JPWO2014178427A1 - Semaphorin 3A expression regulation method - Google Patents
Semaphorin 3A expression regulation method Download PDFInfo
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- JPWO2014178427A1 JPWO2014178427A1 JP2015514873A JP2015514873A JPWO2014178427A1 JP WO2014178427 A1 JPWO2014178427 A1 JP WO2014178427A1 JP 2015514873 A JP2015514873 A JP 2015514873A JP 2015514873 A JP2015514873 A JP 2015514873A JP WO2014178427 A1 JPWO2014178427 A1 JP WO2014178427A1
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Abstract
本願発明者らは、鋭意研究の結果、セマフォリン3A遺伝子の発現調節を担う転写因子を同定した。これらの転写因子を阻害又は促進することにより、セマフォリン3Aの発現を調節（抑制又は増強）することができる。また、これら転写因子の阻害活性又は促進活性を指標として化合物をスクリーニングすることにより、セマフォリン3Aの発現を調節する化合物を得ることができる。Sema3Aの発現を調節する化合物は、皮膚疾患におけるそう痒、アレルギー性鼻炎、骨粗鬆症、及び神経損傷の治療に、とりわけそう痒の治療に有用である。As a result of intensive studies, the inventors of the present application have identified a transcription factor responsible for regulating the expression of the semaphorin 3A gene. By inhibiting or promoting these transcription factors, the expression of semaphorin 3A can be regulated (suppressed or enhanced). In addition, a compound that regulates the expression of semaphorin 3A can be obtained by screening a compound using the inhibitory activity or promoting activity of these transcription factors as an index. Compounds that modulate the expression of Sema3A are useful in the treatment of pruritus, allergic rhinitis, osteoporosis, and nerve damage in skin diseases, especially in the treatment of pruritus.
Description
本発明は、転写因子を利用したセマフォリン3Aの発現を増強又は抑制する剤、及び特定の転写因子の阻害又は促進を指標とした、セマフォリン3Aの発現を増強又は抑制する化合物のスクリーニング方法に関する。 The present invention relates to an agent that enhances or suppresses the expression of semaphorin 3A using a transcription factor, and a method for screening a compound that enhances or suppresses the expression of semaphorin 3A using the inhibition or promotion of a specific transcription factor as an index. .
セマフォリン(Semaphorin: Sema) は神経軸索の伸長などの細胞移動を制御するガイダンス因子として同定され、免疫応答や器官形成、骨代謝制御、がんの進展などの様々な生体機能や病態に関与する分子群であることが知られている。Semaファミリータンパク質はドメイン構造の違いからSema1〜Sema7のクラスに分類され、さらにサブクラスに分類される（非特許文献１）。 Semaphorin (Semaphorin) has been identified as a guidance factor that controls cell migration such as nerve axon elongation and is involved in various biological functions and pathologies such as immune response, organogenesis, bone metabolism control, and cancer progression It is known that it is a molecular group. Sema family proteins are classified into Sema1 to Sema7 classes based on the difference in domain structure, and further subclassified (Non-patent Document 1).
その中のひとつであるセマフォリン３Ａ（Sema3A）は神経軸索の伸長方向を決定する神経ガイダンス因子の代表的な分子として知られ、Sema3Aが存在すると神経軸索の伸長が阻害される。このことは両面的な薬理作用につながると考えられる。すなわち、Sema3Aを阻害することによって神経再生を促進させることと、Sema3Aを増強して神経伸長を抑制することである。実際、前者については、神経再生促進剤としての技術が開示され（特許文献１）、後者については、特に皮膚組織で痒み知覚の軽減に利用する可能性が報告されている（非特許文献２）。 One of them, semaphorin 3A (Sema3A), is known as a typical molecule of a nerve guidance factor that determines the elongation direction of nerve axons, and the presence of Sema3A inhibits nerve axon elongation. This is thought to lead to a two-sided pharmacological action. That is, it is to promote nerve regeneration by inhibiting Sema3A and to enhance Sema3A to suppress nerve elongation. In fact, for the former, a technique as a nerve regeneration promoting agent is disclosed (Patent Document 1), and for the latter, there is a report that it may be used to reduce itchiness, particularly in skin tissue (Non-Patent Document 2). .
皮膚バリア機能の破綻を伴うアトピー性皮膚炎や乾皮症患者では、正常皮膚に内在するSema3A発現の低下に伴って表皮内神経線維が緻密化し、抗ヒスタミン薬が奏功しない難治性の痒みを発症しやすくすることが明らかにされ（非特許文献３）、組換えSema3Aタンパク質を含む軟膏をアトピー性皮膚炎モデルNC/Ngaマウスに外用することで痒みや炎症を有意に抑制するという試み（非特許文献４）やSema3Aタンパク質そのものをアトピー性皮膚炎等のそう痒性皮膚疾患治療薬とする技術が開発されている（特許文献２）。 In patients with atopic dermatitis and psoriasis with disruption of skin barrier function, intraepidermal nerve fibers become dense with the decrease in Sema3A expression in normal skin, and intractable itching that does not respond to antihistamines develops (Non-patent document 3), an attempt to significantly suppress itching and inflammation by externally applying an ointment containing recombinant Sema3A protein to NC / Nga mice with atopic dermatitis (Non-patent document 3) Document 4) and a technique for using Sema3A protein itself as a therapeutic agent for pruritic skin diseases such as atopic dermatitis have been developed (Patent Document 2).
このような、Sema3Aを補充して皮膚内の神経軸索の誘導を抑え、結果として痒みを抑えるというアプローチは、痒み治療の有望な手法となる可能性があるものの、実用化には、Sema3Aタンパク質の調製と安定的な製剤開発が鍵となる。Sema3Aは、セマフォリンの分泌型タンパク質メンバーで、500アミノ酸残基のセマフォリン（Sema）ドメイン、Ｃ−２型免疫グロブリン（Ig）ドメイン、及び塩基性末端ドメインの３つの構造ドメインを有し、Sema3Aが生理活性を示すにはホモ二量体化が必須であることが示されている（非特許文献４）。Sema3Aを医薬品として製剤化する場合、500アミノ酸残基からなるSemaドメインのホモ二量体で分子量10万を超える高分子量タンパク質が有効成分となるが、高分子量二量体タンパク質を安定的に生産し、治療薬として妥当なコストと安定的品質を確保し得る製剤を開発することは決して容易ではない。 Although this approach of supplementing Sema3A to suppress the induction of nerve axons in the skin and consequently suppress itching may be a promising technique for itching treatment, for practical application, Sema3A protein Preparation and stable formulation development are key. Sema3A is a secreted protein member of semaphorin, which has three structural domains, a semaphorin (Sema) domain of 500 amino acid residues, a C-2 type immunoglobulin (Ig) domain, and a basic terminal domain. It has been shown that homodimerization is indispensable in order to exhibit physiological activity (Non-patent Document 4). When Sema3A is formulated as a pharmaceutical product, a high molecular weight protein with a molecular weight of over 100,000, which is a homodimer of a Sema domain consisting of 500 amino acid residues, is an active ingredient, but it can stably produce a high molecular weight dimeric protein. It is never easy to develop a preparation that can ensure reasonable cost and stable quality as a therapeutic agent.
こうした製剤上の課題を解決する技術のひとつとして、生体内のSema3Aを発現増強する物質で治療効果を誘導するアプローチが考えられる。この手法の１例としては、ヒノキ科植物であるアスナロ（Thujopsisdolabrata）の抽出液にその作用があることが見出され、その技術が開示されている（特許文献３）。 As one of the techniques for solving such problems in preparation, an approach of inducing a therapeutic effect with a substance that enhances the expression of Sema3A in vivo can be considered. As an example of this technique, it has been found that an extract of Asunalo (Thujopsisdolabrata), which is a cypress family, has its action, and its technique is disclosed (Patent Document 3).
