAC5.

抑制HDAC5的表現或活性的物質可係，例如，與至少一部分HDAC5 mRNA結合的寡核苷酸或對抗HDAC5的中和抗體。 Substances that inhibit the expression or activity of HDAC5 can be, for example, oligonucleotides that bind to at least a portion of HDAC5 mRNA or neutralizing antibodies against HDAC5.

寡核苷酸可係siRNA、shRNA、及miRNA中的一或多者。siRNA、shRNA、及miRNA係如上所述，因此將省略其描述。 Oligonucleotides can be one or more of siRNA, shRNA, and miRNA. siRNA, shRNA, and miRNA are as described above, so their description will be omitted.

對抗HDAC5之中和抗體可係抗HDAC5抗體。 The anti-HDAC5 neutralizing antibody can be an anti-HDAC5 antibody.

ITAC的mRNA序列可由NM_005409、NM_001302123、或SEQ ID NO：1表示。CXCR3的mRNA序列可由NM_001142797、NM_001504、或SEQ ID NO：2表示。CXCR7的mRNA序列可由NM_001047841、NM_020311、或SEQ ID NO：3表示。HDAC5 mRNA序列可由NP_001015053、NM_005474、NM_139205、或SEQ ID NO：4表示。 The mRNA sequence of ITAC can 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 HDAC5 mRNA sequence can be represented by NP_001015053, NM_005474, NM_139205, or SEQ ID NO:4.

根據本發明之實施例，組成物可提供作為各種類型的食品添加劑或功能性食品。例如，可將其加工成發酵乳、乾酪、酸酪乳、果汁、益生菌、保健食品等，以及各種食品添加劑。 According to embodiments of the present invention, the composition can be provided as various types of food additives or functional foods. For example, it can be processed into fermented milk, cheese, yogurt, fruit juice, probiotics, health food, etc., as well as various food additives.

根據本發明之實施例，組成物可係用於保健食品之組成物。用於保健食品之組成物壓制ITAC的作用，以抑制黑色素細胞遷移及展現色素過少效應。 此外，用於保健食品之組成物可抑制ITAC的作用，以緩解或改善白斑病、或白斑病的症狀或擴散。 According to an embodiment of the present invention, the composition can be a composition for health food. The composition used in health food suppresses the action of ITAC to inhibit the migration of melanocytes and exhibit the effect of hypopigmentation. In addition, the composition for health food can inhibit the action of ITAC to relieve or improve vitiligo, or the symptoms or spread of vitiligo.

在一具體實施例中，用於保健食品之組成物可以配製成丸劑(pill)、膠囊(capsule)、片劑(tablet)、顆粒(granule)、焦糖(caramel)、或飲料(drink)等。在另一具體實施例中，其可加工成液體、粉末(powder)、顆粒、片劑、或茶包等。 In a specific embodiment, the composition for health food can be formulated into pill, capsule, tablet, granule, caramel, or drink Wait. In another specific embodiment, it can be processed into a liquid, powder, granule, tablet, or tea bag or the like.

組成物可根據各種方法施用，諸如簡單飲用、注射、噴霧、或擠壓。 The composition can be administered according to various methods, such as simply drinking, injecting, spraying, or squeezing.

食物組成物可包含提供協同效應(synergic effect)的其他成分而不會負面地影響主效應。例如，為了提高性能，可進一步包含添加劑，諸如調味劑(flavoring)、色素(pigment)、殺菌劑(sterilizer)、抗氧化劑(antioxidant)、防腐劑(preservative)、保濕劑(humectant)、增稠劑(thickener)、礦物鹽(mineral salt)、乳化劑(emulsifier)、合成聚合物(synthetic polymer)等。此外，還可進一步包含輔助成分，諸如水溶性維生素、油溶性維生素、聚合物肽(polymer peptide)、聚合物多醣(polymer polysaccharide)、海藻萃取物(seaweed extract)等。考慮到製劑或用途，所屬技術領域中具有通常知識者可適當地選擇這些成分。可在不負面影響本發明的目的及效應的範圍內決定這些成分的量。例如，以該組成物之總重量計，成分量可係0.01wt%至5wt%，例如0.01wt%至3wt%。 The food composition may contain other ingredients that provide a synergic effect without negatively affecting the main effect. For example, in order to improve performance, additives such as flavoring, pigment, sterilizer, antioxidant, preservative, humectant, thickener may be further included (thickener), mineral salt (mineral salt), emulsifier (emulsifier), synthetic polymer (synthetic polymer) and the like. In addition, auxiliary components such as water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, seaweed extracts, and the like may be further included. Those of ordinary skill in the art can appropriately select these ingredients in consideration of formulation or use. The amounts of these components can be determined within a range that does not adversely affect the objects and effects of the present invention. For example, based on the total weight of the composition, the amount of the ingredients may be 0.01 wt % to 5 wt %, eg, 0.01 wt % to 3 wt %.

根據本發明之實施例，組成物可係藥物組成物。藥物組成物可壓制ITAC的作用，以抑制黑色素細胞遷移、展現色素過少效應，以及預防、治療或、減輕白斑病或白斑病的症狀或轉移。 According to an embodiment of the present invention, the composition may be a pharmaceutical composition. The pharmaceutical composition can suppress the action of ITAC to inhibit melanocyte migration, exhibit hypopigmentation effects, and prevent, treat or reduce symptoms or metastasis of vitiligo or vitiligo.

在一具體實施例中，藥物組成物可進一步包含根據習知方法常用的無機材料或無機載體，以及可製備成用於口服(oral)或非經腸(parenteral)給藥的各種製劑，其形式為固體、半固體(semi-solid)、或液體等。 In a specific embodiment, the pharmaceutical composition may further comprise inorganic materials or inorganic carriers commonly used according to conventional methods, and may be prepared into various formulations for oral or parenteral administration in the form of For solid, semi-solid (semi-solid), or liquid and the like.

用於口服給藥的製劑可包括，例如片劑、丸劑、顆粒、硬或軟膠囊、粉末、細粒(fine particle)，粉劑(dust)、乳劑(emulsion)、糖漿、小丸(pellet)等。用於非經腸給藥的製劑可包括，例如注射劑(injection)、滴劑(drop)、軟膏(ointment)、洗劑(lotion)、噴霧劑(spray)、懸浮劑(suspension)、乳劑、栓劑(suppository)等。藥物組成物可進一步包含藥用佐劑(adjuvant)，例如防腐劑、安定劑(stabilizer)、保濕劑(hydrating agent)、乳化促進劑(emulsifying accelerator)、用於控制滲透壓(osmotic pressure)的鹽或緩衝劑(buffer)等、以及其他治療上有用的物質。 Formulations for oral administration may include, for example, tablets, pills, granules, hard or soft capsules, powders, fine particles, dusts, emulsions, syrups, pellets, and the like. Formulations for parenteral administration may include, for example, injections, drops, ointments, lotions, sprays, suspensions, emulsions, suppositories (support) and so on. The pharmaceutical composition may further comprise pharmaceutical adjuvants such as preservatives, stabilizers, hydrating agents, emulsifying accelerators, salts for controlling osmotic pressure Or buffer (buffer), etc., and other therapeutically useful substances.

施用藥物組成物可口服、非經腸、外用(topically)、經皮(transdermally)、皮下(subcutaneously)、直腸(rectally)、靜脈內(intravenously)、肌內(intravenously)、腹膜內(intraperitoneally)等。 The pharmaceutical composition can be administered orally, parenterally, topically, transdermally, subcutaneously, rectally, intravenously, intramuscularly, intraperitoneally, etc. .

活性成分的劑量將根據各種相關因子而變化，諸如症狀的嚴重性、給藥途徑、年齡、性別、體重、及受試者的健康狀態等。活性成分的一般劑量係0.001mg/kg/天至2000mg/kg/天，例如0.5mg/kg/天至1500mg/kg/天。 The dosage of the active ingredient will vary depending upon various relevant factors, such as the severity of symptoms, route of administration, age, sex, weight, and the state of health of the subject, among others. Typical dosages of active ingredient range from 0.001 mg/kg/day to 2000 mg/kg/day, eg, 0.5 mg/kg/day to 1500 mg/kg/day.

在另一態樣中，本發明之一實施例提供一種用於檢測白斑病的組成物，其包含用於ITAC基因表現量之檢測的試劑。 In another aspect, an embodiment of the present invention provides a composition for detecting vitiligo, comprising a reagent for detecting the expression level of ITAC gene.

用於檢測ITAC基因表現量的試劑可係能夠測量ITAC基因轉錄量之一試劑。在一實例中，用於檢測的試劑可係特異性結合至ITAC轉錄本(transcript)之引子對(primer pair)或探子(probe)。 The reagent for detecting the expression level of the ITAC gene may be one capable of measuring the transcription level of the ITAC gene. In one example, the reagents used for detection may be primer pairs or probes that specifically bind to the ITAC transcript.

當ITAC表現與正常群組相比係因組成物而增加時，可判定係有白斑病或白斑病轉移。 Vitiligo or vitiligo metastasis can be determined when the ITAC expression is increased due to the composition compared to the normal group.

引子可包括但不限於與基因的mRNA互補及能夠擴增(amplifying)mRNA的引子對。 Primers can include, but are not limited to, primer pairs complementary to the mRNA of the gene and capable of amplifying the mRNA.

探子可包含一或多種由基因的mRNA序列組成的多核苷酸(polynucleotide)；而多核苷酸，其係該多核苷酸的片段且包含10或更多個連續的核苷酸，但不限於此。 A probe may comprise one or more polynucleotides consisting of the mRNA sequence of a gene; and a polynucleotide, which is a fragment of the polynucleotide and comprises 10 or more consecutive nucleotides, but is not limited thereto .

在上述實施例中，基因表現量可藉由PCR(聚合酶連鎖反應，polymerase chain reaction)、RT-PCR(反轉錄酶-聚合酶連鎖反應，reverse transcriptase polymerase chain reaction)、北方墨點分析(Northern blot analysis)、西方墨點分析(Western blot analysis)、點墨法(dot blot assay)、ELISA(酵素結合免疫吸附法，enzyme-linked immunosorbent assay)、或微陣列(microarray)測定，例如，使用對偶基因特異性雜合(allele-specific hybridization)的TaqManTM探子方法、一種透過聚合使用對偶基因特異性雜合的探子方法、使用限制酶的限制片段長度多型性法(restriction fragment length polymorphism，RFLP)、使用融解溫度(melting temperature，Tm)的動態對偶基因特異性雜合(dynamic allele-specific hybridization，DASH)、使用聚合的焦磷酸根定序法(pyrosequencing)、使用對偶基因特異性延伸的微陣列方法等，但不限於此。 In the above-mentioned embodiment, the gene expression level can be analyzed by PCR (polymerase chain reaction, polymerase chain reaction), RT-PCR (reverse transcriptase polymerase chain reaction, reverse transcriptase polymerase chain reaction), Northern blot analysis (Northern blot analysis). blot analysis), Western blot analysis, dot blot assay, ELISA (enzyme-linked immunosorbent assay), or microarray assay, e.g., using dual TaqMan ™ probe method for allele-specific hybridization, a probe method using paired gene-specific hybridization by aggregation, restriction fragment length polymorphism (RFLP) using restriction enzymes , Dynamic allele-specific hybridization (DASH) using melting temperature (Tm), Pyrosequencing using polymerization, Microarray using dual-gene specific extension method, etc., but not limited thereto.

樣品可係從人類分離而來的上皮細胞、角質細胞(keratinocytes)、黑色素細胞、諸如肥大細胞的免疫細胞、或纖維母細胞，例如角質細胞。 Samples can be epithelial cells isolated from humans, keratinocytes, melanocytes, immune cells such as mast cells, or fibroblasts, such as keratinocytes.

可根據本領域中習知方法自受試者獲取樣品。可用非侵入性的方式進行，而不會對皮膚造成刺激，諸如在皮膚上藉由膠帶剝離(tape stipping)，但不限於此。在一實例中，可進行膠帶剝離，例如1至20次，例如5次。在上述範圍內，可獲得樣品而不引起皮膚刺激。 A sample can be obtained from a subject according to methods known in the art. It can be done in a non-invasive manner without causing irritation to the skin, such as, but not limited to, tape stripping on the skin. In one example, tape stripping can be performed, eg, 1 to 20 times, eg, 5 times. Within the above range, a sample can be obtained without causing skin irritation.

基因表現量的測定可包括測量樣品中來自基因或蛋白質表現的轉錄量。當使用引子對或探子時，可藉由測量與一或多個引子對及探子雜合(hybridized) 的樣品中的轉錄量來判定基因轉錄量。當使用抗體時，可藉由測量與抗體雜合的樣品中的蛋白質量來判定經表現之蛋白質的量。 Determination of the amount of gene expression can include measuring the amount of transcription from gene or protein expression in the sample. When primer pairs or probes are used, they can be hybridized with one or more primer pairs and probes by measuring The amount of transcription in the sample was used to determine the amount of gene transcription. When an antibody is used, the amount of expressed protein can be determined by measuring the amount of protein in the sample to which the antibody is hybridized.

根據另一實施例，本發明提供用於白斑病診斷的套組(kit)，其包含特異性結合至ITAC、CXCR3、及CXCR7中的一或多者之轉錄本之一或多種引子對或探子，以及特異性結合至經一或多種基因表現的蛋白質之抗體。 According to another embodiment, the present invention provides a kit for vitiligo diagnosis comprising one or more primer pairs or probes for transcripts that specifically bind to one or more of ITAC, CXCR3, and CXCR7 , and antibodies that specifically bind to proteins expressed by one or more genes.

在另一態樣中，本發明之實施例提供一種用於診斷白斑病或基於ITAC表現之白斑病轉移的方法。 In another aspect, embodiments of the present invention provide a method for diagnosing vitiligo or vitiligo metastasis based on ITAC manifestations.

該方法允許檢測ITAC在樣品中的表現量，以及當ITAC過度表現時(與正常量相比)診斷白斑病。樣品可係受試者的表皮組織。在一實例中，樣品可係從受試者分離而來的一或多種的上皮細胞、角質細胞、黑色素細胞、及免疫細胞，諸如肥大細胞。 This method allows detection of the amount of ITAC expressed in a sample, and diagnosis of vitiligo when ITAC is overexpressed (compared to normal amounts). The sample can be epidermal tissue of a subject. In one example, the sample can be one or more of epithelial cells, keratinocytes, melanocytes, and immune cells, such as mast cells, isolated from a subject.