Sema3A発現を内在性に抑制できれば神経再生促進作用を、他方、増強できれば神経軸索伸長阻害作用を、それぞれ誘導することができる。しかしながら、Sema3Aの発現を制御する物質を簡便に探索あるいは検査する方法は十分に開発されていない。特に、皮膚そう痒性疾患では表皮内のSema3Aの低下が知られており、それが痒み発症につながっていることが示されていることから、内在性のSema3Aを増強する物質を探索するための簡便なSema3A発現調節物質検索方法を提供できれば、難治性のそう痒症治療薬の開発に非常に有用である。 If Sema3A expression can be suppressed endogenously, nerve regeneration promoting action can be induced, and if it can be enhanced, nerve axon elongation inhibiting action can be induced. However, a method for simply searching for or examining a substance that controls the expression of Sema3A has not been sufficiently developed. In particular, it has been known that skin pruritus is associated with a decrease in Sema3A in the epidermis, which has been shown to lead to the development of itch, so it is necessary to search for substances that enhance endogenous Sema3A. If a simple Sema3A expression regulator search method can be provided, it will be very useful for the development of refractory pruritus therapeutics.
本発明は、Sema3Aの発現を調節する物質のスクリーニングや、発現調節作用の評価に有用な新規な手段を提供すること、およびそう痒の治療や神経損傷の治療等に有用な新規な手段を提供することを目的とする。 The present invention provides a novel means useful for screening a substance that regulates the expression of Sema3A, evaluation of the expression regulating action, and a novel means useful for treating pruritus and treating nerve damage. The purpose is to do.
本願発明者らは、ヒトSema3A遺伝子プロモーターの単離を行い、その特性を決定してプロモーター領域の転写因子群を同定した。これら転写因子群について、鋭意検討を行ない、Sema3Aの発現増強に関わる転写因子群および発現抑制に関わる転写因子群を同定することに成功し、さらに、これら転写因子の阻害等によりSema3A遺伝子の発現を調節できることを確認し、本願発明を完成した。 The inventors of the present application isolated a human Sema3A gene promoter, determined its characteristics, and identified a transcription factor group in the promoter region. These transcription factors have been intensively studied and succeeded in identifying a transcription factor group that is related to Sema3A expression enhancement and a transcription factor group that is related to suppression of expression. Furthermore, the expression of the Sema3A gene is inhibited by inhibiting these transcription factors. After confirming that it can be adjusted, the present invention was completed.
すなわち、本発明は、NF-κB、c-Rel、CREB、Nkx2.5、Brn-2、Elk-1、HNF-4α1、XBP-1、HLF、NF-E2、RORα、ER、AP-1、Sox-4、Sox-5、Sox-9、Pbx1b、Jun、Fos、GABP、STATx、AREB6、GABPα、JunB、JunD、Fra1、及びFra2から選択されるいずれかの転写因子を調節する少なくとも１種の物質を有効成分として含有する、セマフォリン3Aの発現調節剤を提供する。また、本発明は、NF-κB、c-Rel、CREB、Nkx2.5、Brn-2、Elk-1、HNF-4α1、XBP-1、HLF、NF-E2、RORα、ER、AP-1、Sox-4、Sox-5、Sox-9、Pbx1b、Jun、Fos、GABP、STATx、AREB6、GABPα、JunB、JunD、Fra1、及びFra2から選択される少なくとも１種の転写因子の調節を指標として化合物をスクリーニングすることを含む、セマフォリン3Aの発現を調節する化合物のスクリーニング方法を提供する。さらに、本発明は、セマフォリン3Aプロモーター領域及びレポーター遺伝子を含む核酸構築物を含む細胞と、転写因子を調節する物質を接触させ、レポーター遺伝子の発現量を測定することを含む、前記物質によるセマフォリン3Aの発現調節作用の評価方法を提供する。さらに、本発明は、培養細胞と、NF-κB、c-Rel、CREB、Nkx2.5、Brn-2、Elk-1、HNF-4α1、XBP-1、HLF、NF-E2、RORα、ER、AP-1、Sox-4、Sox-5、Sox-9、Pbx1b、Jun、Fos、GABP、STATx、AREB6、GABPα、JunB、JunD、Fra1、及びFra2から選択される転写因子を調節する物質を接触させ、前記細胞におけるセマフォリン3Aの発現量を測定することを含む、前記物質によるセマフォリン3Aの発現調節作用の評価方法を提供する。 That is, the present invention is NF-κB, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, RORα, ER, AP-1, At least one transcription factor that regulates any transcription factor selected from Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, JunB, JunD, Fra1, and Fra2. A semaphorin 3A expression regulator comprising a substance as an active ingredient is provided. The present invention also includes NF-κB, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, RORα, ER, AP-1, Compounds with the regulation of at least one transcription factor selected from Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, JunB, JunD, Fra1, and Fra2 A method for screening a compound that modulates the expression of semaphorin 3A is provided. Furthermore, the present invention provides a cell containing a nucleic acid construct containing a semaphorin 3A promoter region and a reporter gene, and a substance that regulates a transcription factor, and measuring the expression level of the reporter gene, and then measuring the expression level of the reporter gene. A method for evaluating the expression-regulating action of 3A is provided. Furthermore, the present invention relates to cultured cells, NF-κB, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, RORα, ER, Contact with a substance that regulates a transcription factor selected from AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, JunB, JunD, Fra1, and Fra2. And a method for evaluating an action of regulating the expression of semaphorin 3A by the substance, comprising measuring the expression level of semaphorin 3A in the cell.
本発明により、Sema3Aの遺伝子発現を調節する転写因子を利用した、Sema3Aの発現調節剤が提供される。Sema3Aの発現を増強する剤は、アトピー性皮膚炎等のそう痒性皮膚疾患におけるそう痒の治療及び予防等に有用である。Sema3Aの発現を抑制する剤は、神経損傷部位における神経再生の促進等に有用である。また、本発明により、Sema3Aの発現を調節する化合物をスクリーニングする方法、及びSema3Aの発現増強作用又は発現抑制作用の強さを評価するための方法も提供される。本発明のスクリーニング方法及び評価方法によれば、Sema3Aの発現調節により優れた治療効果を奏する新規な医薬を提供することが可能になる。 According to the present invention, there is provided a Sema3A expression regulator using a transcription factor that regulates Sema3A gene expression. An agent that enhances the expression of Sema3A is useful for the treatment and prevention of pruritus in pruritic skin diseases such as atopic dermatitis. An agent that suppresses the expression of Sema3A is useful for promoting nerve regeneration at the site of nerve injury. The present invention also provides a method for screening a compound that regulates Sema3A expression and a method for evaluating the strength of Sema3A expression enhancing action or expression inhibiting action. According to the screening method and the evaluation method of the present invention, it is possible to provide a novel pharmaceutical that exhibits an excellent therapeutic effect by regulating the expression of Sema3A.
「転写因子」とは、ＤＮＡに特異的に結合するタンパク質の一群で、ＤＮＡ上のプロモーターやエンハンサーといった、遺伝子の転写領域の5'上流に存在する転写を制御する領域に結合し、ＤＮＡの遺伝情報をメッセンジャーＲＮＡに転写する過程を促進、あるいは逆に抑制する役割をもつタンパク質である。転写因子は、１つのタンパク質分子が単独で、または複数のタンパク質分子と複合体を形成することによって転写調節（促進又は抑制）機能を発揮する。本発明における「転写因子」との語には、単独のタンパク質分子からなるもののほか、同一又は異なる複数のタンパク質が複合した多量体ないしは複合体の形態のものが包含され、さらにはそのような多量体ないしは複合体を構成する個々のタンパク質分子も包含される。ヒトのゲノム上には、転写因子をコードする遺伝子がおよそ1,800前後存在するとの推定がなされているが、標的タンパク質に対する転写因子はまちまちである。 A “transcription factor” is a group of proteins that specifically bind to DNA, and binds to a region that regulates transcription 5 ′ upstream of the transcription region of the gene, such as a promoter or enhancer on the DNA, thereby inheriting the DNA. It is a protein that plays a role in promoting or repressing the process of transcription of information into messenger RNA. A transcription factor exerts a transcriptional regulation (promotion or repression) function when one protein molecule is used alone or forms a complex with a plurality of protein molecules. The term “transcription factor” in the present invention includes not only a single protein molecule but also a multimer or complex form in which a plurality of identical or different proteins are complexed. Also included are individual protein molecules that make up the body or complex. It is estimated that there are about 1,800 genes encoding transcription factors on the human genome, but there are various transcription factors for target proteins.
本発明において、「転写因子の調節」とは、転写因子の阻害又は促進をいう。 In the present invention, “regulation of a transcription factor” refers to inhibition or promotion of a transcription factor.
「転写因子の阻害」とは、転写因子をコードする遺伝子からのmRNAの転写に始まり、生産された転写因子がSema3A遺伝子のプロモーター領域に結合してSema3Aの発現を調節するに至る過程のいずれかのステップを阻害することをいう。転写因子に対するアンチセンスRNA、マイクロRNA、siRNA等の利用は、転写因子の阻害の一例である。 “Transcription factor inhibition” refers to any process that begins with transcription of mRNA from a gene that encodes a transcription factor and leads to regulation of Sema3A expression by binding to the promoter region of the Sema3A gene. Inhibiting this step. Use of antisense RNA, microRNA, siRNA and the like for transcription factors is an example of inhibition of transcription factors.
「転写因子の促進」とは、転写因子をコードする遺伝子からのmRNAの転写に始まり、生産された転写因子がSema3A遺伝子のプロモーター領域に結合してSema3Aの発現を調節するに至る過程のいずれかのステップを促進することをいう。患部に転写因子タンパク質そのものを適用すること、あるいは患部において転写因子遺伝子の発現を増強することは、転写因子の促進の一例である。 “Transcription factor promotion” refers to any process that begins with transcription of mRNA from a gene that encodes a transcription factor and leads to regulation of Sema3A expression when the produced transcription factor binds to the promoter region of the Sema3A gene. It means to promote the step. Applying the transcription factor protein itself to the affected area, or enhancing the expression of the transcription factor gene in the affected area is an example of promoting the transcription factor.
図２に示した推定転写因子結合部位は、本願発明者らが新規にクローニングしたSema3A遺伝子上流のプロモーター領域の塩基配列（配列番号１）を元に、Match-1.0 Publicプログラム及びP-Match-1.0 Publicプログラム（BIOBASE）による検索を行なって同定されたものである。本プログラムは転写因子や実験的に証明されている転写因子結合配列に関する情報を網羅的に収載したデータベースで、新規の転写因子結合配列を予測できるソフトである。データベース解析を行うことで、Sema3A遺伝子上流に存在する未知の転写因子結合配列を推定し、Sema3Aの発現に関わる転写因子を予測できた。 The putative transcription factor binding site shown in FIG. 2 is based on the base sequence (SEQ ID NO: 1) of the promoter region upstream of the Sema3A gene newly cloned by the present inventors, and the Match-1.0 Public program and the P-Match-1.0 It was identified by performing a search using the Public program (BIOBASE). This program is a database that comprehensively contains information on transcription factors and experimentally proven transcription factor binding sequences, and is software that can predict new transcription factor binding sequences. By analyzing the database, it was possible to estimate an unknown transcription factor binding sequence upstream of the Sema3A gene and to predict transcription factors involved in Sema3A expression.
転写因子結合配列の予測結果、プロモーター領域の5'末端欠失変異体のレポーターアッセイ、及びsiRNAによる転写因子ノックダウン実験の結果に基づき、Sema3A遺伝子の発現調節を担う転写因子として以下の転写因子が同定された。これらの転写因子の調節によってSema3Aの発現を調節することができる。
NF-κB、Nkx2.5、Brn-2、Elk-1、HNF-4α1、XBP-1、HLF、NF-E2（Nrf2ともいう）、Fra1（Fosl1ともいう）、RORα、ER（Esr1ともいう）、AP-1、Sox-4、Sox-5、Sox-9、Pbx1b、Jun、Fos、GABP、STATx、AREB6、GABPα、c-Rel、CREB（Atf2ともいう）、JunB、JunD、Fra2Based on the prediction results of transcription factor binding sequences, reporter assays of 5 'end deletion mutants in the promoter region, and the results of transcription factor knockdown experiments with siRNA, the following transcription factors are responsible for regulating Sema3A gene expression. Identified. Regulation of these transcription factors can regulate Sema3A expression.
NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2 (also referred to as Nrf2), Fra1 (also referred to as Fosl1), RORα, ER (also referred to as Esr1) , AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, c-Rel, CREB (also called Atf2), JunB, JunD, Fra2
上記の転写因子は、以下の群に分類することができる。
(A) Sema3Aの発現を抑制する転写因子群
NF-κB、Nkx2.5、Brn-2、Elk-1、HNF-4α1、XBP-1、HLF、NF-E2、Fra1
(B) Sema3Aの発現を促進する転写因子群
RORα、ER、AP-1、Sox-4、Sox-5、Sox-9、Pbx1b、Jun、Fos、GABP、STATx、AREB6、GABPα、c-Rel、CREB、JunB、JunD、Fra2The above transcription factors can be classified into the following groups.
(A) Transcription factors that suppress Sema3A expression
NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, Fra1
(B) Transcription factors that promote Sema3A expression
RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, c-Rel, CREB, JunB, JunD, Fra2
(A)の転写因子群は、好ましくはNF-κBであり得る。なお、NF-κBの調節は、そのサブユニットであるNF-κB1の調節により行なってよい。 The transcription factor group (A) may preferably be NF-κB. The regulation of NF-κB may be performed by the regulation of its subunit NF-κB1.
(B)の転写因子群は、好ましくはRORα、AP-1、Sox-4、Jun、Fos、STATx（特にSTAT3）、GABPα、Sox-9、CREB、JunB、JunD、及びFra2からなる転写因子群、より好ましくはRORα、AP-1、Sox-4、Jun、Fos、CREB、及びJunBからなる転写因子群、さらに好ましくはRORα、AP-1、Jun、Fos、及びCREBからなる転写因子群、あるいはRORα、AP-1、Jun、及びFosからなる転写因子群であり得る。なかでも、RORαは、Sema3Aの発現調節のための標的として特に好ましい転写因子である。なお、Jun及びFosはAP-1を構成するタンパク質であり、これらも本発明における「転写因子」に包含される。 The transcription factor group (B) is preferably a transcription factor group consisting of RORα, AP-1, Sox-4, Jun, Fos, STATx (especially STAT3), GABPα, Sox-9, CREB, JunB, JunD, and Fra2. More preferably a transcription factor group consisting of RORα, AP-1, Sox-4, Jun, Fos, CREB, and JunB, more preferably a transcription factor group consisting of RORα, AP-1, Jun, Fos, and CREB, or It may be a transcription factor group consisting of RORα, AP-1, Jun, and Fos. Among these, RORα is a particularly preferred transcription factor as a target for regulating expression of Sema3A. Jun and Fos are proteins constituting AP-1, and these are also included in the “transcription factor” in the present invention.