在過度表現的情況下，基因表現量係正常群組表現量的1.1倍或以上、1.2倍或以上、1.3倍或以上、1.4倍或以上、1.5倍或以上、1.6倍或以上、1.7倍或以上、1.8倍或以上、1.9倍或以上、2.0倍或以上、2.1倍或以上、2.2倍或以上、2.3倍或以上、2.4倍或以上、2.5倍或以上、2.6倍或以上、2.7倍或以上、2.8倍或以上、2.9倍或以上、3.0倍或以上、3.1倍或以上、3.2倍或以上、3.3倍或以上、3.4倍或以上、3.5倍或以上、3.6倍或以上、3.7倍或以上、3.8倍或以上、3.9倍或以上、4.0倍或以上、4.1倍或以上、4.2倍或以上、4.3倍或以上、4.4倍或以上、4.5倍或以上、4.6倍或以上、4.7倍或以上、4.8倍或以上、4.9倍或以上、或5.0倍或以上，及20.0倍或以下、19.5倍或以下、19.0倍或以下、18.5倍或以下、18.0倍或以下、17.5倍或以下、17.0倍或以下、16.5倍或以下、16.0倍或以下、15.5倍或以下、15.0倍或以下、14.5倍或以下、14.0倍或以下、13.5 倍或以下、13.0倍或以下、12.5倍或以下、12.0倍或以下、11.5倍或以下、11.0倍或以下、10.5倍或以下、10.0倍或以下、9.5倍或以下、9.0倍或以下、8.5倍或以下、8.0倍或以下、7.5倍或以下、7.0倍或以下、6.5倍或以下、6.0倍或以下、5.5倍或以下、或5.0倍或以下。例如，當基因表現量係正常群組表現量的1.1至20.0倍，例如1.5至10.0倍、例如2至8倍時，可診斷白斑病。 In the case of overexpression, the expression level of the gene is 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more than that of the normal group. more, 1.8 times or more, 1.9 times or more, 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more, 2.5 times or more, 2.6 times or more, 2.7 times or more more, 2.8 times or more, 2.9 times or more, 3.0 times or more, 3.1 times or more, 3.2 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 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 More, 4.8 times or more, 4.9 times or more, or 5.0 times or more, and 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 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, 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, or 5.0 times or less. For example, leukoplakia can be diagnosed when the gene expression level is 1.1 to 20.0 times, eg, 1.5 to 10.0 times, eg, 2 to 8 times, the expression level of the normal group.

該方法除了ITAC表現量之外，可進一步包含測量作為ITAC受體的CXCR3或CXCR7的表現量、或兩者的表現量。與ITAC表現一樣，當CXCR3或CXCR7過度表現時，可診斷白斑病或白斑病轉移。 The method may further comprise measuring the amount of expression of CXCR3 or CXCR7, or both, as an ITAC receptor, in addition to the amount of ITAC expression. As with ITAC manifestations, vitiligo or vitiligo metastases can be diagnosed when CXCR3 or CXCR7 is overexpressed.

在過度表現的情況下，基因表現量係正常群組表現量的1.1倍或以上、1.2倍或以上、1.3倍或以上、1.4倍或以上、1.5倍或以上、1.6倍或以上、1.7倍或以上、1.8倍或以上、1.9倍或以上、2.0倍或以上、2.1倍或以上、2.2倍或以上、2.3倍或以上、2.4倍或以上、2.5倍或以上、2.6倍或以上、2.7倍或以上、2.8倍或以上、2.9倍或以上、3.0倍或以上、3.1倍或以上、3.2倍或以上、3.3倍或以上、3.4倍或以上、3.5倍或以上、3.6倍或以上、3.7倍或以上、3.8倍或以上、3.9倍或以上、4.0倍或以上、4.1倍或以上、4.2倍或以上、4.3倍或以上、4.4倍或以上、4.5倍或以上、4.6倍或以上、4.7倍或以上、4.8倍或以上、4.9倍或以上、或5.0倍或以上，及20.0倍或以下、19.5倍或以下、19.0倍或以下、18.5倍或以下、18.0倍或以下、17.5倍或以下、17.0倍或以下、16.5倍或以下、16.0倍或以下、15.5倍或以下、15.0倍或以下、14.5倍或以下、14.0倍或以下、13.5倍或以下、13.0倍或以下、12.5倍或以下、12.0倍或以下、11.5倍或以下、11.0倍或以下、10.5倍或以下、10.0倍或以下、9.5倍或以下、9.0倍或以下、8.5倍或以下、8.0倍或以下、7.5倍或以下、7.0倍或以下、6.5倍或以下、6.0倍或以 下、5.5倍或以下、或5.0倍或以下。例如，當基因表現量係正常群組表現量的1.1至20.0倍，例如1.5至10.0倍、例如2至8倍時，可診斷白斑病。 In the case of overexpression, the expression level of the gene is 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more than that of the normal group. more, 1.8 times or more, 1.9 times or more, 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more, 2.5 times or more, 2.6 times or more, 2.7 times or more more, 2.8 times or more, 2.9 times or more, 3.0 times or more, 3.1 times or more, 3.2 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 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 More, 4.8 times or more, 4.9 times or more, or 5.0 times or more, and 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, 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 more lower, 5.5 times or less, or 5.0 times or less. For example, leukoplakia can be diagnosed when the gene expression level is 1.1 to 20.0 times, eg, 1.5 to 10.0 times, eg, 2 to 8 times, the expression level of the normal group.

根據又另一實施例，本發明提供用於篩選對於白斑病的預防性或治療性試劑的方法。 According to yet another embodiment, the present invention provides methods for screening prophylactic or therapeutic agents for vitiligo.

在一實例中，用於篩選白斑病的預防性或治療性試劑的方法，可包含用測試物質處理人類表皮組織，以及鑑定該測試物質是否抑制人類表皮組織中ITAC、CXCR3、及CXCR7基因中的一或多者之表現。 In one example, a method for screening a prophylactic or therapeutic agent for vitiligo may include treating human epidermal tissue with a test substance, and identifying whether the test substance inhibits the ITAC, CXCR3, and CXCR7 genes in the human epidermal tissue. performance of one or more.

表皮組織可係從受試者分離而來的一或多種上皮細胞、角質細胞、黑色素細胞、及免疫細胞，諸如肥大細胞。 Epidermal tissue can be one or more of epithelial cells, keratinocytes, melanocytes, and immune cells, such as mast cells, isolated from a subject.

在一實例中，當測試物質顯著地抑制表現時(與未經處理的群組相比)，可將其判定為預防性或治療性試劑。例如，當表現被抑制時，與未經處理的群組相比，表現量會降低為P<0.05。 In one example, a test substance can be judged as a prophylactic or therapeutic agent when it significantly inhibits performance (compared to the untreated group). For example, when performance was suppressed, the amount of performance was reduced to P<0.05 compared to the untreated cohort.

實施例 Example

以下，將透過測試實例、實例、及比較實例詳細描述本發明的特徵及效應。然而，提供以下測試實例、實例、及比較實例，僅為了說明目的，並不意圖限制本發明之範疇。 Hereinafter, the features and effects of the present invention will be described in detail through test examples, examples, and comparative examples. However, the following test examples, examples, and comparative examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

透過經由HDAC5將p53去穩定化，ITAC誘導黑色素細胞遷移及色素過少：ITAC在色素相關病症中的可能角色ITAC induces melanocyte migration and hypopigmentation by destabilizing p53 via HDAC5: a possible role for ITAC in pigment-related disorders

關於這個主題已知為何？What is known about this topic?

●趨化激素的重要角色之一係經由誘導細胞遷移以介導免疫反應及腫瘤發生。 • One of the important roles of chemokines is to mediate immune responses and tumorigenesis by inducing cell migration.

●一些趨化激素參與小鼠白斑病。 ● Some chemokines are involved in vitiligo in mice.

●干擾素誘導性T細胞α趨化因子(ITAC)有著比其他配體對CXCR3更高的結合親和力，從免疫細胞激發強烈的趨化性反應。 • Interferon-inducible T cell alpha chemokine (ITAC) has a higher binding affinity for CXCR3 than other ligands, eliciting a strong chemotactic response from immune cells.

●ITAC對黑色素細胞遷移及色素沉著的影響以及其在相關病症的參與是未知的。 • The effect of ITAC on melanocyte migration and pigmentation and its involvement in related disorders is unknown.

●第二類(HDAC)，特別是HDAC5，將p53蛋白去乙醯化，該p53蛋白參與各種細胞過程其包括細胞遷移以及調控蛋白質穩定性及活性。 • The second class (HDACs), in particular HDAC5, deacetylates the p53 protein, which is involved in various cellular processes including cell migration and regulation of protein stability and activity.

這項研究增加了什麼？What does this study add?

●在CXCR3靶向趨化激素中，僅ITAC有效地誘導黑色素細胞及黑色素瘤細胞的遷移以及色素過少。 ● Among the CXCR3-targeted chemokines, only ITAC effectively induces melanocyte and melanoma cell migration and hypopigmentation.

●ITAC表現在白斑病皮膚表皮中增加。 ●ITAC manifestations are increased in vitiligo skin epidermis.

●HDAC5藉由去乙醯化及去穩定化其基質p53而參與ITAC介導的細胞過程。 • HDAC5 is involved in ITAC-mediated cellular processes by deacetylating and destabilizing its substrate p53.

●本研究經由HDAC5藉由p53的去穩定化，提供ITAC對黑色素細胞的移動效應及色素過少效應的活體外證據以及ITAC在病理狀況諸如黑色素瘤及白斑病中的角色轉譯上的洞察。 • This study provides in vitro evidence of the migratory and hypopigmented effects of ITAC on melanocytes via destabilization of p53 by HDAC5 and translational insight into the role of ITAC in pathological conditions such as melanoma and vitiligo.

縮寫abbreviation

CXCR3：第3型CXC-趨化激素受體；HDAC：組蛋白去乙醯酶；IP-10：干擾素γ誘導蛋白10(interferon-gamma-induced protein 10)；ITAC：干擾素誘導性T細胞α趨化因子；MIG：干擾素-γ誘導之單核細胞因子(monokine induced by interferon-gamma)；MMP1：基質金屬蛋白酶1(matrix metallopeptidase 1)；NHEM：正常人類表皮黑色素細胞；TYR：酪胺酸酶(tyrosinase)；MITF：小眼症相關轉錄因子(microphthalmia-associated transcription factor)；TRP1：酪胺酸酶相關蛋白 1(tyrosinase-related protein-1)；DCT：多巴色素異構酶(dopachrome tautomerase)；HMB45：人類黑色素瘤45(human melanoma black-45) CXCR3: CXC-chemohormone receptor type 3; HDAC: histone deacetylase; IP-10: interferon-gamma-induced protein 10; ITAC: interferon-induced T cells Alpha chemokine; MIG: monokine induced by interferon-gamma; MMP1: matrix metallopeptidase 1; NHEM: normal human epidermal melanocytes; TYR: tyramine tyrosinase; MITF: microphthalmia-associated transcription factor; TRP1: tyrosinase-related protein 1 (tyrosinase-related protein-1); DCT: dopachrome tautomerase; HMB45: human melanoma black-45

藉由抑制ITAC及其受體調控細胞遷移及護理皮膚病Regulation of cell migration and skin care by inhibiting ITAC and its receptors

●ITAC(趨化激素)：誘導黑色素細胞、黑色素瘤、及免疫細胞的遷移 ITAC (Chemokine): induces migration of melanocytes, melanoma, and immune cells

●透過ITAC受體CXCR3、CXCR7之信號轉移 ●Signal transfer through ITAC receptors CXCR3 and CXCR7

●透過較低的信號因子HDAC5活化、p53抑制來誘導細胞遷移 ●Induction of cell migration through lower signaling factor HDAC5 activation, p53 inhibition

●治療中和抗體(抗CXCR3、抗CXCR7)抑制ITAC誘導的細胞遷移 Therapeutic neutralizing antibodies (anti-CXCR3, anti-CXCR7) inhibit ITAC-induced cell migration

●細胞遷移相關皮膚病：黑色素瘤、白斑病 Skin diseases associated with cell migration: melanoma, vitiligo

●ITAC及受體的表現在轉移性黑色素瘤組織中增加 Increased expression of ITAC and receptors in metastatic melanoma tissue

●ITAC的表現在白斑病病灶中增加 The manifestations of ITAC are increased in vitiligo lesions

●因此，據認為抑制ITAC及其受體可有助於黑色素瘤及白斑病的改善 ● Therefore, inhibition of ITAC and its receptors is thought to contribute to the improvement of melanoma and vitiligo

●在這方面，用於照護/治療特定皮膚病之組成物使用ITAC及ITAC受體的中和抗體。 - In this regard, compositions for the care/treatment of specific skin diseases use ITAC and neutralizing antibodies to ITAC receptors.

改善各種趨化激素/細胞激素(cytokine)的細胞遷移效應 Improve cell migration effects of various chemokines/cytokines

使用趨化激素來改善黑色素細胞的遷移 Using chemokines to improve melanocyte migration

彙總 Summary

背景 細胞遷移在免疫反應和腫瘤發生中扮演主要角色。干擾素誘導性T細胞α趨化因子(ITAC)從免疫細胞激發強烈的趨化性反應。 Background Cell migration plays a major role in immune responses and tumorigenesis. Interferon-inducible T cell alpha chemokine (ITAC) elicits a strong chemotactic response from immune cells.

目標 檢查ITAC對黑色素細胞遷移及色素沉著的效應以及其在相關病症的參與，以及調查在這些過程中潛在的關鍵參與者。 Objectives To examine the effects of ITAC on melanocyte migration and pigmentation and its involvement in related disorders, and to investigate potential key players in these processes.