(A)の転写因子を阻害することで、Sema3A遺伝子の発現が増強する。従って、(A)の転写因子のいずれかを阻害する物質は、Sema3Aの発現増強剤として用いることができる。そのような剤は、Sema3Aの発現増強が望まれる疾患又は状態の治療又は予防剤として有用である。そのような疾患又は状態の具体例としては、アトピー性皮膚炎、乾皮症等の皮膚疾患におけるそう痒、アレルギー性鼻炎、骨粗鬆症を挙げることができる。アトピー性皮膚炎等のそう痒性皮膚疾患においては、正常皮膚に内在するSema3Aの発現が低下していることが知られており、Sema3Aタンパク質自体を患部に補うことでそう痒を抑制できることが知られている（非特許文献３、４及び特許文献２等）。アレルギー性鼻炎においても、鼻粘膜上皮でSema3Aの発現が低下し、上皮層内の末梢神経線維及び鼻甲介の粘膜固有層が増加していること、Sema3Aタンパク質を鼻腔内投与することで鼻炎の症状を軽減できることが知られている（Sawaki et al., J PharmacolSci 117, 34-44 (2011)）。また、Sema3Aが骨量を増加させる因子であり、骨粗鬆症の治療にも有用であることが知られている（林ら、実験医学、Vol. 31, No. 4, 2013年）。従って、Sema3A遺伝子の発現を抑制する転写因子の阻害によりSema3Aの発現を増強することで、皮膚疾患におけるそう痒、アレルギー性鼻炎、骨粗鬆症等を治療及び予防することができる。すなわち、(A)の転写因子のいずれかを阻害する物質は、皮膚疾患におけるそう痒、アレルギー性鼻炎、骨粗鬆症等の治療又は予防剤として、とりわけ皮膚疾患におけるそう痒又はアレルギー性鼻炎の治療又は予防剤として、特には皮膚疾患におけるそう痒の治療又は予防剤として好ましく用いることができる。もっとも、Sema3Aの発現増強が望まれる疾患又は状態は上記の具体例に限定されない。 Inhibiting the transcription factor (A) enhances the expression of the Sema3A gene. Therefore, a substance that inhibits any of the transcription factors (A) can be used as a Sema3A expression enhancer. Such an agent is useful as a therapeutic or prophylactic agent for a disease or condition for which enhanced expression of Sema3A is desired. Specific examples of such diseases or conditions include pruritus, allergic rhinitis and osteoporosis in skin diseases such as atopic dermatitis and xeroderma. In pruritic skin diseases such as atopic dermatitis, it is known that the expression of Sema3A inherent in normal skin is decreased, and it is known that pruritus can be suppressed by supplementing the affected area with Sema3A protein itself (Non-patent Documents 3 and 4 and Patent Document 2 etc.). In allergic rhinitis, the expression of Sema3A decreases in the nasal mucosal epithelium, peripheral nerve fibers in the epithelial layer and the lamina propria of the nasal turbinate increase, and symptoms of rhinitis caused by intranasal administration of Sema3A protein (Sawaki et al., J PharmacolSci 117, 34-44 (2011)). Sema3A is a factor that increases bone mass and is known to be useful for the treatment of osteoporosis (Hayashi et al., Experimental Medicine, Vol. 31, No. 4, 2013). Therefore, it is possible to treat and prevent pruritus, allergic rhinitis, osteoporosis and the like in skin diseases by enhancing Sema3A expression by inhibiting transcription factors that suppress Sema3A gene expression. That is, the substance that inhibits any of the transcription factors in (A) is used as a therapeutic or preventive agent for pruritus, allergic rhinitis, osteoporosis, etc. in skin diseases, and in particular, treatment or prevention of pruritus or allergic rhinitis in skin diseases. It can be preferably used as an agent, particularly as an agent for treating or preventing pruritus in skin diseases. However, the disease or condition for which enhanced expression of Sema3A is desired is not limited to the above specific examples.
Sema3Aの発現増強はまた、(B)の転写因子を促進することによっても可能である。従って、(B)の転写因子のいずれかを促進する物質は、(A)の転写因子の阻害剤と同様に、Sema3Aの発現増強剤として用いることができ、Sema3Aの発現増強が望まれる疾患又は状態の治療又は予防剤として有用である。Sema3Aの発現増強が望まれる疾患又は状態の好ましい具体例は上記の通りである。とりわけ、RORαを促進する物質は、Sema3Aの発現増強のために使用可能な物質の特に好ましい例である。 Sema3A expression can also be enhanced by promoting the transcription factor (B). Therefore, a substance that promotes any of the transcription factors of (B) can be used as an Sema3A expression enhancer, as well as an inhibitor of the transcription factor of (A). It is useful as an agent for treating or preventing a condition. Preferred examples of diseases or conditions for which enhanced expression of Sema3A is desired are as described above. In particular, substances that promote RORα are particularly preferred examples of substances that can be used to enhance the expression of Sema3A.
Sema3Aの発現増強剤は、(A)の転写因子を阻害する物質を１種又は２種以上含んでいてもよいし、(B)の転写因子を促進する物質を１種又は２種以上含んでいてもよく、あるいは、(A)の転写因子を阻害する物質の１種以上及び(B)の転写因子を促進する物質の１種以上の両者を含んでいてもよい。 The expression enhancer of Sema3A may contain one or more substances that inhibit the transcription factor (A), or one or more substances that promote the transcription factor (B). Alternatively, it may contain one or more substances that inhibit the transcription factor (A) and one or more substances that promote the transcription factor (B).
一方、(B)の転写因子を阻害することで、Sema3A遺伝子の発現が抑制される。従って、(B)の転写因子のいずれかを阻害する物質は、Sema3Aの発現抑制剤として用いることができる。そのような剤は、Sema3Aの発現抑制が望まれる疾患又は状態の治療又は予防剤として有用である。そのような疾患又は状態の具体例としては、外傷性神経傷害、嗅覚異常症、脳梗塞性神経障害、顔面神経麻痺、糖尿病性神経症、緑内障、網膜色素変性症、アルツハイマー病、パーキンソン病、筋発育不全性側索硬化症、筋萎縮性側索硬化症、ハンチントン病、脳梗塞、外傷性神経変性疾患等における、脊髄神経又は末梢神経の損傷等を挙げることができる。Sema3Aを阻害することによって神経再生を促進できることが知られており、これを利用した神経再生促進剤が知られている（特許文献１）。従って、Sema3A遺伝子の発現を促進する転写因子の阻害によりSema3Aの発現を抑制することで、神経損傷部における神経再生を促進することができる。すなわち、(B)の転写因子のいずれかを阻害する物質は、神経再生促進剤として好ましく用いることができる。もっとも、Sema3Aの発現抑制が望まれる疾患又は状態は上記の具体例に限定されない。 On the other hand, inhibition of the transcription factor (B) suppresses Sema3A gene expression. Therefore, a substance that inhibits any of the transcription factors (B) can be used as a Sema3A expression inhibitor. Such an agent is useful as a therapeutic or prophylactic agent for a disease or condition for which suppression of Sema3A expression is desired. Specific examples of such diseases or conditions include traumatic nerve injury, olfactory dysfunction, cerebral infarction neuropathy, facial paralysis, diabetic neuropathy, glaucoma, retinitis pigmentosa, Alzheimer's disease, Parkinson's disease, muscle Examples include spinal nerve injury or peripheral nerve damage in developmental lateral sclerosis, amyotrophic lateral sclerosis, Huntington's disease, cerebral infarction, traumatic neurodegenerative disease, and the like. It is known that nerve regeneration can be promoted by inhibiting Sema3A, and a nerve regeneration promoter using this is known (Patent Document 1). Therefore, by suppressing the expression of Sema3A by inhibiting a transcription factor that promotes the expression of the Sema3A gene, nerve regeneration in the nerve damage site can be promoted. That is, a substance that inhibits any of the transcription factors (B) can be preferably used as a nerve regeneration promoter. However, the disease or condition for which Sema3A expression suppression is desired is not limited to the above specific examples.
Sema3Aの発現抑制はまた、(A)の転写因子を促進することによっても可能である。従って、(A)の転写因子のいずれかを促進する物質は、(B)の転写因子の阻害剤と同様に、Sema3Aの発現抑制剤として用いることができ、Sema3Aの発現抑制が望まれる疾患又は状態の治療又は予防剤、例えば神経再生促進剤として有用である。 Sema3A expression can also be suppressed by promoting the transcription factor (A). Therefore, a substance that promotes any of the transcription factors of (A) can be used as an inhibitor of Sema3A expression, as well as an inhibitor of the transcription factor of (B), It is useful as an agent for treating or preventing a condition, such as a nerve regeneration promoter.
Sema3Aの発現抑制剤は、(A)の転写因子を促進する物質を１種又は２種以上含んでいてもよいし、(B)の転写因子を阻害する物質を１種又は２種以上含んでいてもよく、あるいは、(A)の転写因子を促進する物質の１種以上及び(B)の転写因子を阻害する物質の１種以上の両者を含んでいてもよい。 The expression inhibitor of Sema3A may contain one or more substances that promote the transcription factor (A), or one or more substances that inhibit the transcription factor (B). Alternatively, it may contain one or more substances that promote the transcription factor (A) and one or more substances that inhibit the transcription factor (B).
本発明が提供する医薬（皮膚疾患におけるそう痒、アレルギー性鼻炎、又は骨粗鬆症の治療又は予防剤等の、Sema3Aの発現増強が望まれる疾患又は状態の治療又は予防剤、および神経再生促進剤等の、Sema3Aの発現抑制が望まれる疾患又は状態の治療又は予防剤）は、主として局所投与で用いられる。例えば、神経再生が望まれる患部への注射等による投与、痒みを抑制すべき患部への塗布による投与、鼻炎症状の抑制のための鼻腔内投与等が挙げられる。骨粗鬆症の治療又は予防剤の投与方法としては、例えば、骨組織内で特異的に発現するプロモーターを利用した核酸医薬（例えば、(A)の転写因子に対するsiRNA又は(B)の転写因子自体を骨組織内特異的プロモーター制御下で発現するベクター等）の形態での全身投与を挙げることができ、これにより骨組織局所的にSema3Aの発現を増強させることができる。具体的な投与経路に応じて、注射剤、軟膏等の適当な剤形に製剤することができる。投与量は、患部の大きさ、症状の強さ等に応じて適宜選択され、特に限定されないが、有効成分量として、１回の投与当たり0.01μg〜1g程度、例えば0.01mg〜10mg程度であり得る。 Drugs provided by the present invention (such as pruritus, allergic rhinitis or osteoporosis treatment or prevention agent for skin diseases, treatment or prevention agent for diseases or conditions in which expression enhancement of Sema3A is desired, and nerve regeneration promoting agent, etc. Therapeutic or preventive agent for diseases or conditions for which suppression of Sema3A expression is desired) is mainly used by local administration. For example, administration by injection or the like to the affected area where nerve regeneration is desired, administration by application to the affected area where itching should be suppressed, intranasal administration for suppressing nasal inflammation, and the like can be mentioned. As a method of administering an agent for treating or preventing osteoporosis, for example, a nucleic acid drug using a promoter that is specifically expressed in bone tissue (for example, siRNA for (A) transcription factor or (B) transcription factor itself is used as bone. Systemic administration in the form of a vector or the like that is expressed under the control of a promoter specific in the tissue), and thereby the expression of Sema3A can be locally enhanced in the bone tissue. Depending on the specific route of administration, it can be formulated into an appropriate dosage form such as an injection or ointment. The dose is appropriately selected according to the size of the affected area, the strength of the symptom, etc., and is not particularly limited. However, the amount of the active ingredient is about 0.01 μg to 1 g, for example, about 0.01 mg to 10 mg per administration. obtain.
Sema3Aの発現増強剤として用いる、(A)の転写因子のいずれかを阻害する物質としては、例えば上記で定義した通り、各転写因子の発現を阻害するアンチセンスRNA、マイクロRNA、siRNA等を用いることができる。この場合、対象となる転写因子に対するそのようなRNA分子を生産可能な発現ベクターを公知の手法により作製し、該ベクターを有効成分としてSema3A発現増強剤を製剤することができる。各種の転写因子のアミノ酸配列及びこれをコードする遺伝子配列は公知であり、またsiRNAやmiRNAを生体細胞内で生産させるためのベクターも公知で市販品も存在するので、当業者であればアンチセンスRNA、マイクロRNA、siRNA等を利用したSema3A発現増強剤を調製することができる。あるいは、各転写因子の阻害剤として既に知られている物質であってもよい。(A)の転写因子の阻害剤としては、IMD-0354(Tanaka et al,Blood, 105:2324-31, 2005)、DHMEQ(Ariga et al, J BiolChem, 277:24625-30, 2002)(いずれもNF-κBを阻害)等が知られている。あるいはまた、(A)の転写因子のいずれかを阻害することを指標として、化合物群をスクリーニングし、Sema3Aの発現増強剤として提供することもできる。 As a substance that inhibits any of the transcription factors of (A) used as an expression enhancer of Sema3A, for example, as defined above, antisense RNA, microRNA, siRNA, etc. that inhibit the expression of each transcription factor are used. be able to. In this case, an expression vector capable of producing such an RNA molecule for the transcription factor of interest can be prepared by a known technique, and a Sema3A expression enhancer can be formulated using the vector as an active ingredient. The amino acid sequences of various transcription factors and the gene sequences encoding them are known, and vectors for producing siRNA and miRNA in living cells are known and commercially available. A Sema3A expression enhancer using RNA, microRNA, siRNA or the like can be prepared. Alternatively, it may be a substance already known as an inhibitor of each transcription factor. (A) Transcription factor inhibitors include IMD-0354 (Tanaka et al, Blood, 105: 2324-31, 2005), DHMEQ (Ariga et al, J BiolChem, 277: 24625-30, 2002) Is also known to inhibit NF-κB). Alternatively, the compound group can be screened using the inhibition of any of the transcription factors of (A) as an index and provided as an expression enhancer of Sema3A.
(A)の転写因子のいずれかを阻害することを指標とした化合物のスクリーニングは、例えば次のようにして実施することができる。 The screening of compounds using as an index the inhibition of any of the transcription factors of (A) can be performed, for example, as follows.
工程１：転写因子と化合物を接触させ、結合の有無を調べる。アッセイロボットを用いてマイクロプレート内で結合の有無を調べることで、ハイスループットのスクリーニングが可能である。 Step 1: A transcription factor and a compound are contacted, and the presence or absence of binding is examined. High throughput screening is possible by examining the presence or absence of binding in a microplate using an assay robot.