方法 用ITAC處理人類黑色素細胞或黑色素瘤細胞，以及進行遷移試驗(migration assay)。進行整體基因表現分析(Global gene expression analysis)以找 出藉由ITAC處理所調控的基因。參與ITAC誘導的細胞過程之關鍵參與者的功能係結合ITAC處理使用減弱或過度表現實驗來解決。檢查ITAC在炎症相關的色素過少病症(白斑症)中的表現。 Methods Human melanocytes or melanoma cells were treated with ITAC and a migration assay was performed. Global gene expression analysis was performed to identify genes regulated by ITAC treatment. The functions of key players involved in ITAC-induced cellular processes were addressed using attenuation or overexpression experiments in conjunction with ITAC treatment. Examining the performance of ITAC in an inflammation-related hypopigmentation disorder (Vitiligo).

結果 在CXCR3配體中，僅ITAC誘導黑色素細胞遷移。ITAC處理上調了組蛋白去乙醯酶5(HDAC5)的表現，以及下調了p53(HDAC5的已知標靶)的表現。透過HDAC5及p53的減弱或過度表現，我們證實了藉由降低p53的表現量(經由去乙醯化)，HDAC5介導ITAC誘導的遷移。此外，ITAC處理可降低p53依賴及HDAC5依賴方式的色素沉著。最後，驗證了藉由ITAC處理增加的人類黑色素瘤細胞的遷移以及在白斑病皮膚表皮中增加的ITAC表現。 Results Among CXCR3 ligands, only ITAC induced melanocyte migration. ITAC treatment upregulated the expression of histone deacetylase 5 (HDAC5) and downregulated the expression of p53, a known target of HDAC5. Through attenuation or overexpression of HDAC5 and p53, we demonstrated that HDAC5 mediates ITAC-induced migration by reducing the amount of expression of p53 (via deacetylation). Furthermore, ITAC treatment reduced pigmentation in a p53-dependent and HDAC5-dependent manner. Finally, increased migration of human melanoma cells by ITAC treatment and increased ITAC performance in vitiligo skin epidermis were demonstrated.

結論 本研究提供ITAC對黑色素細胞的移動效應及色素過少效應的活體外證據及ITAC在病理狀況中的角色轉譯上的洞察，以及意味著HDAC5及其基質p53係用於調控ITAC誘導的細胞過程的潛在標靶。 Conclusions This study provides in vitro evidence for the migratory and hypopigmented effects of ITAC on melanocytes and translational insights into the role of ITAC in pathological conditions, and implies that HDAC5 and its substrate p53 are involved in regulating ITAC-induced cellular processes. potential target.

關鍵字：HDAC5、p53、ITAC、黑色素細胞、遷移、黑色素瘤、白斑病 Keywords: HDAC5, p53, ITAC, melanocytes, migration, melanoma, vitiligo

導論 introduction

離胺酸(lysines)乙醯化係一種轉譯後修飾(post-translational modification)，可影響人類細胞中超過1750種蛋白質1，意味著乙醯化/去乙醯化係生理蛋白活性重要的調控因子。組蛋白乙醯轉移酶(acetyltransferases)藉由將乙醯部分從乙醯輔酶A(coenzyme A)轉移到標靶蛋白上的離胺酸殘基的ε-胺來催化組蛋白上的離胺酸乙醯化，而組蛋白去乙醯酶(HDAC)則催化反向過程。不像組蛋白乙醯移轉酶具有不同結構和功能，HDAC可基於與其酵母對應物的序列同源性(sequence homology)分成四個不同的類別：第I類(HDAC1、2、3、及8)、第II 類(IIa：HDAC4、5、7、9、及MITR；IIb：HDAC6及10)、第III類(SIRT1-7)、以及第IV類(HDAC11)。2第I類HDAC組成型表現於細胞核中且發現於所有組織中，而第II類HDAC顯示組織特異性表現模式並進行細胞核質(nucleocytoplasmic)穿梭，意味著它們參與非組蛋白的去乙醯化。在非組蛋白中，一種在細胞生長停滯、細胞凋亡(apoptosis)、及老化中起作用的轉錄因子p53，是一種代表性的蛋白質，其穩定性係透過HDAC藉由離胺酸去乙醯化來調控。3-4最近，亦報導HDAC5藉由將離胺酸120(K120)去乙醯化而作用於p53上。5 Lysine acetylation is a post-translational modification that affects more than 1750 proteins in human cells 1 , implying that acetylation/deacetylation is an important regulator of physiological protein activity . Histone acetyltransferases catalyze lysine acetylation on histones by transferring the acetyl moiety from acetyl coenzyme A (coenzyme A) to the ε-amine of lysine residues on target proteins. acetylation, while histone deacetylases (HDACs) catalyze the reverse process. Unlike histone acetyltransferases, which have distinct structures and functions, HDACs can be divided into four distinct classes based on sequence homology to their yeast counterparts: Class I (HDACs 1, 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 constitutively expressed in the nucleus and are found in all tissues, whereas class II HDACs show tissue-specific patterns of expression and undergo nucleocytoplasmic shuttling, implying that they are involved in the deacetylation of non-histone proteins . Among non-histone proteins, p53, a transcription factor that plays a role in cell growth arrest, apoptosis, and aging, is a representative protein whose stability is mediated by lysine deacetylation through HDACs to control. 3-4 Recently, HDAC5 was also reported to act on p53 by deacetylation of lysine 120 (K120). 5

儘管HDAC及其可能的標靶已被相對地好好研究，但是活化特定HDAC的因子了解甚少。因為HDAC參與各種細胞過程，因此其失調與癌症及炎症疾病的致病機制有關。儘管臨床試驗已經研究了用於治療這些疾病的HDAC抑制劑，但在患者身上HDAC抑制的分子基礎尚未完全了解。6先前透過移動性相關蛋白(諸如α-微管蛋白及皮質肌動蛋白(cortactin))的去乙醯化，顯示了HDAC對於在各種細胞及腫瘤中的細胞移動性(cell motility)是重要的。7-8此發現及HDAC在轉移及炎症中的有力角色意味著HDAC與疾病過程之間的關係涉及細胞遷移。 Although HDACs and their possible targets are relatively well studied, the factors that activate specific HDACs are poorly understood. Because HDACs are involved in various cellular processes, their dysregulation has been implicated in the pathogenesis of cancer and inflammatory diseases. Although clinical trials have investigated HDAC inhibitors for the treatment of these diseases, the molecular basis of HDAC inhibition in patients is not fully understood. 6 HDACs were previously shown to be important for cell motility in various cells and tumors through deacetylation of mobility-related proteins such as alpha-tubulin and cortactin . 7-8 This finding and the potent role of HDACs in metastasis and inflammation implies that the relationship between HDACs and the disease process involves cell migration.

在皮膚中，免疫細胞及皮膚細胞在正常條件下以及回應環境壓力而分泌各種細胞激素及趨化激素。尤其是經由這些分泌因子(secreted factor)，暴露於UVB波長導致黑色素細胞微環境(microenvironment)的失調(dysregulation)，造成黑色素細胞向異常區域過度增殖(hyper-proliferation)及遷移。9然而，關於趨化激素對成熟黑色素細胞遷移的影響報導很少。我們先前證明了皮膚衍生的趨化因子SDF1在成熟的黑色素細胞中誘導趨化作用(chemotaxis)。10其他趨化激素，特別是藉由干擾素-g(MIG)、干擾素-g誘導的蛋白10(IP-10)、及干擾素誘導性T細胞α趨化因子(ITAC)所誘導的第3型CXC-趨化激素受體(CXCR3)靶向趨化激 素單體激素(monokine)，在將不同亞型(subtypes)的免疫細胞募集(recruitment)至炎症部位中發揮重要作用。11有趣地，這些趨化激素的表現模式及結合親和力(binding affinities)有所不同：IP-10及ITAC在基底角質細胞中表現，但MIG在真皮滲入物(infiltrates)中表現，12以及ITAC以高於IP-10或MIG兩者之一的親和力與CXCR3結合。13MIG及IP-10在慢性UVB曝露下參與延遲色素沉著14以及在角質細胞及黑色素細胞的共培養物中誘導色素沉著，15但是ITAC在黑色素細胞遷移及色素沉著中的作用仍然係未知的。基於ITAC相對於其他CXCR3配體的不同表現模式及受體結合活性，我們預測ITAC在這些過程中扮演不同的角色。 In the skin, immune cells and skin cells secrete various cytokines and chemokines under normal conditions and in response to environmental stress. In particular, through these secreted factors, exposure to UVB wavelengths leads to dysregulation of the melanocyte microenvironment, resulting in hyper-proliferation and migration of melanocytes to abnormal areas. 9 However, little has been reported on the effect of chemokines on the migration of mature melanocytes. We previously demonstrated that the skin-derived chemokine SDF1 induces chemotaxis in mature melanocytes. 10 Other chemokines, in particular by interferon-g (MIG), interferon-g-induced protein 10 (IP-10), and interferon-inducible T cell alpha chemokine (ITAC). Type 3 CXC-chemokine receptor (CXCR3) targets chemokine monomeric hormones (monokines) and plays an important role in the recruitment of different subtypes of immune cells to sites of inflammation. 11 Interestingly, the patterns of expression and binding affinities of these chemokines differ: IP-10 and ITAC are expressed in basal keratinocytes, but MIG is expressed in dermal infiltrates, 12 and ITAC are expressed in dermal infiltrates. Binds to CXCR3 with higher affinity than either IP-10 or MIG. 13 MIG and IP- 10 are involved in delayed pigmentation under chronic UVB exposure14 and in induction of pigmentation in co-cultures of keratinocytes and melanocytes, 15 but the role of ITAC in melanocyte migration and pigmentation remains unknown. Based on the different expression patterns and receptor-binding activities of ITAC relative to other CXCR3 ligands, we predict that ITAC plays different roles in these processes.

在本研究中，我們確立了ITAC對人類黑色素細胞及黑色素瘤遷移的影響。我們鑑定了HDAC5(第IIA類HDAC)，在ITAC誘導的黑色素細胞遷移中作為介質以及p53作為HDAC5的去乙醯化標靶。我們進一步驗證了ITAC治療降低了p53依賴及HDAC5依賴方式的色素沉著，以及在白斑病皮膚的表皮中增加了ITAC表現。 In the present study, we established the effect of ITAC on human melanocyte and melanoma migration. We identified HDAC5 (class IIA HDAC) as a mediator in ITAC-induced melanocyte migration and p53 as a deacetylation target of HDAC5. We further demonstrated that ITAC treatment reduced pigmentation in a p53- and HDAC5-dependent manner, and increased ITAC expression in the epidermis of vitiligo skin.

材料及方法Materials and Methods

細胞培養cell culture

購買來自中等膚色的NHEM(Cascade Biologics,Portland,OR)，以及在含有5% CO2、補充有人類黑色素細胞生長補充劑(HMGS；Life Technologies,Carlsbad,CA)的培養基254中的潮濕培養箱中培養。用各種濃度的經指示趨化激素處理NHEM(表S1；R&D system,Minneapolis,MN)。 NHEM was purchased from Medium Skin Tone (Cascade Biologics, Portland, OR) and in a humidified incubator in Medium 254 with 5% CO2 supplemented with Human Melanocyte Growth Supplement (HMGS; Life Technologies, Carlsbad, CA) nourish. NHEMs were treated with various concentrations of the indicated chemokines (Table S1; R&D system, Minneapolis, MN).

細胞遷移試驗(Cell migration assay)Cell migration assay

根據已發表的程序，在所示細胞處理後於博伊登室(Boyden chamber)中進行遷移試驗。10在所選擇的實驗中，用對抗CXCR3(R&D system)、CXCR7(Biolegend,San Diego,CA)、或個別的同型匹配(isotype-matched)的對照抗體之50vg/mL中和抗體預處理NHEM。博伊登室試驗(Boyden chamber assays)的詳細計劃書提供於輔助資料中。 Migration assays were performed in Boyden chambers after cell treatment as indicated, according to published procedures. 10 In selected experiments, NHEMs were pretreated with 50 vg/mL neutralizing antibody against CXCR3 (R&D system), CXCR7 (Biolegend, San Diego, CA), or individual isotype-matched control antibodies. Detailed protocols for Boyden chamber assays are provided in the Supplementary Information.

西方墨點分析及瞬時轉染(transient transfection)Western blot analysis and transient transfection

使用25mg細胞溶胞產物(cell lysates)進行西方墨點分析。有關抗體的詳細計劃書及資訊描述於輔助資料中。根據製造商的說明書，使用Lipofectamine RNAiMAX或Lipofectamine 2000(Invitrogen,Washington,Dc)將用於HDAC5、HDAC9、及p53(Dharmacon,Lafayette,CO)的各50nM siRNA的等分試樣(aliquot)，或用於HDAC5-Myc及p53-Flag(GeneCopoeia,Rockville,Md)的2mg DNA質體轉染到細胞中，分別進行48小時。 Western blot analysis was performed using 25 mg of cell lysates. Detailed protocols and information about antibodies are described in the Supplementary Information. Aliquots (aliquots) of each 50 nM siRNA for HDAC5, HDAC9, and p53 (Dharmacon, Lafayette, CO) were performed using Lipofectamine RNAiMAX or Lipofectamine 2000 (Invitrogen, Washington, Dc) according to the manufacturer's instructions, or with 2 mg of DNA plasmids in HDAC5-Myc and p53-Flag (GeneCopoeia, Rockville, Md) were transfected into cells for 48 hours, respectively.

微陣列分析(Microarray analysis)Microarray analysis

用50ng/mL ITAC處理NHEM 48小時，以及使用TRIzol試劑(Gibco-BRL,Gaithersburg,MD)分離總RNA。使用五十奈克之總RNA進行分析。在輔助資料中詳細介紹微陣列分析程序。 NHEM was treated with 50 ng/mL ITAC for 48 hours, and total RNA was isolated using TRIzol reagent (Gibco-BRL, Gaithersburg, MD). Fifty ng of total RNA was used for analysis. Microarray analysis procedures are described in detail in the Supplementary Information.

定量即時PCR分析quantitative real-time PCR analysis

按照製造商的說明，藉由使用隨機六聚體(random hexamers)及反轉錄酶(Promega,Madison,WI)使用二微克總RNA以合成cDNA。在輔助資料中提供詳細的程序。 cDNA was synthesized using two micrograms of total RNA by using random hexamers and reverse transcriptase (Promega, Madison, WI) according to the manufacturer's instructions. Detailed procedures are provided in the supplementary material.

黑色素試驗(Melanin assay)Melanin assay

用50或200ng/mL ITAC處理4天、或用各種濃度的HDAC5-Myc表現質體轉染細胞2天。細胞裂解及黑色素試驗的詳細程序給於輔助資料中。 Cells were treated with 50 or 200 ng/mL ITAC for 4 days, or transfected with various concentrations of HDAC5-Myc expressing plastids for 2 days. Detailed procedures for cell lysis and melanin assays are given in the Supplementary Information.

人類白斑病皮膚的免疫組織化學分析(Immunohistochemical analysis)Immunohistochemical analysis of human vitiligo skin

診斷患有白斑病的兩名患者在提供書面知情同意書後參加了本研究。使用抗ITAC(R&D系統)及抗HMB45(黑色素細胞標記)的抗體(GeneTex,Irvine,Ca)，用3毫米直徑的穿刺針(punch)將三對色素過少及正常色素的皮膚樣品切片(biopsied)以用於免疫組織化學分析。該研究得到了東國大學伊山醫院(Dongguk University Ilsan Hospital)機構審查委員會(Institutional Review Board)的核准，並根據「赫爾辛基宣言(Declaration of Helsinki Principles)」進行。 Two patients diagnosed with vitiligo participated in this study after providing written informed consent. Three pairs of hypopigmented and normopigmented skin samples were biopsied using anti-ITAC (R&D Systems) and anti-HMB45 (melanocyte marker) antibodies (GeneTex, Irvine, Ca) with a 3 mm diameter punch. for immunohistochemical analysis. The study was approved by the Institutional Review Board of Dongguk University Ilsan Hospital and was conducted in accordance with the Declaration of Helsinki Principles.

統計分析Statistical Analysis

所有數據表示為平均值±SD。雙尾司圖頓t-檢驗(two-tailed Student's t-test)用於分析兩組之間的差異。 All data are expressed as mean ± SD. A two-tailed Student's t -test was used to analyze differences between the two groups .

結果result

ITAC誘導人類表皮黑色素細胞的遷移。ITAC induces migration of human epidermal melanocytes.

皮膚細胞，例如巨噬細胞(macrophages)、蘭格漢氏細胞(Langerhans cells)、γδ-T細胞、肥大細胞、角質細胞、及黑色素細胞產生各種趨化激素(表S1，參見輔助資料)。趨化激素的重要作用之一係介導細胞遷移。我們使用博伊登室試驗篩選了皮膚衍生的趨化激素誘導正常人類表皮黑色素細胞(NHEM)遷移的能力。NHEM朝向FK506及SDF1遷移，FK506係一種已知的黑色素細胞遷移誘導因子、SDF1已被證明可誘導NHEM遷移(表1，參見輔助資料)。10相較於未處理的對照組，許多其他趨化激素諸如CTACK、PARC、ITAC、RANTES、TARC、 及Lptn與SDF1在相同的濃度下亦顯著增加遷移(圖8，參見輔助資料)。我們專注於ITAC，因為CXCR3靶向趨化激素其重要地參與不同亞型的免疫細胞募集到炎症部位，僅ITAC而非MIG和IP-10顯著地誘導了NHEM遷移(圖8、圖1A、圖1B)。ITAC的遷移效應也在三種不同的人類黑色素瘤細胞系中得到驗證，導致這些細胞的遷移顯著地增加(圖9)。鑑於先前已知MIG及IP-10參與色素沉著14-15但ITAC並沒有，ITAC可能發揮與黑色素生成有關的MIG與IP-10不同的作用，也就是，藉由調控黑色素細胞遷移。為了解決ITAC誘導的NHEM遷移的機制，我們首先使用特異性抗體評估了ITAC的受體(啟動ITAC相關訊號16)CXCR3及CXCR7的表現量，並發現兩種蛋白質均在NHEM中表現(圖1C)。為了判定每個在ITAC誘導遷移的參與程度，在使用特異性中和抗體消耗內源性CXCR3或CXCR7蛋白後，我們執行了遷移試驗。相較於每個同型對照，用任一中和抗體處理後，ITAC誘導的遷移顯著降低(圖1D；泳道4與5及泳道6與7)，意味著CXCR3和CXCR7二者均參與ITAC誘導的黑色素細胞遷移。為了研究ITAC和兩種受體在病理狀況如黑色素瘤中的表現是否發生改變，我們進行了可公開取得的微陣列基因表現資料庫(Gene Expression Omnibus，GEO)的綜合分析，並發現相較於黑色素細胞(GSE29377)，ITAC及兩種受體的表現在黑色素瘤組織中顯示增加趨勢。儘管患者之間存在差異，但該分析結果意味著ITAC及其受體可能相互關聯以及參與某些黑色素瘤病理學。 Skin cells such as macrophages, Langerhans cells, γδ-T cells, mast cells, keratinocytes, and melanocytes produce various chemokines (Table S1, see Supplementary Information). One of the important roles of chemokines is to mediate cell migration. We screened for the ability of skin-derived chemokines to induce migration of normal human epidermal melanocytes (NHEMs) using a Boyden chamber assay. NHEM migrates towards FK506, a known melanocyte migration inducer, and SDF1, SDF1 has been shown to induce NHEM migration (Table 1, see Supplementary Information). 10 Many other chemokines such as CTACK, PARC, ITAC, RANTES, TARC, and Lptn and SDF1 also significantly increased migration at the same concentrations compared to untreated controls (Figure 8, see Supplementary Information). We focused on ITACs because CXCR3 targets chemokines that are critically involved in the recruitment of different subtypes of immune cells to sites of inflammation, and only ITACs but not MIG and IP-10 significantly induced NHEM migration (Fig. 8, Fig. 1A, Fig. 1B). The migratory effect of ITAC was also validated in three different human melanoma cell lines, resulting in a marked increase in the migration of these cells (Figure 9). Given that MIG and IP-10 were previously known to be involved in pigmentation14-15 but not ITAC , ITAC may play a different role for MIG than IP-10 in relation to melanogenesis, that is, by regulating melanocyte migration. To address the mechanism of ITAC-induced NHEM migration, we first assessed the expression of ITAC's receptors (initiating ITAC-related signaling 16 ), CXCR3 and CXCR7, using specific antibodies, and found that both proteins were expressed in NHEM (Fig. 1C). . To determine the degree of involvement of each in ITAC-induced migration, we performed migration assays after depletion of endogenous CXCR3 or CXCR7 proteins using specific neutralizing antibodies. ITAC-induced migration was significantly reduced after treatment with either neutralizing antibody compared to each isotype control (Figure ID; lanes 4 and 5 and lanes 6 and 7), implying that both CXCR3 and CXCR7 are involved in ITAC-induced migration. Melanocyte migration. To investigate whether the expression of ITAC and both receptors is altered in pathological conditions such as melanoma, we performed a comprehensive analysis of the publicly available Gene Expression Omnibus (GEO) and found that compared with The expression of melanocytes (GSE29377), ITAC and both receptors showed an increasing trend in melanoma tissues. Despite the differences between patients, the results of this analysis imply that ITACs and their receptors may be interrelated and involved in certain melanoma pathologies.

ITAC上調NHEM中遷移相關基因的mRNA表現。ITAC upregulates the mRNA expression of migration-related genes in NHEM.

為了瞭解潛在的ITAC誘導NHEM遷移的訊息傳遞途徑，我們使用對照組或ITAC處理的NHEM進行整體基因表現分析。IITAC處理上調了391個基因及下調了350個基因，相較於未處理的對照組，變化大於2倍(表2)。基於 功能分類分析，我們觀察到許多細胞移動性相關基因被上調，而一些黑色素生成相關基因諸如酪氨酸酶(TYR)及多巴色素異構酶(DCT)被下調(圖2A；表2)。從細胞移動性相關基因(23)中，我們選出了兩個基因HDAC5及HDAC9，其作為第IIa類成員，可藉由去乙醯化非組蛋白來調控蛋白活性，並且已被認為參與細胞移動性。7-8我們證實了在RT-qPCR分析中，藉由ITAC處理顯著地增加了這兩個基因的mRNA表現量(圖2B、圖2C)。 To understand the underlying signaling pathways for ITAC-induced NHEM migration, we performed global gene expression analysis using control or ITAC-treated NHEMs. IITAC treatment up-regulated 391 genes and down-regulated 350 genes, a greater than 2-fold change compared to untreated controls (Table 2). Based on functional classification analysis, we observed that many cell mobility-related genes were up-regulated, while some melanogenesis-related genes such as tyrosinase ( TYR ) and dopachrome isomerase ( DCT ) were down-regulated (Fig. 2A; Table 2) . From cell mobility-related genes (23), we selected two genes, HDAC5 and HDAC9 , which, as class IIa members, regulate protein activity by deacetylating non-histone proteins, and have been implicated in cell motility sex. 7-8 We confirmed that the mRNA expression levels of these two genes were significantly increased by ITAC treatment in RT-qPCR analysis (Fig. 2B, Fig. 2C).

HDAC5介導ITAC誘導的NHEM遷移。HDAC5 mediates ITAC-induced NHEM migration.

為了檢查這些基因是否參與ITAC誘導的黑色素細胞遷移，我們使用各種特定的siRNA抑制了HDAC5及HDAC9，以及在ITAC存在或不存在的情況下執行博伊登室試驗。藉由用RT-qPCR測量mRNA表現量來證實每個基因減弱(圖10)。NHEM對FK506的反應如預期，顯示遷移量增加約2倍(圖3A，泳道2)。相較於沒有ITAC的情況下(泳道3)誘導NHEM遷移的凌亂siRNA，HDAC5的減弱減少了遷移，但HDAC9的抑制卻沒有影響(泳道4、5)。在ITAC存在下，相較於凌亂對照(泳道6與7)，只有HDAC5的減弱顯著抑制了遷移，並與沒有ITAC之HDAC5減弱顯示了相似的遷移量(泳道4與7)，表示ITAC誘導的遷移主要由HDAC5介導。因此，我們聚焦於判定HDAC5在黑色素細胞遷移中的角色。使用HDAC5-Myc表現質體(plasmid)之HDAC5的過度表現大幅增加了NHEM遷移(圖3B，前兩個泳道)。透過ITAC處理，對照組細胞的遷移顯著增加到可與HDAC5過度表現相較的表現量，而過度表現HDAC5的細胞顯示出添加的遷移效應(圖3B，最後兩條泳道)，表示藉由過度表現或ITAC處理的HDAC5上調有效地介導NHEM遷移。我們證實了在ITAC處理後，HDAC5的蛋白質量隨著HDAC5 mRNA的上調而增加(圖3C)。 To examine whether these genes are involved in ITAC-induced melanocyte migration, we inhibited HDAC5 and HDAC9 using various specific siRNAs and performed Boyden chamber assays in the presence or absence of ITAC. Attenuation of each gene was confirmed by measuring mRNA expression with RT-qPCR (Figure 10). The NHEM response to FK506 was as expected, showing an approximately 2-fold increase in migration (Fig. 3A, lane 2). Attenuation of HDAC5 reduced migration compared to scrambled siRNA that induced NHEM migration in the absence of ITAC (lane 3), but inhibition of HDAC9 had no effect (lanes 4, 5). In the presence of ITAC, only attenuation of HDAC5 significantly inhibited migration compared to scrambled controls (lanes 6 and 7) and showed a similar amount of migration to attenuation of HDAC5 without ITAC (lanes 4 and 7), indicating that ITAC-induced Migration is mainly mediated by HDAC5. Therefore, we focused on determining the role of HDAC5 in melanocyte migration. Overexpression of HDAC5 using HDAC5-Myc expression plasmid greatly increased NHEM migration (Fig. 3B, first two lanes). By ITAC treatment, the migration of control cells was significantly increased to an expression level comparable to HDAC5 overexpression, while HDAC5 overexpressed cells showed an added migration effect (Fig. 3B, last two lanes), indicating that by overexpression or ITAC-treated HDAC5 upregulation efficiently mediates NHEM migration. We confirmed that the protein amount of HDAC5 increased with the upregulation of HDAC5 mRNA after ITAC treatment (Fig. 3C).

HDAC5影響p53在ITAC處理的NHEM中的乙醯化狀態。HDAC5 affects the acetylation status of p53 in ITAC-treated NHEM.

我們鑑定了HDAC5在ITAC誘導的遷移中之下游標靶。我們首先檢查了遷移相關蛋白，α-微管蛋白，其在神經元中藉由HDAC5去乙醯化。7然而，α-微管蛋白的乙醯化狀態不受ITAC處理(圖11A)或NHEM中HDAC5缺失的影響(圖11B)，表示在ITAC誘導的黑色素細胞遷移中，HDAC5並未靶向α-微管蛋白。在藉由乙醯化及去乙醯化調節的非組蛋白中，p53在細胞遷移中發揮作用。17此外，p53係HDAC5交互作用蛋白，其藉由在過度表現HDAC5的細胞中之免疫親和分離而鑑定，18而HDAC5直接將p53的K120去乙醯化，5導致MDM2依賴之泛素化及降解途徑。19因此，當HDAC5 mRNA及蛋白質表現量升高時，我們檢查了乙醯化或總p53的量是否受到ITAC處理的影響(圖2B、圖3C)。在ITAC處理的NHEM中，乙醯化p53的量隨著總p53略為減少而降低，該乙醯化p53的量藉由抗體識別p53之373及382處的至少兩個乙醯化離胺酸殘基來證實(圖4A)。在人類黑色素瘤細胞系SK-MEL-28中(其藉由ITAC處理顯示了遷移增加(圖9B))，藉由ITAC處理顯著降低了總p53蛋白量(圖11C)。為了探討HDAC5對p53表現量的影響，我們將HDAC5過度表現或減弱。HDAC5過度表現對應於乙醯化的及總p53的表現量的降低(圖4B)，以及當siRNA抑制了HDAC5時，兩種p53物種都增加(圖4C)。這些數據表示p53的乙醯化狀態係藉由HDAC5調控，且p53的下調參與ITAC誘導的細胞活性。 We identified downstream targets of HDAC5 in ITAC-induced migration. We first examined the migration-associated protein, α-tubulin, which is deacetylated by HDAC5 in neurons. 7 However, the acetylation status of α-tubulin was not affected by ITAC treatment (Fig. 11A) or HDAC5 depletion in NHEM (Fig. 11B), indicating that HDAC5 did not target α- in ITAC-induced melanocyte migration tubulin. Among non-histone proteins regulated by acetylation and deacetylation, p53 plays a role in cell migration. 17 In addition, p53 is an HDAC5-interacting protein that was identified by immunoaffinity isolation in cells overexpressing HDAC5,18 whereas HDAC5 directly deacetylates K120 of p53,5 leading to MDM2-dependent ubiquitination and degradation way. 19 Therefore, we examined whether the amount of acetylated or total p53 was affected by ITAC treatment when the amount of HDAC5 mRNA and protein expression was elevated (Fig. 2B, Fig. 3C). In ITAC-treated NHEM, the amount of acetylated p53 decreased with a slight decrease in total p53 by antibodies that recognize at least two acetylated lysine residues at 373 and 382 of p53 base to confirm (Figure 4A). In the human melanoma cell line SK-MEL-28, which showed increased migration by ITAC treatment (FIG. 9B), total p53 protein amount was significantly reduced by ITAC treatment (FIG. 11C). To explore the effect of HDAC5 on the amount of p53 expression, we overexpressed or attenuated HDAC5. HDAC5 overexpression corresponded to a decrease in the expression of acetylated and total p53 (Figure 4B), and an increase in both p53 species when HDAC5 was inhibited by siRNA (Figure 4C). These data indicate that the acetylation status of p53 is regulated by HDAC5 and that downregulation of p53 is involved in ITAC-induced cellular activity.

p53的過度表現抑制ITAC誘導及HDAC5介導的NHEM遷移。Overexpression of p53 inhibits ITAC-induced and HDAC5-mediated NHEM migration.