工程２：結合が確認された化合物について、Sema3A遺伝子の発現量を上昇させる作用があるか否かを調べる。この工程は、例えば、Sema3Aプロモーター領域及びレポーター遺伝子を含む核酸構築物でトランスフェクトした細胞を用いたレポーターアッセイにより実施できる。該核酸構築物を含む細胞と、工程１で結合が確認された化合物を、マイクロプレートのウェル内等で接触させ、レポーター遺伝子の発現量を調べればよい。レポーターアッセイ自体は周知の常法であり、市販のキット等を用いて容易に行なうことができる。Sema3Aプロモーター領域の配列は、配列番号１に示したものを好ましく用いることができる。(A)の転写因子が結合する領域は配列番号１中の1位〜471位である。このようなスクリーニングにより、Sema3Aの発現増強が望まれる疾患又は状態（好ましい具体例は上記した通り）の治療又は予防剤として有用な、Sema3Aの発現を増強する化合物を新たに同定することができる。 Step 2: It is examined whether or not a compound that has been confirmed to have a binding action has an effect of increasing the expression level of the Sema3A gene. This step can be performed, for example, by a reporter assay using cells transfected with a nucleic acid construct containing a Sema3A promoter region and a reporter gene. The expression level of the reporter gene may be examined by bringing the cell containing the nucleic acid construct into contact with the compound confirmed to be bound in step 1 in a well of a microplate. The reporter assay itself is a well-known conventional method and can be easily performed using a commercially available kit or the like. As the sequence of the Sema3A promoter region, the sequence shown in SEQ ID NO: 1 can be preferably used. The region to which the transcription factor of (A) binds is from position 1 to position 471 in SEQ ID NO: 1. Such screening makes it possible to newly identify a compound that enhances the expression of Sema3A, which is useful as a therapeutic or prophylactic agent for a disease or condition in which Sema3A expression enhancement is desired (preferred specific examples are as described above).
Sema3Aの発現増強剤として用いる、(B)の転写因子のいずれかを促進する物質は、例えば上記で定義した通り、各転写因子タンパク質そのものであってもよい。具体的には、例えば医薬として用いる場合には、Sema3Aの発現を増強したい患部に転写因子タンパク質そのものを、又は転写因子タンパク質を発現可能なベクターを適用することができる。あるいは、各転写因子の促進剤として既に知られている物質であってもよい。(B)の転写因子の促進剤としては、SR1078(Wang et al, ACS Chem Biol. 5:1029-34, 2010), コレステロール硫酸（Bitsch et al, Anal Biochem. 323:139-49, 2003）（いずれもRORαを促進）等が知られている。RORα活性化作用を有するRORαアゴニストは、(B)の転写因子の促進剤として特に有望である。あるいはまた、(B)の転写因子のいずれかを促進することを指標として、化合物群をスクリーニングし、Sema3Aの発現増強剤として提供することもできる。上記した通り、Sema3A発現増強剤は、そう痒、アレルギー性鼻炎、骨粗鬆症等の治療又は予防、とりわけそう痒の治療又は予防剤として有用である。特に注目されるRORαアゴニストの医薬用途として、例えばWO2011/115892にはSR1078等を中枢神経疾患、代謝病、がん等の治療に用いることが記載されているが（ただしWO2011/115892にはRORの逆作動薬を中枢神経疾患、代謝病、がん等の治療に用いることも記載されており、RORの阻害と促進のどちらがこれらの疾患の治療に有用であるかは具体的に開示されていない）、皮膚疾患であるそう痒の治療用途に関する報告はなく、本発明で初めて提案する新しい用途である。 The substance that promotes any of the transcription factors (B) used as an expression enhancer of Sema3A may be each transcription factor protein itself as defined above, for example. Specifically, for example, when used as a medicine, the transcription factor protein itself or a vector capable of expressing the transcription factor protein can be applied to the affected area where Sema3A expression is to be enhanced. Alternatively, it may be a substance already known as a promoter for each transcription factor. As a promoter of the transcription factor (B), SR1078 (Wang et al, ACS Chem Biol. 5: 1029-34, 2010), cholesterol sulfate (Bitsch et al, Anal Biochem. 323: 139-49, 2003) ( All of them promote RORα). RORα agonists having RORα activation action are particularly promising as promoters of the transcription factor (B). Alternatively, the compound group can be screened and provided as an Sema3A expression enhancer using as an index the promotion of any of the transcription factors of (B). As described above, the Sema3A expression enhancer is useful as a therapeutic or prophylactic agent for pruritus, allergic rhinitis, osteoporosis, etc., particularly as a therapeutic or prophylactic agent for pruritus. For example, WO2011 / 115892 describes the use of SR1078 for the treatment of central nervous diseases, metabolic diseases, cancers, etc. as a medicinal use of RORα agonists of particular interest (however, WO2011 / 115892 describes the use of ROR It also describes the use of inverse agonists for the treatment of central nervous disease, metabolic diseases, cancer, etc., and it is not specifically disclosed which of ROR inhibition or promotion is useful for the treatment of these diseases ), There is no report on the therapeutic use of pruritus, which is a skin disease, and this is a new use proposed for the first time in the present invention.
(B)の転写因子のいずれかを促進することを指標とした化合物のスクリーニングは、例えば、Sema3Aプロモーター領域及びレポーター遺伝子を含む核酸構築物でトランスフェクトした細胞を用いたレポーターアッセイにより、レポーター遺伝子の発現量を増大させる化合物をスクリーニングすることで実施することができる。マイクロプレートのウェル内等で、Sema3Aプロモーター領域及びレポーター遺伝子を含む核酸構築物を含む細胞と化合物を接触させ、レポーター遺伝子の発現量を調べればよい。この場合、Sema3Aプロモーター領域としては、配列番号１に示した塩基配列を用いることもできるし、あるいは、(B)の転写因子が結合する領域が存在する、Sema3A遺伝子の-134位〜-24位の領域（配列番号１中の1311位〜1421位、この領域の配列を取り出して配列番号２に示す）のみを用いることもできる。このようなスクリーニングにより、Sema3Aの発現増強が望まれる疾患又は状態（好ましい具体例は上記した通り）の治療又は予防剤として有用な、Sema3Aの発現を増強する化合物を新たに同定することができる。 (B) Screening of a compound using any of the transcription factors as an index is performed by, for example, reporter gene expression by reporter assay using a cell transfected with a nucleic acid construct containing a Sema3A promoter region and a reporter gene. This can be done by screening for compounds that increase the amount. The expression level of the reporter gene may be examined by bringing the compound into contact with a cell containing a nucleic acid construct containing the Sema3A promoter region and the reporter gene in a well of the microplate. In this case, as the Sema3A promoter region, the nucleotide sequence shown in SEQ ID NO: 1 can be used, or the region to which the transcription factor (B) binds exists, -134 to -24 positions of the Sema3A gene It is also possible to use only the region (positions 1311 to 1421 in SEQ ID NO: 1, the sequence of this region is taken out and shown in SEQ ID NO: 2). Such screening makes it possible to newly identify a compound that enhances the expression of Sema3A, which is useful as a therapeutic or prophylactic agent for a disease or condition in which Sema3A expression enhancement is desired (preferred specific examples are as described above).
Sema3Aの発現抑制剤として用いる、(A)の転写因子のいずれかを促進する物質については、上記した(B)の転写因子のいずれかを促進する物質と同様のことがいえる。各転写因子タンパク質そのものであってもよいし、各転写因子の促進剤として既に知られている物質であってもよく、あるいは、(A)の転写因子のいずれかを促進することを指標として化合物群をスクリーニングし、Sema3Aの発現抑制剤として提供することもできる。(A)の転写因子の促進剤としては、イソフムロン類（WO 2006/043671）やスルフォラファン（Thimmulappa et al, Cancer Res,62:5196-203,2002）（いずれもNF-E2を活性化）等が知られている。化合物のスクリーニングは、例えば、Sema3Aプロモーター領域及びレポーター遺伝子を含む核酸構築物でトランスフェクトした細胞を用いたレポーターアッセイにより、レポーター遺伝子の発現量を増大させる化合物をスクリーニングすることで実施することができる。(A)の転写因子が結合する領域は配列番号１中の1位〜471位であり、配列番号１に示した塩基配列をSema3Aプロモーター領域として用いることができる。このようなスクリーニングにより、Sema3Aの発現抑制が望まれる疾患又は状態の治療又は予防剤、好ましくは神経再生促進剤として有用な、Sema3Aの発現を抑制する化合物を新たに同定することができる。 About the substance which accelerates | stimulates any of the transcription factor of (A) used as a Sema3A expression inhibitor, it can say the same thing as the substance which promotes any of the transcription factor of the above-mentioned (B). Each transcription factor protein itself may be a substance that is already known as a promoter of each transcription factor, or a compound that uses any of the transcription factors of (A) as an index. The group can also be screened and provided as an Sema3A expression inhibitor. (A) Transcription factor promoters include isohumulones (WO 2006/043671) and sulforaphane (Thimmulappa et al, Cancer Res, 62: 5196-203, 2002) (all of which activate NF-E2). Are known. Compound screening can be carried out, for example, by screening a compound that increases the expression level of a reporter gene by a reporter assay using a cell transfected with a nucleic acid construct containing a Sema3A promoter region and a reporter gene. The region to which the transcription factor of (A) binds is from position 1 to position 471 in SEQ ID NO: 1, and the base sequence shown in SEQ ID NO: 1 can be used as the Sema3A promoter region. By such screening, it is possible to newly identify a compound that suppresses the expression of Sema3A, which is useful as a therapeutic or preventive agent for a disease or condition for which suppression of Sema3A expression is desired, preferably as a nerve regeneration promoter.
Sema3Aの発現抑制剤として用いる、(B)の転写因子のいずれかを阻害する物質については、上記した(A)の転写因子のいずれかを阻害する物質と同様のことがいえる。各転写因子に対するアンチセンスRNA、マイクロRNA、siRNA等を用いることができるし、各転写因子の阻害剤として既に知られている物質であってもよく、あるいは、(B)の転写因子のいずれかを阻害することを指標として化合物をスクリーニングして得ることもできる。(B)の転写因子の阻害剤としては、T-5224（Uchihashi et al, Drug Metab Dispos.39:803-13, 2011.AP-1を阻害）、SR1001（Crumbley et al, PLoS One. 7:e33804, 2012. RORαの逆作動薬）、SR3335（Solt and Burris, Trends in Endocrinology and Metabolism. 23:619-627, 2012. 及びKumar et al., ACS Chem Biol. 18:218-22, 2011. RORαの逆作動薬）等が知られている。化合物のスクリーニングは、(A)の転写因子を阻害する物質のスクリーニングと同様に、上記工程１及び工程２に準じた工程を行えばよい。核酸構築物に含まれるSema3Aプロモーター領域としては、配列番号１に示した塩基配列を用いることもできるし、あるいは、(B)の転写因子が結合する領域が存在する、配列番号１中の1311位〜1421位の領域（この領域の配列を取り出して配列番号２に示す）のみを用いることもできる。このようなスクリーニングにより、Sema3Aの発現抑制が望まれる疾患又は状態の治療又は予防剤、好ましくは神経再生促進剤として有用な、Sema3Aの発現を抑制する化合物を新たに同定することができる。 About the substance which inhibits any of the transcription factors of (B) used as a Sema3A expression inhibitor, the same thing as the substance which inhibits any of the transcription factors of (A) described above can be said. Antisense RNA, microRNA, siRNA, etc. for each transcription factor can be used, and it can be a substance already known as an inhibitor of each transcription factor, or any of the transcription factors of (B) It can also be obtained by screening a compound using as an index the inhibition. As an inhibitor of the transcription factor of (B), T-5224 (inhibits Uchihashi et al, Drug Metab Dispos. 39: 803-13, 2011.AP-1), SR1001 (Crumbley et al, PLoS One. 7: e33804, 2012. RORα inverse agonist), SR3335 (Solt and Burris, Trends in Endocrinology and Metabolism. 23: 619-627, 2012. and Kumar et al., ACS Chem Biol. 18: 218-22, 2011. RORα Inverse agonists) are known. The compound screening may be performed in accordance with the above-described steps 1 and 2, similarly to the screening of the substance that inhibits the transcription factor (A). As the Sema3A promoter region contained in the nucleic acid construct, the base sequence shown in SEQ ID NO: 1 can be used, or the region to which the transcription factor (B) binds exists, from position 1311 to SEQ ID NO: 1. Only the region at position 1421 (the sequence of this region is extracted and shown in SEQ ID NO: 2) can be used. By such screening, it is possible to newly identify a compound that suppresses the expression of Sema3A, which is useful as a therapeutic or preventive agent for a disease or condition for which suppression of Sema3A expression is desired, preferably as a nerve regeneration promoter.
なお、配列番号１に示した塩基配列は、本願発明者らが同定したSema3A遺伝子のプロモーター領域の配列であるが、配列番号１中の少数の塩基、特にSema3A遺伝子調節のシスエレメントである-1444位〜-974位（配列番号１中の1位〜471位）及び-134位〜-24位（配列番号１中の1311位〜1421位）以外の部位の塩基であれば、配列番号１に示した塩基配列と多少の相違があっても、スクリーニング方法においてSema3Aプロモーター領域として使用可能である。従って、Sema3Aの発現を増強又は抑制する化合物のスクリーニングにおいては、配列番号１に示す塩基配列又は配列番号２に示す塩基配列と同一の塩基配列のほか、配列番号１又は配列番号２と高い同一性（９０％以上、好ましくは９５％以上、より好ましくは９８％以上の同一性）を有する塩基配列も、Sema3Aプロモーター領域として使用することができる。 The base sequence shown in SEQ ID NO: 1 is the sequence of the promoter region of the Sema3A gene identified by the inventors of the present application, but is a small number of bases in SEQ ID NO: 1, particularly a cis element of Sema3A gene regulation. If it is a base other than position -974 (positions 1 to 471 in SEQ ID NO: 1) and positions -134 to -24 (positions 1311 to 1421 in SEQ ID NO: 1), Even if there is some difference from the base sequence shown, it can be used as the Sema3A promoter region in the screening method. Therefore, in screening for compounds that enhance or suppress the expression of Sema3A, in addition to the nucleotide sequence shown in SEQ ID NO: 1 or the nucleotide sequence shown in SEQ ID NO: 2, it is highly identical to SEQ ID NO: 1 or SEQ ID NO: 2. A base sequence having (identity of 90% or more, preferably 95% or more, more preferably 98% or more) can also be used as the Sema3A promoter region.
ここで、塩基配列の「同一性」とは、比較すべき２つの塩基配列の塩基ができるだけ多く一致するように両配列を整列させ、一致した塩基数を、全塩基数で除したものを百分率で表したものである。上記整列の際には、必要に応じ、比較する２つの配列の一方又は双方に適宜ギャップを挿入する。このような配列の整列化は、例えばBLAST、FASTA、CLUSTAL W等の周知のプログラムを用いて行なうことができる。ギャップが挿入される場合、上記全塩基数は、１つのギャップを１つの塩基として数えた塩基数となる。このようにして数えた全塩基数が、比較する２つの配列間で異なる場合には、同一性（％）は、長い方の配列の全塩基数で、一致した塩基数を除して算出される。ただし、比較すべき配列が他の任意の配列と連結された状態にある場合には（例えば、発現ベクターに組み込まれた状態、発現させたい所望の遺伝子と連結させた状態など）、プロモーターに相当する領域のみを取り出して配列を対比し、同一性を算出する。 Here, the “identity” of the base sequences is a percentage obtained by aligning both sequences so that the bases of the two base sequences to be compared match as much as possible, and dividing the number of matched bases by the total number of bases. It is represented by. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like. When gaps are inserted, the total number of bases is the number of bases obtained by counting one gap as one base. When the total number of bases counted in this way differs between the two sequences to be compared, the identity (%) is calculated by dividing the total number of bases of the longer sequence and dividing the number of matched bases. The However, if the sequence to be compared is in a state linked to any other sequence (for example, a state incorporated in an expression vector, a state linked to a desired gene to be expressed, etc.), it corresponds to a promoter. Only the region to be extracted is taken out, the sequences are compared, and the identity is calculated.