為了探討在HDAC5依賴方式中的ITAC治療後，p53是否影響NHEM遷移，我們在NHEM中過度表現HDAC5、p53、或兩者，並進行博伊登室遷移 法。在沒有ITAC的情況下，HDAC5過度表現顯大幅增加了細胞遷移(圖5A，泳道2)，但是p53過度表現取消了這種影響(泳道4)，表示在HDAC5誘導的細胞遷移中之p53參與。p53過度表現亦抑制了在細胞遷移中ITAC誘導的增加(泳道5與7)。與模擬處理的對照組相比，在ITAC處理的細胞中HDAC5過度表現進一步增加了細胞遷移(泳道5與6)，而p53過度表現消除了此種作用(泳道6與8)。這些數據意味著p53係HDAC5的下游效應子，並且參與ITAC及HDAC5誘導的細胞遷移。此外，已知由p53負調控的p53轉錄標靶，遷移相關基因基質金屬蛋白酶1(matrix metallopeptidase 1)(MMP1)20及膠原蛋白(Collagens) 21藉由ITAC處理反向上調，如微陣列分析(表2)及MMP1的RT-qPCR(圖5B)所測定，表示在ITAC處理的細胞中p53的轉錄活性降低。 To explore whether p53 affects NHEM migration following ITAC treatment in an HDAC5-dependent manner, we overexpressed HDAC5, p53, or both in NHEM and performed the Boyden chamber migration assay. In the absence of ITAC, HDAC5 overexpression significantly increased cell migration (Figure 5A, lane 2), but p53 overexpression abolished this effect (lane 4), indicating the involvement of p53 in HDAC5-induced cell migration. p53 overexpression also inhibited the ITAC-induced increase in cell migration (lanes 5 and 7). HDAC5 overexpression further increased cell migration in ITAC-treated cells compared to mock-treated controls (lanes 5 and 6), whereas p53 overexpression abolished this effect (lanes 6 and 8). These data imply that p53 is a downstream effector of HDAC5 and is involved in ITAC and HDAC5-induced cell migration. In addition, p53 transcriptional targets known to be negatively regulated by p53, migration-related genes matrix metallopeptidase 1 ( MMP1 ) 20 and collagen (Collagens) 21 were inversely up-regulated by ITAC treatment, as shown by microarray analysis ( Table 2) and RT-qPCR of MMP1 (Fig. 5B) showed that the transcriptional activity of p53 was reduced in ITAC-treated cells.

ITAC處理經由p53下調導致在NHEM中色素過少。ITAC treatment resulted in hypopigmentation in NHEM via p53 downregulation.

一些CXCR3配體，諸如IP-10及MIG，在延遲的色素斑點中上調。14在ITAC處理的SK-MEL-28細胞中，TYR蛋白的量隨著p53表現量的降低而顯著地降低(圖11C)。為了評估這些CXCR3配體(包括ITAC)是否對黑色素生成有影響，我們各用200ng/mL處理NHEM。ITAC降低了酪胺酸酶相關蛋白-1(TRP1，黑色素生成相關基因TYR)及DCT的mRNA表現量(圖12)，此與整體基因表現分析結果一致，其中TYR及DCT在ITAC處理的細胞中下調(圖2A；表2)。然而，MIG及IP-10並未改變這些基因的mRNA量(圖12)，表示與細胞遷移結果相似，在CXCR3配體之間對黑色素生成的影響不同(圖1A)。此外，我們用200ng/mL ITAC處理NHEM四天後，測得了黑色素含量顯著的劑量依賴性(dose-dependent)降低(圖6A)。先前，據報導藉由上調色素沉著相關基因如TYR及TRP1，p53在UV照射後，在皮膚色素沉著中發揮作用。22-23該發明人檢驗了 ITAC是否可在UV照射後降低p53介導的色素沉著。在UV處理的NHEM中，黑色素含量增加，並且總的或乙醯化的p53蛋白的量隨之增加(圖6B，泳道1與2)。藉由ITAC處理減少了UV照射後的這些效應，導致黑色素含量及p53蛋白量降低(圖6B，泳道2與3)。我們進一步藉由在ITAC存在下分析p53 siRNA處理後之黑色素生成相關基因的表現，進一步證實了p53在ITAC誘導的色素過少中的影響。結果，藉由p53 siRNA處理(相對於凌亂的對照組)(圖13，泳道1和2)及ITAC處理(TYR及TRP1；圖13，泳道3)，這些基因的表現顯著地降低，但是在ITAC處理前後，p53減弱的效果相似(圖13，泳道2與4)。此外，我們發現HDAC5在NHEM中的過度表現誘導了色素過少(圖6C)。總之，這些結果意味著ITAC誘導的色素過少係部分藉由p53介導，透過HDAC5對p53的去乙醯化，從而導致黑色素細胞的不穩定。 Some CXCR3 ligands, such as IP-10 and MIG, are upregulated in delayed pigmented spots. 14 In ITAC-treated SK-MEL-28 cells, the amount of TYR protein was significantly decreased with decreasing p53 expression (Fig. 11C). To assess whether these CXCR3 ligands, including ITAC, have an effect on melanogenesis, we treated NHEM with 200 ng/mL each. ITAC reduced the mRNA expression levels of tyrosinase-related protein-1 ( TRP1 , melanogenesis-related gene TYR ) and DCT (Fig. 12), which was consistent with the results of the overall gene expression analysis, in which TYR and DCT were expressed in ITAC-treated cells downregulated (Figure 2A; Table 2). However, MIG and IP-10 did not alter the mRNA levels of these genes (FIG. 12), indicating that similar to the cell migration results, the effects on melanogenesis differed between CXCR3 ligands (FIG. 1A). Furthermore, we measured a significant dose-dependent reduction in melanin content after treating NHEM with 200 ng/mL ITAC for four days (Fig. 6A). Previously, p53 was reported to play a role in skin pigmentation after UV irradiation by up-regulating pigmentation-related genes such as TYR and TRP1 . 22-23 The inventors examined whether ITAC could reduce p53-mediated pigmentation after UV irradiation. In UV-treated NHEM, melanin content was increased, with a concomitant increase in the amount of total or acetylated p53 protein (Fig. 6B, lanes 1 and 2). These effects after UV irradiation were attenuated by ITAC treatment, resulting in a decrease in melanin content and p53 protein content (Fig. 6B, lanes 2 and 3). We further confirmed the effect of p53 in ITAC-induced hypopigmentation by analyzing the expression of melanogenesis-related genes after p53 siRNA treatment in the presence of ITAC. As a result, the expression of these genes was significantly reduced by p53 siRNA treatment (relative to the scrambled control) (Figure 13, lanes 1 and 2) and ITAC treatment ( TYR and TRP1 ; Figure 13, lane 3), but not in ITAC The effect of p53 attenuation was similar before and after treatment (Figure 13, lanes 2 and 4). Furthermore, we found that overexpression of HDAC5 in NHEM induced hypopigmentation (Fig. 6C). Taken together, these results imply that ITAC-induced hypopigmentation is partially mediated by p53 through deacetylation of p53 by HDAC5, resulting in melanocyte destabilization.

儘管ITAC可以在p53關聯的色素沉著中帶來抗黑色素生成的效果，但由於其對於免疫及黑色素細胞的遷移能力，ITAC的過度產生可能與色素沉著失調有關。白斑病係一種炎症相關的色素過少病症常見的形式。本發明人通過使用抗ITAC抗體的免疫組織化學分析研究了ITAC表現是否在白斑病病灶中發生改變。一如預期，僅在正常皮膚中沿著表皮的基底膜觀察到黑色素(箭頭)及黑色素細胞(箭頭)(圖7，N#1)。有趣的是，與正常皮膚相比(幾乎沒有檢測到ITAC)，在白斑病病灶的表皮中ITAC表現大幅地上調且富集(圖7，L#1、L#2、與N#1)。與在人類黑色素瘤組織(GSE29377)中的ITAC表現分析結果一致，此結果表示ITAC在病理狀況中包括白斑病及黑色素瘤可能上調，並藉由影響細胞遷移及黑色素生成而參與其致病機制(pathogenesis)。 Although ITAC may confer anti-melanogenesis effects in p53-associated pigmentation, overproduction of ITAC may be associated with dyspigmentation due to its ability to im- mune and migrate melanocytes. Vitiligo is a common form of an inflammation-related hypopigmentation disorder. The inventors investigated whether ITAC manifestations were altered in vitiligo lesions by immunohistochemical analysis using anti-ITAC antibodies. As expected, melanin (arrows) and melanocytes (arrows) were observed along the basement membrane of the epidermis only in normal skin (Figure 7, N#1). Interestingly, ITACs were significantly up-regulated and enriched in the epidermis of vitiligo lesions compared to normal skin (where almost no ITACs were detected) (Figure 7, L#1, L#2, and N#1). Consistent with the analysis of ITAC expression in human melanoma tissue (GSE29377), this result suggests that ITAC may be up-regulated in pathological conditions including vitiligo and melanoma, and participate in its pathogenic mechanism by affecting cell migration and melanin production ( pathogenesis).

討論discuss

在本研究中，我們證實了ITAC，一種由皮膚細胞產生的趨化激素，藉由上調HDAC5的表現(其反過來藉由去乙醯化下調p53的量)，來誘導黑色素細胞遷移。ITAC誘導的遷移效應亦顯示在人類黑色素瘤細胞系中。用ITAC處理增加了黑色素細胞的遷移，如同以FK506處理一樣有效，而此現象是由其已知的受體CXCR3及CXCR7所介導。基於GEO資料庫的整體基因表現分析，比較黑色素細胞與黑色素瘤組織，ITAC及兩受體CXCR3與CXCR7的表現顯示了在黑色素瘤組織中增加的趨勢。CXCR3或CXCR7的消耗完全阻斷NHEM遷移的發現，表明CXCR3及CXCR7形成異二聚體(heterodimer)以介導黑色素細胞遷移。先前已經研究了CXCR類(例如CXCR4和CXCR7)之間的異二聚化(heterodimerization)；24然而，黑色素細胞中CXCR3及CXCR7之間是否發生異二聚化仍有待闡明。 In the present study, we demonstrated that ITAC, a chemokine produced by skin cells, induces melanocyte migration by upregulating the expression of HDAC5, which in turn downregulates the amount of p53 by deacetylation. ITAC-induced migratory effects were also shown in human melanoma cell lines. Treatment with ITAC increased melanocyte migration as effectively as treatment with FK506, a phenomenon mediated by its known receptors CXCR3 and CXCR7. Based on an overall gene expression analysis of the GEO database, comparing melanocytes with melanoma tissue, the expression of ITAC and the two receptors CXCR3 and CXCR7 showed an increased trend in melanoma tissue. The finding that depletion of CXCR3 or CXCR7 completely blocks NHEM migration suggests that CXCR3 and CXCR7 form a heterodimer to mediate melanocyte migration. Heterodimerization between CXCR classes such as CXCR4 and CXCR7 has been previously studied; 24 However, whether heterodimerization between CXCR3 and CXCR7 occurs in melanocytes remains to be elucidated.

與未經處理的對照組相比，我們發現在ITAC處理的細胞中HDAC5及HDAC9的量增加。在這些基因之間，僅HDAC5與ITAC誘導的NHEM遷移相關聯(圖3A)。根據以前的報告，7我們最初測試了α-微管蛋白作為HDAC5的標靶，以解釋ITAC的遷移效應。ITAC處理及HDAC5的siRNA減弱與乙醯化的α-微管蛋白量的變化無關(圖11A、圖11B)，表示另一個標靶參與ITAC誘導的黑色素細胞遷移。p53係各種訊息傳遞途徑中的重要調控蛋白，包括DNA損傷反應25以及各種細胞類型的遷移，其包括腫瘤細胞、26纖維母細胞、27和角質形成細胞。28p53的活性及穩定性在很大程度上取決於其乙醯化狀態，其被含有HDAC1或SIRT1的特異性HDAC蛋白質複合物所拮抗。因為HDAC1主要存在於細胞核中，其他HDAC則可在細胞質中調節p53乙醯化。4最近，據報導HDAC5在K120調控p53乙醯化。5基於在ITAC誘導的黑色素細胞遷移中p53可被 HDAC5去乙醯化的假說，我們顯示了HDAC5降低乙醯化及總p53蛋白(圖4B、圖4C)，從而介導了黑色素細胞遷移(圖5A)。雖然本研究並未鑑定藉由HDAC5去乙醯化所靶向的特異性離胺酸殘基，但我們認為在373/382處的離胺酸殘基及p53的120處可能是標靶位置(targetsite)。5為了檢測乙醯化的p53蛋白，我們使用抗乙醯化p53抗體，據認為其在373及382處識別至少兩個乙醯化的離胺酸殘基，以及發現這些殘基上的乙醯化p53被HDAC5過度表現或減弱分別地下調或上調(圖4B、圖C)，意味著這些殘基作為HDAC5去乙醯化的可能標靶位置。 We found increased amounts of HDAC5 and HDAC9 in ITAC-treated cells compared to untreated controls. Among these genes, only HDAC5 was associated with ITAC-induced NHEM migration (Fig. 3A). Based on previous reports, 7 we initially tested α-tubulin as a target of HDAC5 to explain the migratory effects of ITAC. ITAC treatment and siRNA attenuation of HDAC5 were not associated with changes in the amount of acetylated alpha-tubulin (FIG. 11A, FIG. 11B), indicating that another target is involved in ITAC-induced melanocyte migration. p53 is an important regulatory protein in various signaling pathways, including the DNA damage response 25 and the migration of various cell types, including tumor cells, 26 fibroblasts, 27 and keratinocytes. 28 The activity and stability of p53 is largely dependent on its acetylation state, which is antagonized by specific HDAC protein complexes containing HDAC1 or SIRT1. Because HDAC1 resides primarily in the nucleus, other HDACs regulate p53 acetylation in the cytoplasm. 4 Recently, it was reported that HDAC5 regulates p53 acetylation at K120. 5 Based on the hypothesis that p53 can be deacetylated by HDAC5 in ITAC-induced melanocyte migration, we showed that HDAC5 reduced acetylation and total p53 protein (Fig. 4B, Fig. 4C), thereby mediating melanocyte migration (Fig. 5A). Although this study did not identify specific lysine residues targeted by HDAC5 deacetylation, we believe that lysine residues at 373/382 and 120 of p53 may be the target positions ( targetsite). 5 To detect acetylated p53 protein, we used an anti-acetylated p53 antibody, which is thought to recognize at least two acetylated lysine residues at 373 and 382, and found acetylated Hap53 was overexpressed or attenuated by HDAC5 down- or up-regulated, respectively (Fig. 4B, Panel C), implying that these residues serve as possible target positions for HDAC5 deacetylation.

除了遷移，我們觀察到ITAC處理誘導了正常及紫外線處理的黑色素細胞之色素過少(圖6A、圖6B)。ITAC處理下調黑色素生成相關基因及p53蛋白量的表現(圖2、圖4A、圖6B)。p53藉由紫外線照射而上調，以及藉由增加TYR及TRP1表現誘導黑色素生成。22-23我們發現p53參與ITAC誘導的色素過少(圖6B)。因為HDAC5係p53穩定性的主要調控因子，係透過在ITAC誘導的黑色素細胞遷移中之去乙醯化來調控，以及HDAC5過度表現與脫色作用(depigmentation)相關聯(圖6C)，因此HDAC5可能透過p53的去乙醯化直接參與ITAC誘導的色素過少，其反過來下調黑色素生成相關(melanogenesis-related)的基因。受p53下調影響的基因可能係在ITAC治療後黑色素細胞遷移、色素過少、或兩者的原因。我們發現在ITAC處理的細胞中上調了膠原蛋白及MMP1(由p53負調控之細胞遷移相關基因)(圖5B；表2)。這些上調基因亦可參與ITAC誘導的色素過少。總之，這些數據意味著經由第IIa類HDAC(特別是HDAC5)對p53的去乙醯化，p53的去穩定化在ITAC誘導的細胞過程中諸如遷移及色素過少發揮關鍵作用。 In addition to migration, we observed that ITAC treatment induced hypopigmentation in normal and UV-treated melanocytes (Fig. 6A, Fig. 6B). ITAC treatment down-regulated the expression of melanogenesis-related genes and p53 protein levels ( FIG. 2 , FIG. 4A , and FIG. 6B ). p53 is upregulated by UV irradiation, and melanogenesis is induced by increasing TYR and TRP1 expression. 22-23 We found that p53 was involved in ITAC-induced hypopigmentation (Fig. 6B). Because HDAC5 is a major regulator of p53 stability, regulated through deacetylation in ITAC-induced melanocyte migration, and HDAC5 overexpression is associated with depigmentation (Fig. 6C), HDAC5 may be Deacetylation of p53 is directly involved in ITAC-induced hypopigmentation, which in turn downregulates melanogenesis-related genes. Genes affected by p53 downregulation may be responsible for melanocyte migration, hypopigmentation, or both after ITAC treatment. We found that collagen and MMP1 , a cell migration-related gene negatively regulated by p53, were up-regulated in ITAC-treated cells (Fig. 5B; Table 2). These up-regulated genes may also be involved in ITAC-induced hypopigmentation. Taken together, these data imply that p53 destabilization plays a key role in ITAC-induced cellular processes such as migration and hypopigmentation via the deacetylation of p53 by class IIa HDACs, in particular HDAC5.

急性及慢性皮膚炎症可能導致色素過少或色素過多(hyperpigmentation)，取決於環境和遺傳因子之間的交互作用。29在CXCR3配體中，MIG及IP-10在炎症細胞累積的延遲色素斑點中上調，導致黑色素細胞的活化及慢性黑色素合成。14相反地，ITAC在色素過少的失調白斑病皮膚中上調(圖7)，其中IP-10與健康的對照組相比沒有放鬆管制，30意味著CXCR3配體之間具有不同的功能以及ITAC在人類白斑病致病機制中可能的參與度。先前，據稱MIG和IP-10對於小鼠白斑病至關重要，而其中ITAC未發揮主要作用。31我們認為這種分歧(discrepancy)可能是由於小鼠與人類之間的表皮結構的差異，例如，1)人類表皮由多層組成，與小鼠的薄表皮產生不同的環境，2)人類表皮黑色素細胞同時存在於濾泡(follicular)及濾泡間(interfollicular)之區域，但是小鼠黑色素細胞僅存在於濾泡區中。也許，自基底膜上的角質形成細胞分泌的ITAC可直接影響濾泡間區域的黑色素細胞，該區域並不存在小鼠黑色素細胞。 Acute and chronic skin inflammation may lead to hypopigmentation or hyperpigmentation, depending on interactions between environmental and genetic factors. 29 Among CXCR3 ligands, MIG and IP-10 are upregulated in delayed pigmented spots accumulated by inflammatory cells, leading to melanocyte activation and chronic melanin synthesis. 14 Conversely, ITAC was upregulated in hypopigmented dysregulated vitiligo skin (Figure 7), where IP-10 was not deregulated compared to healthy controls, 30 implying different functions between CXCR3 ligands and ITAC in Possible involvement in the pathogenesis of human vitiligo. Previously, MIG and IP-10 were said to be critical for vitiligo in mice, in which ITAC did not play a major role. 31 We believe that this discrepancy may be due to differences in epidermal structure between mice and humans, e.g., 1) the human epidermis is composed of multiple layers and produces a different environment from the thin mouse epidermis, 2) the human epidermal melanin Cells are present in both follicular and interfollicular regions, but mouse melanocytes are present only in the follicular region. Perhaps, ITAC secreted from keratinocytes on the basement membrane can directly affect melanocytes in the interfollicular region, where mouse melanocytes are absent.

在人類角質形成細胞及黑色素細胞的共培養系統中，低濃度(10ng/mL)的MIG、IP-10、及ITAC增加了黑色素含量。15然而，在黑色素細胞單一培養系統中，這三種趨化激素的低濃度對黑色素生成相關基因的表現沒有影響(數據未顯示)，而且只有ITAC以高濃度(200ng/mL)下調了這些基因表現(圖12)。這些不同的觀察結果可歸因於，在共培養系統中，ITAC處理(其可能間接影響黑色素細胞活化)後，角質形成細胞釋放的可溶性因子(soluble factors)。MIG及IP-10即使在高濃度下，對色素沉著或黑色素細胞遷移都沒有影響，意味著它們並不直接影響黑色素細胞，而是必須發揮不同於ITAC的作用。雖然MIG、IP-10、及ITAC與干擾素-g-反應趨化激素密切相關，並透過CXCR3介導信號以吸引T淋巴細胞(T lymphocytes)，但它們對黑色素細胞遷移及黑色素生成的影響似乎根 據細胞環境(cellular context)而不同。此觀察結果可部分地藉由它們對CXCR3的不同結合親和力、其它ITAC輔助受體如CXCR7在黑色素細胞上的存在、以及它們在病理生理(pathophysiological)狀況下表現量的差異來解釋。考慮到ITAC主要由疾病斑點周圍的基底膜上的角質形成細胞及數種類型的炎性皮膚疾病中的皮膚細胞所分泌，12其下游效應子(downstream effectors)HDAC5及p53對於調控成熟黑色素細胞行為可能是重要的。許多色素沉著相關疾病諸如日光性小痣(solar lentigo)、黑皮病(melasma)、及黑色素瘤係由黑色素細胞凝結(condensation)代表及惡化。我們的研究強調透過HDAC5刺激的p53下調是用來減輕這些疾病的策略之一，其係藉由同時誘導凝結的黑色素細胞遷移及色素過少。 In a co-culture system of human keratinocytes and melanocytes, low concentrations (10 ng/mL) of MIG, IP-10, and ITAC increased melanin content. 15 However, in the melanocyte monoculture system, low concentrations of these three chemokines had no effect on the expression of melanogenesis-related genes (data not shown), and only ITAC at high concentrations (200 ng/mL) down-regulated the expression of these genes. (Figure 12). These different observations can be attributed to soluble factors released by keratinocytes following ITAC treatment (which may indirectly affect melanocyte activation) in the co-culture system. Neither MIG nor IP-10 had any effect on pigmentation or melanocyte migration, even at high concentrations, meaning that they do not directly affect melanocytes, but must play a different role than ITAC. Although MIG, IP-10, and ITAC are closely related to interferon-g-responsive chemokines and mediate signaling through CXCR3 to attract T lymphocytes, their effects on melanocyte migration and melanogenesis appear to be Varies according to the cellular context. This observation may be explained in part by their differing binding affinities for CXCR3, the presence of other ITAC coreceptors such as CXCR7 on melanocytes, and differences in their expression under pathophysiological conditions. Considering that ITAC is mainly secreted by keratinocytes on the basement membrane around disease spots and by skin cells in several types of inflammatory skin diseases, 12 its downstream effectors, HDAC5 and p53, are important in regulating mature melanocyte behavior. may be important. Many pigmentation-related diseases such as solar lentigo, melasma, and melanoma are represented and exacerbated by melanocyte condensation. Our study highlights that p53 downregulation through HDAC5 stimulation is one of the strategies used to alleviate these diseases by simultaneously inducing coagulated melanocyte migration and hypopigmentation.

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圖1. ITAC誘導人類表皮黑色素細胞的遷移。Figure 1. ITAC induces migration of human epidermal melanocytes.

在以200ng/mL MIG、IP-10、或ITAC(a)、或各種濃度的ITAC(b)處理後，使用博伊登室試驗評估正常人類表皮黑色素細胞(新生兒的(NHEM))的遷移。允許細胞遷移24小時。數據代表三項獨立實驗(*P<0.05,**P<0.01)。(c)藉由西方墨點法測定CXCR3及CXCR7的蛋白表現量。重複執行西方墨點分析，並顯示代表性圖像。(d)用對抗CXCR3或CXCR7的中和抗體預處理NHEM 30分鐘，以及在ITAC存在下執行遷移試驗。抗IgG(CXCR3)及抗IgG(CXCR7)抗體分別用作抗CXCR3及抗CXCR7中和抗體的同型對照。數據代表三項獨立實驗(***P<0.001)。 Migration of normal human epidermal melanocytes (neonatal (NHEM)) was assessed using the 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. Data are representative of three independent experiments (* P < 0.05, ** P < 0.01). (c) The protein expression levels of CXCR3 and CXCR7 were determined by Western blotting method. Western blot analysis was performed in duplicate and representative images are shown. (d) NHEMs were pretreated with neutralizing antibodies against CXCR3 or CXCR7 for 30 minutes, and migration assays were performed in the presence of ITAC. Anti-IgG (CXCR3) and anti-IgG (CXCR7) antibodies were used as isotype controls for anti-CXCR3 and anti-CXCR7 neutralizing antibodies, respectively. Data are representative of three independent experiments (*** P < 0.001).

圖2. ITAC上調NHEM中遷移相關基因的mRNA表現。Figure 2. ITAC upregulates the mRNA expression of migration-related genes in NHEM.

以50ng/mL ITAC處理NHEM 48小時，以及用50ng總RNA執行微陣列分析。(a)藉由使用DAVID進行基因本體(GO)分析將ITAC處理顯著上調或下調的生物過程分類。B-H P值：Benjamini-Hochberg多重測試校正的p值。(b-c)藉由RT-qPCR測定NHEM中HDAC5(b)及HDAC9(c)的mRNA量。數據代表三項獨立實驗(*P<0.05)。 NHEMs were treated with 50 ng/mL ITAC for 48 hours, and microarray analysis was performed with 50 ng of total RNA. (a) Classification of biological processes significantly up- or down-regulated by ITAC treatment by Gene Ontology (GO) analysis using DAVID. BH P-value: Benjamini-Hochberg multiple test corrected p-value. (bc) Determination of HDAC5 (b) and HDAC9 (c) mRNA levels in NHEM by RT-qPCR. Data are representative of three independent experiments (* P < 0.05).

圖3. HDAC5介導在NHEM中ITAC誘導的遷移。Figure 3. HDAC5 mediates ITAC-induced migration in NHEM.

(a)將siRNA轉染到NHEM中48小時。在50ng/mL ITAC不存在或存在的情況下，使細胞在轉染後遷移24小時，然後固定以計數。凌亂siRNA(Scrambled siRNA)及FK506分別用作陰性及陽性對照。數據代表三項獨立實驗(*P<0.05,***P<0.001)。(b)將HDAC5-Myc質體導入NHEM中48小時。在50ng/mL ITAC不存在或存在的情況下允許細胞遷移24小時。計算遷移的細胞。數據代表三項獨立實驗(*P<0.05,**P<0.01)。(c)以50ng/mL ITAC處理48小時後藉由西方墨點法測定HDAC5蛋白表現量。重複執行西方墨點分析，並顯示代表性的圖像。示出相對帶強度。 (a) siRNA was transfected into NHEM for 48 hours. Cells were allowed to migrate 24 hours post-transfection in the absence or presence of 50 ng/mL ITAC and then fixed for enumeration. Scrambled siRNA and FK506 were used as negative and positive controls, respectively. Data are representative of three independent experiments (* P < 0.05, *** P < 0.001). (b) Introduction of HDAC5-Myc plastids into NHEM for 48 hours. Cells were allowed to migrate for 24 hours in the absence or presence of 50 ng/mL ITAC. Counting migrated cells. Data are representative of three independent experiments (* P < 0.05, ** P < 0.01). (c) The expression of HDAC5 protein was determined by Western blotting method after 48 hours of treatment with 50 ng/mL ITAC. Western blot analysis was performed in duplicate and representative images are shown. Relative band intensities are shown.

圖4. HDAC5影響p53在ITAC處理的NHEM中的乙醯化狀態。Figure 4. HDAC5 affects the acetylation status of p53 in ITAC-treated NHEM.

(a)用50ng/mL ITAC處理NHEM 48小時。乙醯化及總p53蛋白的量以所示抗體藉由西方墨點法測定。將HDAC5-Myc過度表現構建體(construct)(b)或對抗HDAC5之siRNA(c)轉染到NHEM中48小時。模擬及凌亂siRNA分別用作HDAC5-Myc及HDAC5特異性siRNA的對照。乙醯化及總p53蛋白量藉由使用所示抗體之西方墨點法測定。GAPDH用作加載控制。重複執行西方墨點分析，並顯示代表性圖像。示出相對帶強度。a-Ac-p53，抗乙醯化p53抗體。 (a) NHEM was treated with 50 ng/mL ITAC for 48 hours. The amount of acetylated and total p53 protein was determined by Western blotting with the indicated antibodies. HDAC5-Myc overexpression constructs (b) or siRNA against HDAC5 (c) were transfected into NHEM for 48 hours. Mock and scrambled siRNA were used as controls for HDAC5-Myc and HDAC5 specific siRNA, respectively. Acetylation and total p53 protein amounts were determined by Western blotting using the indicated antibodies. GAPDH is used as loading control. Western blot analysis was performed in duplicate and representative images are shown. Relative band intensities are shown. a-Ac-p53, anti-acetylated p53 antibody.

圖5. p53的過度表現抑制ITAC誘導及HDAC5介導的NHEM遷移。Figure 5. Overexpression of p53 inhibits ITAC-induced and HDAC5-mediated NHEM migration.

(a)模擬轉染NHEM(泳道1、5)或用表現質體轉染HDAC5-Myc(泳道2、6)、p53-Flag(泳道3、7)、或兩者(泳道4、8。在轉染後48小時，允許細胞在50ng/mL ITAC存在或不存在的情況下遷移24小時，然後固定以計數。數據代表三項獨立實驗(* P<0.05)。(b)在ITAC處理前後，藉由RT-qPCR測定NHEM中 MMP1的mRNA量。將值標準化(normalized)至GAPDH。數據代表三項獨立實驗(*P<0.05)。 (a) Mock transfection of NHEM (lanes 1, 5) or transfection of HDAC5-Myc (lanes 2, 6), p53-Flag (lanes 3, 7), or both with expressing plastids (lanes 4, 8. In Forty-eight hours after transfection, cells were allowed to migrate in the presence or absence of 50 ng/mL ITAC for 24 hours and then fixed for enumeration. Data are representative of three independent experiments ( * P < 0.05). (b) Before and after ITAC treatment, The mRNA amount of MMP1 in NHEM was determined by RT-qPCR. Values were normalized to GAPDH. Data are representative of three independent experiments (* P < 0.05).

圖6. ITAC處理經由p53下調導致在NHEM中色素過少。Figure 6. ITAC treatment leads to hypopigmentation in NHEM via p53 downregulation.

(a)將NHEM用不同濃度的ITAC處理4天。觀察每個沉澱物(pellet)，測定黑色素含量。數據代表三項獨立實驗(* P<0.05)。(b)藉由UV(20mJ/cm2)照射NHEM兩次以及維持兩週。照射後每三天處理一次ITAC(50ng/ml)。測定黑色素含量。數據代表三項獨立實驗(* P<0.05)。乙醯化及總p53蛋白量藉由使用所示抗體之西方墨點法測定。GAPDH用作加載控制。重複執行西方墨點分析，並顯示代表性圖像。示出相對帶強度。a-Ac-p53，抗乙醯化p53抗體。(c)模擬轉染或用不同濃度的HDAC5-Myc表現質粒轉染NHEM 48小時。離心後觀察每個沉澱物，確定黑色素含量。數據代表三項獨立實驗(*P<0.05)。 (a) NHEMs were treated with different concentrations of ITAC for 4 days. Each pellet was observed and the melanin content was determined. Data are representative of three independent experiments ( * P < 0.05). (b) NHEM was irradiated by UV (20 mJ/cm 2 ) twice and maintained for two weeks. ITAC (50ng/ml) was treated every three days after irradiation. Determination of melanin content. Data are representative of three independent experiments ( * P < 0.05). Acetylation and total p53 protein amounts were determined by Western blotting using the indicated antibodies. GAPDH is used as loading control. Western blot analysis was performed in duplicate and representative images are shown. Relative band intensities are shown. a-Ac-p53, anti-acetylated p53 antibody. (c) Mock transfection or transfection of NHEM with different concentrations of HDAC5-Myc expression plasmid for 48 hours. Each pellet was observed after centrifugation to determine the melanin content. Data are representative of three independent experiments (* P < 0.05).

圖7. ITAC富含於白斑病皮膚表皮中。Figure 7. ITAC is enriched in vitiligo skin epidermis.

從兩位白斑病患者(#1及#2)獲得正常(N)或色素過少病灶(L)的皮膚樣品，用抗ITAC(綠色)和抗HMB45(紅色)抗體染色;細胞核用DAPI(藍色)染色。拍攝明亮的野外圖像以分析(visualize)組織中的黑色素(箭頭)。箭頭表示黑色素細胞。HMB45，人類黑色素瘤45比例尺，50μm。 Normal (N) or hypopigmented lesions (L) skin samples were obtained from two vitiligo patients (#1 and #2), stained with anti-ITAC (green) and anti-HMB45 (red) antibodies; nuclei were stained with DAPI (blue) )dyeing. Bright field images were taken to visualize melanin in tissue (arrow). Arrows indicate melanocytes. HMB45, human melanoma 45 Scale bar, 50 μm.

<輔助資料><Supporting Information>

透過經由HDAC5將p53去穩定化，ITAC誘導黑色素細胞遷移及色素過少:ITAC在色素相關病症中的可能角色ITAC induces melanocyte migration and hypopigmentation by destabilizing p53 via HDAC5: a possible role for ITAC in pigment-related disorders

補充材料及方法:Supplementary materials and methods:

細胞培養cell culture

人類黑色素瘤MNT-1細胞系係由東國大學的Ai-Young Lee博士所提供，他最初係收到來自國家衛生研究院(National Institutes of Health,Bethesda,Maryland,USA)的Vincent J.Hearing博士送的禮物。細胞在37℃、含有20% FBS及抗生素的DMEM(Lonza,Basel,Switzerland)中培養。WM266-4人類黑色素瘤細胞系得自美國菌種保存中心(American Type Culture Collection,ATCC,Manassas,VA,USA)。SK-MEL-28人類黑色素瘤細胞系係得自韓國細胞系銀行(Korean Cell Line Bank,KCLB,Seoul,Korea)。將那些細胞保持在37℃、含有10% FBS及抗生素的EMEM培養基(ATCC)中。 The human melanoma MNT-1 cell line was provided by Dr. Ai-Young Lee of Dongguk University, who originally received Dr. Vincent J. Hearing from the National Institutes of Health (Bethesda, Maryland, USA) gift. Cells were cultured at 37°C in DMEM (Lonza, Basel, Switzerland) containing 20% FBS and antibiotics. The WM266-4 human melanoma cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The SK-MEL-28 human melanoma cell line was obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). Those cells were maintained at 37°C in EMEM medium (ATCC) containing 10% FBS and antibiotics.

西方墨點分析Western Ink Dot Analysis

藉由在4℃下於溶解緩衝液(lysis buffer)(20mM Tris[pH7.5]、0.1% Triton X-100、0.5%去氧膽酸鹽(deoxycholate)、1mM PMSF、10μg/ml抑胰肽酶(aprotinin)、及10μg/ml亮抑酶肽(leupeptin))中培育(incubation)10分鐘來萃取細胞(2 x 106)，並以13,000rpm離心5分鐘。根據製造商的說明，使用二辛可寧酸試劑套組(BCA assay kit；Sigma,St.Louis,MO)測量蛋白質濃度。藉由SDS/PAGE分離25mg總蛋白，並轉移到PVDF膜(Invitrogen,Washington,DC)。用含有0.3%酪蛋白(casein)的PBS在室溫下封閉(blocking))0.5小時後，在4℃下將膜與3-5μg/ml一級抗體在含有0.1% Tween 20的PBS中的培育過夜。使用ImagePro Plus軟體版本4.0定量信號強度，並將其標準化至GAPDH的強度。各種抗體資訊如下：抗CXCR3、抗CXCR7(Abcam,Cambridge,UK)、抗-HDAC5、抗p53(Cell Signaling,Danvers,MA)，、抗乙醯化p53(Millipore,MA)、抗α-微管蛋白、抗乙醯化α-微管蛋白(Sigma-Aldrich,St.Louis,MO)、抗ITAC(R&D system,Minneapolis, MN)、抗HMB45(GeneTex,Irvine,CA)、抗GAPDH抗體(Santa Cruz Biotechnology,Dollas,TX)。所有數據均得自兩次以上的獨立實驗，每個實驗重複進行三次。 by lysis buffer (20 mM Tris [pH 7.5], 0.1% Triton X-100, 0.5% deoxycholate, 1 mM PMSF, 10 μg/ml aprotinin) at 4°C Cells were extracted (2 x 10 6 ) by incubation in aprotinin, and 10 μg/ml leupeptin for 10 minutes, and centrifuged at 13,000 rpm for 5 minutes. Protein concentrations were measured using a bicinchoninic acid reagent kit (BCA assay kit; Sigma, St. Louis, MO) according to the manufacturer's instructions. 25 mg of total protein were separated by SDS/PAGE and transferred to PVDF membranes (Invitrogen, Washington, DC). After blocking with 0.3% casein in PBS for 0.5 h at room temperature, the membrane was incubated with 3-5 μg/ml primary antibody in PBS containing 0.1% Tween 20 overnight at 4°C . Signal intensities were quantified using ImagePro Plus software version 4.0 and normalized to the intensity of GAPDH. Various 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-α-microtubule Protein, anti-acetylated α-tubulin (Sigma-Aldrich, St.Louis, MO), anti-ITAC (R&D system, Minneapolis, MN), anti-HMB45 (GeneTex, Irvine, CA), anti-GAPDH antibody (Santa Cruz Biotechnology, Dollars, TX). All data were obtained from more than two independent experiments, each performed in triplicate.

用於原代黑色素細胞(primary melanocytes)及黑色素瘤細胞系(melanoma cell lines)之細胞遷移試驗(Cell migration assay)Cell migration assay for primary melanocytes and melanoma cell lines

在室溫下將博伊登室(孔徑8vm；Corning Inc.,Corning,NY)中的濾器用第IV型膠原蛋白包覆3小時。將5 X 104個細胞置於腔室的上部孔中，並將下部孔用含有0.5%牛血清白蛋白(bovine albumin)的EpiLife培養基(Life Technologies,Carlsbad,CA,USA)及包括不同濃度的ITAC之所示趨化激素填充。於37℃培養24小時後，用甲醇固定過濾***物，用蘇木色素(hematoxylin)及曙紅(eosin，Merck,Darmstadt,Germany)染色。在相位差顯微鏡(phase-contrast microscopy)下拍攝圖像，並計算細胞以定量。所有數據均得自兩次以上的獨立實驗，每個實驗重複進行三次。 Filters in Boyden chambers (8 vm pore size; Corning Inc., Corning, NY) were coated with Type IV collagen for 3 hours at room temperature. 5 x 10 cells were placed in the upper well of the chamber, and the lower well was treated with EpiLife medium (Life Technologies, Carlsbad, CA, USA) containing 0.5% bovine serum albumin (Life Technologies, Carlsbad, CA, USA) and various concentrations of Chemokine filling as indicated by ITAC. After incubation at 37°C for 24 hours, the filter inserts were fixed with methanol and stained with hematoxylin and eosin (Merck, Darmstadt, Germany). Images were taken under phase-contrast microscopy and cells were counted for quantification. All data were obtained from more than two independent experiments, each performed in triplicate.

微陣列分析(Microarray analysis)Microarray analysis

用50ng/mL ITAC處理NHEM 48小時，以及使用TRIzol試劑(Gibco-BRL,Gaithersburg,MD)分離總RNA。按照製造商的計劃書，使用Quick Amp Kit(Agilent Technologies,Palo Alto,CA)將50奈克總RNA轉換成Cy3或Cy5標記的cRNA。使用Agilent Bioanalyzer測定260及280nm處之吸光度比以用於品質評估(quality assessment)及濃度。將來自兩個不同樣本之等量Cy3或Cy5-標記的cRNA與Agilent Human Whole Genome 4 X 44k微陣列雜合。使用Feature Extraction 10.2版(Agilent Technologies)從掃描圖像中提取數據。每個樣本重複雜合反應。 NHEM was treated with 50 ng/mL ITAC for 48 hours, 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 using the Quick Amp Kit (Agilent Technologies, Palo Alto, CA) according to the manufacturer's protocol. Absorbance ratios at 260 and 280 nm were determined for quality assessment and concentration using an Agilent Bioanalyzer. Equal amounts of Cy3 or Cy5-labeled cRNA from two different samples were hybridized to Agilent Human Whole Genome 4 X 44k microarrays. Data were extracted from scanned images using Feature Extraction version 10.2 (Agilent Technologies). Repeat the complex reaction for each sample.

定量即時PCR分析quantitative real-time PCR analysis

使用Superscript II反轉錄酶套組(Superscript II Reverse Transcriptase Kit)(Life Technologies,Grand Island,NY)將來自每個樣本的二vg總RNA用於cDNA合成。購買TaqMan®探子(Themo Fisher Scientific,Waltham,MA)，並使用ABI Fast Real-Time PCR System(Life Technologies)執行qPCR。將樣本中每個基因的相對表現量標準化(normalized)至管家基因(housekeeping gene)(甘油醛-3-磷酸去氫酶(glyceraldehyde 3-phosphate dehydrogenase，GAPDH))，以及使用式2-△△Ct計算樣本之間的基因表現差異。 Two vg total RNA from each sample was used for cDNA synthesis using the Superscript II Reverse Transcriptase Kit (Life Technologies, Grand Island, NY). TaqMan® probes (Themo Fisher Scientific, Waltham, MA) were purchased and qPCR was performed using the ABI Fast Real-Time PCR System (Life Technologies). The relative expression level of each gene in the sample was normalized to a housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), and using the formula 2- △△Ct Calculate the difference in gene performance between samples.

黑色素試驗(Melanin assay)Melanin assay

在含有1% Nonidet P-40、0.01% SDS，及蛋白酶抑制劑混合物(protease inhibitor cocktail，Roche Molecular Biochemical,Indianapolis,IN)的0.1M Tris-HCl(pH 7.2)緩衝液中，藉由超音波震盪(sonicating)正常的或UV處理的NHEM來製備細胞溶胞產物。黑色素在溶解於1N NaOH之前，藉由離心(centrifugation)自細胞溶胞產物中分離。藉由使用Synergy H2酶標儀(microplate reader，BioTek,Winooski,VT,USA)測量在490nm的吸光度來測定黑色素含量，並將其標準化至蛋白質輸入。執行了三次獨立實驗。 in 0.1 M Tris-HCl (pH 7.2) buffer containing 1% Nonidet P-40, 0.01% SDS, and protease inhibitor cocktail (Roche Molecular Biochemical, Indianapolis, IN) by sonication (sonicating) normal or UV-treated NHEM to prepare cell lysates. Melanin was isolated from cell lysates by centrifugation prior to solubilization in 1 N NaOH. Melanin content was determined by measuring absorbance at 490 nm using a Synergy H2 microplate reader (microplate reader, BioTek, Winooski, VT, USA) and normalized to protein input. Three independent experiments were performed.

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Menezes-Souza, D., Guerra-Sa, R., Carneiro, C.M., Vitoriano-Souza, J., Giunchetti, R.C., Teixeira-Carvalho, A., Silveira-Lemos, D., Oliveira, G.C., Correa-Oliveira, R., and Reis, A.B. (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 Menezes-Souza, D., Guerra-Sa, R., Carneiro, C.M., Vitoriano-Souza, J., Giunchetti, R.C., Teixeira-Carvalho, A., Silveira-Lemos, D., Oliveira, G.C., Correa-Oliveira , R., and Reis, A.B. (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 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 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 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 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

圖8. NHEM對皮膚細胞衍生的趨化因子的遷移能力Figure 8. Migration ability of NHEM to skin cell-derived chemokines

藉由博伊登室試驗分析細胞遷移，如補充材料及方法中所述。對於博伊登室試驗，將細胞加至上部孔中，而所示趨化激素加至下部孔中(200ng/ml)。允許細胞遷移24小時以及固定、染色、成像、及定量。這裡顯示的結果在兩次獨立實驗中是可再現的(*** P<0.001).。 Cell migration was analyzed by Boyden chamber assay as described in Supplementary Materials and Methods. For the Boyden chamber assay, cells were added to the upper wells and the indicated chemokines were added to the lower wells (200ng/ml). Cells were allowed to migrate for 24 hours and were fixed, stained, imaged, and quantified. The results shown here are reproducible in two independent experiments (*** P < 0.001).

圖9. ITAC對人類黑色素瘤細胞系的遷移效應Figure 9. Migration effect of ITAC on human melanoma cell lines

藉由博伊登室試驗分析細胞遷移。將人類黑色素瘤細胞系WM266-4(a)、SK-MEL-28(b)、及MNT-1(c)在不含I-TAC(對照)的培養基或含200ng/ml的I-TAC(I-TAC)遷移24h。將細胞固定、染色、及定量。數據代表三項獨立實驗(*P<0.05,**P<0.01)。 Cell migration was analyzed by Boyden chamber assay. Human melanoma cell lines WM266-4 (a), SK-MEL-28 (b), and MNT-1 (c) were cultured in medium without I-TAC (control) or with 200 ng/ml of I-TAC ( I-TAC) migrated for 24h. Cells were fixed, stained, and quantified. Data are representative of three independent experiments (* P < 0.05, ** P < 0.01).

圖10. HDAC5特異性siRNA及HDAC9特異性siRNA在NHEM中的有效性Figure 10. Effectiveness of HDAC5-specific siRNA and HDAC9-specific siRNA in NHEM

將對抗HDAC5、HDAC9、JUB、或THBS的各個siRNA轉染入NHEM中48小時，並萃取總RNA。凌亂siRNA用作陰性對照。藉由RT-qPCR測定NHEM中HDAC5(a)及HDAC9(b)的mRNA量。藉由GAPDH將各值標準化。數據代表三項獨立實驗(***P<0.001)。 Each siRNA against HDAC5, HDAC9, JUB, or THBS was transfected into NHEM for 48 hours and total RNA was extracted. Scrambled siRNA was used as a negative control. The mRNA levels of HDAC5 (a) and HDAC9 (b) in NHEM were determined by RT-qPCR. Values were normalized by GAPDH. Data are representative of three independent experiments (*** P < 0.001).

圖11. ITAC處理對NHEM或人類黑色素瘤細胞中蛋白質表現的影響Figure 11. Effects of ITAC treatment on protein expression in NHEM or human melanoma cells

(a)用50ng/ml ITAC處理NHEM、或(b)用對抗HDAC5的siRNA轉染48小時。藉由使用所示抗體之西方墨點法測定α-微管蛋白的乙醯化量。凌亂 siRNA用作HDAC5特異性siRNA的對照。(c)人類黑色素瘤細胞系SK-MEL-38用ITAC(50ng/ml)處理48h，並收集以用於使用抗p53及抗酪胺酸酶(TYR)抗體之西方墨點分析。重複執行西方墨點分析，並顯示代表性圖像。GAPDH用作加載控制。a-Ac-p53，抗乙醯化p53抗體。α-Ac-α-微管蛋白，抗乙醯化的α-微管蛋白抗體。 (a) NHEMs were treated with 50ng/ml ITAC, or (b) transfected with siRNA against HDAC5 for 48 hours. The amount of acetylated alpha-tubulin was determined by Western blotting using the indicated antibodies. messy siRNA was used as a control for HDAC5-specific siRNA. (c) The human melanoma cell line SK-MEL-38 was treated with ITAC (50 ng/ml) for 48 h and collected for western blot analysis using anti-p53 and anti-tyrosinase (TYR) antibodies. Western blot analysis was performed in duplicate and representative images are shown. GAPDH is used as loading control. a-Ac-p53, anti-acetylated p53 antibody. α-Ac-α-tubulin, anti-acetylated α-tubulin antibody.

圖12. MIG、IP-10、或ITAC對NHEM中黑色素生成的影響Figure 12. Effects of MIG, IP-10, or ITAC on melanin production in NHEM

用200ng/ml MIG、IP-10、或ITAC處理NHEM 48小時，並萃取總RNA。藉由RT-qPCR測定NHEM中TYR(a)、MITF(b)、TRP1(c)、及DCT(d)的mRNA量。藉由GAPDH將各值標準化。數據代表兩獨立實驗。(*P<0.05) NHEMs were treated with 200ng/ml MIG, IP-10, or ITAC for 48 hours and total RNA was extracted. The mRNA levels of TYR (a), MITF (b), TRP1 (c), and DCT (d) in NHEM were determined by RT-qPCR. Values were normalized by GAPDH. Data are representative of two independent experiments. (* P <0.05)

圖13. p53減弱對在ITAC誘導的色素過少中黑色素生成相關基因表現的影響Figure 13. Effects of p53 attenuation on the expression of melanogenesis-related genes in ITAC-induced hypopigmentation

(a-d)用凌亂siRNA或對抗p53的siRNA轉染NHEM 48小時及用200ng/mL ITAC轉染另外48小時。藉由RT-qPCR測定每個mRNA量。數據代表三項獨立實驗(** P<0.01,*** P<0.001)。 (ad) NHEM was transfected with scrambled siRNA or siRNA against p53 for 48 hours and 200 ng/mL ITAC for another 48 hours. The amount of each mRNA was determined by RT-qPCR. Data are representative of three independent experiments ( ** P < 0.01, *** P < 0.001).

表1.用於細胞遷移試驗的趨化激素列表Table 1. List of chemokines used in cell migration assays

Figure 106123288-A0305-02-0043-1
Figure 106123288-A0305-02-0043-1

Figure 106123288-A0305-02-0044-2
Figure 106123288-A0305-02-0044-2
Figure 106123288-A0305-02-0045-3
Figure 106123288-A0305-02-0045-3
Figure 106123288-A0305-02-0046-4
Figure 106123288-A0305-02-0046-4
Figure 106123288-A0305-02-0047-5
Figure 106123288-A0305-02-0047-5

<110> 愛茉莉太平洋股份有限公司(AMOREPACIFIC CORPORATION) <110> AMOREPACIFIC CORPORATION

<120> 以ITAC基因表現量之檢測試劑作為製備檢測白斑病組成物及其套組 之用途 <120> Use the detection reagent of ITAC gene expression as the preparation and detection composition of vitiligo and its kit the purpose of

<130> 16P414/IND <130> 16P414/IND

<160> 4 <160> 4

<170> KoPatentIn 3.0 <170> KoPatentIn 3.0

<210> 1 <210> 1

<211> 1610 <211> 1610

<212> RNA <212> RNA

<213> 智人(Homo sapiens) <213> Homo sapiens

<400> 1

Figure 106123288-A0305-02-0049-6
Figure 106123288-A0305-02-0050-7
<400> 1
Figure 106123288-A0305-02-0049-6
Figure 106123288-A0305-02-0050-7

<210> 2 <210> 2

<211> 1914 <211> 1914

<212> RNA <212> RNA

<213> 智人(Homo sapiens) <213> Homo sapiens

<400> 2

Figure 106123288-A0305-02-0050-8
Figure 106123288-A0305-02-0051-9
<400> 2
Figure 106123288-A0305-02-0050-8
Figure 106123288-A0305-02-0051-9

<210> 3 <210> 3

<211> 2311 <211> 2311

<212> RNA <212> RNA

<213> 智人(Homo sapiens) <213> Homo sapiens

<400> 3

Figure 106123288-A0305-02-0051-10
Figure 106123288-A0305-02-0052-11
Figure 106123288-A0305-02-0053-12
<400> 3
Figure 106123288-A0305-02-0051-10
Figure 106123288-A0305-02-0052-11
Figure 106123288-A0305-02-0053-12

<210> 4 <210> 4

<211> 5327 <211> 5327

<212> RNA <212> RNA

<213> 智人(Homo sapiens) <213> Homo sapiens

<400> 4

Figure 106123288-A0305-02-0053-13
Figure 106123288-A0305-02-0054-14
Figure 106123288-A0305-02-0055-15
Figure 106123288-A0305-02-0056-16
<400> 4
Figure 106123288-A0305-02-0053-13
Figure 106123288-A0305-02-0054-14
Figure 106123288-A0305-02-0055-15
Figure 106123288-A0305-02-0056-16

Claims (6)

一種以ITAC基因表現量之檢測試劑作為製備檢測白斑病組成物之用途。 The invention relates to the use of a detection reagent for the expression amount of ITAC gene as the preparation of a composition for detecting vitiligo. 如請求項1所述之用途，其中該檢測試劑包含一或多種與該基因的轉錄物特異性地結合之引子對或探子，以及與該基因所表現的蛋白質特異性地結合之抗體。 The use according to claim 1, wherein the detection reagent comprises one or more primer pairs or probes that specifically bind to the transcript of the gene, and an antibody that specifically binds to the protein expressed by the gene. 如請求項2所述之用途，其中該引子對包含與該基因的mRNA互補且能夠擴增該mRNA的引子對；以及該探子包含一或多個由該基因的mRNA序列組成之多核苷酸(polynucleotide)；而多核苷酸，其係包含10或更多個連續核苷酸之該多核苷酸的片段。 The use as claimed in claim 2, wherein the primer pair comprises a primer pair complementary to the mRNA of the gene and capable of amplifying the mRNA; and the probe comprises one or more polynucleotides consisting of the mRNA sequence of the gene ( polynucleotide); and a polynucleotide, which is a fragment of the polynucleotide comprising 10 or more contiguous nucleotides. 一種以ITAC基因表現量之檢測試劑作為製備一偵測白斑病套組之用途。 The invention relates to the use of a detection reagent for the expression of ITAC gene as a use for preparing a kit for detecting vitiligo. 如請求項4所述之用途，其中，該檢測試劑包含一或多種與該基因的轉錄物特異性地結合之引子對或探子，以及與該基因所表現的蛋白質特異性地結合之抗體。 The use according to claim 4, wherein the detection reagent comprises one or more primer pairs or probes that specifically bind to the transcript of the gene, and an antibody that specifically binds to the protein expressed by the gene. 如請求項5所述之用途，其中該引子對包含與mRNA互補且能夠擴增該mRNA的引子對；以及該探子包含一或多個由該基因的mRNA序列組成之多核苷酸(polynucleotide)；而多核苷酸，其係包含10或更多個連續核苷酸之該多核苷酸的片段。 The use of claim 5, wherein the primer pair comprises a primer pair complementary to mRNA and capable of amplifying the mRNA; and the probe comprises one or more polynucleotides consisting of the mRNA sequence of the gene; A polynucleotide is a fragment of the polynucleotide comprising 10 or more contiguous nucleotides.
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KR20130056955A (en) * 2011-11-23 2013-05-31 (주)아모레퍼시픽 Composition for promoting mature melanocyte migration comprising chemokine, detection kit for skin-active ingredients comprising chemokine gene and the method for detecting skin-active ingredients by using chemokine gene
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KR20130056955A (en) * 2011-11-23 2013-05-31 (주)아모레퍼시픽 Composition for promoting mature melanocyte migration comprising chemokine, detection kit for skin-active ingredients comprising chemokine gene and the method for detecting skin-active ingredients by using chemokine gene
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