また、上記した通り、各種の転写因子について、阻害剤及び促進剤が公知である。このような公知の阻害剤及び促進剤の中から、Sema3Aの発現を調節（抑制又は増強）する活性が特に強い化合物を見出す目的で、上記のスクリーニング方法の説明において記載した、Sema3Aプロモーター領域−レポーター遺伝子構築物を含む細胞を用いたレポーターアッセイを実施することができる。すなわち、本発明は、セマフォリン3Aプロモーター領域及びレポーター遺伝子を含む核酸構築物を含む細胞と、NF-κB、c-Rel、CREB、Nkx2.5、Brn-2、Elk-1、HNF-4α1、XBP-1、HLF、NF-E2、RORα、ER、AP-1、Sox-4、Sox-5、Sox-9、Pbx1b、Jun、Fos、GABP、STATx、AREB6、GABPα、JunB、JunD、Fra1、及びFra2から選択されるいずれかの転写因子を調節する物質を接触させ、レポーター遺伝子の発現量を測定することを含む、前記物質によるセマフォリン3Aの発現調節作用の評価方法も提供する。 As described above, inhibitors and promoters are known for various transcription factors. Among these known inhibitors and promoters, the Sema3A promoter region-reporter described in the above description of the screening method for the purpose of finding a compound having particularly strong activity for regulating (suppressing or enhancing) the expression of Sema3A. Reporter assays using cells containing the gene construct can be performed. That is, the present invention includes a cell comprising a nucleic acid construct comprising a semaphorin 3A promoter region and a reporter gene, NF-κB, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP -1, HLF, NF-E2, RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, JunB, JunD, Fra1, and There is also provided a method for evaluating the action of regulating the expression of semaphorin 3A by the substance, which comprises contacting a substance that regulates any transcription factor selected from Fra2 and measuring the expression level of the reporter gene.
レポーターアッセイやSema3Aプロモーター領域については、スクリーニング方法に関する上記の説明と同様であり、Sema3Aプロモーター領域としては、下記(1)〜(3)のいずれかの塩基配列を含むものを用いることができる。
(1) 配列番号１に示す塩基配列
(2) 配列番号２に示す塩基配列
(3) (1)又は(2)と９０％以上、好ましくは９５％以上、より好ましくは９８％以上の同一性を有する塩基配列The reporter assay and the Sema3A promoter region are the same as described above for the screening method. As the Sema3A promoter region, one containing any one of the following base sequences (1) to (3) can be used.
(1) Base sequence shown in SEQ ID NO: 1
(2) Base sequence shown in SEQ ID NO: 2
(3) Base sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity with (1) or (2)
上記(A)の転写因子のいずれかの阻害剤、及び上記(B)の転写因子のいずれかの促進剤について、上記の評価方法を適用した場合には、該阻害剤ないしは該促進剤によるSema3Aの発現増強作用が評価される。一方、上記(A)の転写因子のいずれかの促進剤、及び上記(B)の転写因子のいずれかの阻害剤について、上記の評価方法を適用した場合には、該促進剤ないしは該阻害剤によるSema3Aの発現抑制作用が評価される。配列番号２又はこれと９０％以上の同一性を有する塩基配列を利用したプロモーター領域は、(B)の転写因子の阻害剤又は促進剤を評価する場合に使用可能である。 When the above evaluation method is applied to the inhibitor of any of the transcription factors of (A) and the promoter of any of the transcription factors of (B), Sema3A by the inhibitor or the promoter is used. The expression enhancing action of is evaluated. On the other hand, when the above evaluation method is applied to any promoter of the transcription factor (A) and any inhibitor of the transcription factor (B), the promoter or the inhibitor Sema3A expression-suppressing action is evaluated. A promoter region using SEQ ID NO: 2 or a nucleotide sequence having 90% or more identity to this can be used when evaluating an inhibitor or promoter of the transcription factor (B).
あるいは、レポーターアッセイによらず、培養細胞中でのSema3Aの発現量を指標として、上記の転写因子を調節（阻害又は促進）する物質によるSema3Aの発現調節作用を評価することも可能である。この場合は、培養細胞と、上記(A)の転写因子（NF-κB、Nkx2.5、Brn-2、Elk-1、HNF-4α1、XBP-1、HLF、NF-E2、Fra1）及び上記(B)の転写因子（RORα、ER、AP-1、Sox-4、Sox-5、Sox-9、Pbx1b、Jun、Fos、GABP、STATx、AREB6、GABPα、c-Rel、CREB、JunB、JunD、Fra2）から選択される転写因子を調節（阻害又は促進）する物質を接触させ、細胞内でのSema3Aの発現量を測定する。 Alternatively, it is also possible to evaluate the Sema3A expression-regulating action by a substance that regulates (inhibits or promotes) the above transcription factor using the expression level of Sema3A in cultured cells as an index, without using a reporter assay. In this case, the cultured cells, the transcription factor (A) (NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, Fra1) and the above (B) transcription factors (RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, c-Rel, CREB, JunB, JunD , Fra2) is contacted with a substance that regulates (inhibits or promotes) a transcription factor selected from, and the expression level of Sema3A in the cell is measured.
上記(A)の転写因子のいずれかの阻害剤及び上記(B)の転写因子のいずれかの促進剤について当該評価方法を適用した場合には、該阻害剤ないしは該促進剤によるSema3Aの発現増強作用が評価される。一方、上記(A)の転写因子のいずれかの促進剤及び上記(B)の転写因子のいずれかの阻害剤について当該評価方法を適用した場合には、該促進剤ないしは該阻害剤によるSema3Aの発現抑制作用が評価される。 When the evaluation method is applied to any one of the inhibitors of the transcription factor of (A) and the promoter of any of the transcription factors of (B), the expression of Sema3A by the inhibitor or the promoter is enhanced. The effect is evaluated. On the other hand, when the evaluation method is applied to any promoter of the transcription factor (A) and any inhibitor of the transcription factor (B), the promoter or the Sema3A by the inhibitor Expression inhibitory action is evaluated.
(A)の転写因子の阻害剤の評価と(B)の転写因子の促進剤の評価を組み合わせて実施し、Sema3Aの発現を増強する作用が非常に高い薬剤の組み合わせを同定することができる。そのような薬剤の組み合わせは、そう痒の治療又は予防剤等の、Sema3Aの発現の増強が望まれる疾患又は状態の治療又は予防剤として特に好ましく用いることができる。 The combination of the evaluation of the inhibitor of the transcription factor (A) and the evaluation of the promoter of the transcription factor (B) can be carried out to identify a combination of drugs having a very high effect of enhancing the expression of Sema3A. Such a combination of agents can be particularly preferably used as a therapeutic or prophylactic agent for diseases or conditions in which enhanced expression of Sema3A is desired, such as a therapeutic or prophylactic agent for pruritus.
また、(A)の転写因子の促進剤の評価と(B)の転写因子の阻害剤の評価を組み合わせて実施し、Sema3Aの発現を抑制する作用が非常に高い薬剤の組み合わせを同定することができる。そのような薬剤の組み合わせは、神経再生促進剤等の、Sema3Aの発現の抑制が望まれる疾患又は状態の治療又は予防剤として特に好ましく用いることができる。 In addition, the evaluation of the transcription factor promoter in (A) and the evaluation of the transcription factor inhibitor in (B) can be conducted in combination to identify combinations of drugs that have a very high effect of suppressing Sema3A expression. it can. Such a combination of drugs can be particularly preferably used as a therapeutic or prophylactic agent for a disease or condition for which suppression of Sema3A expression is desired, such as a nerve regeneration promoter.
以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
１．ヒトSema3A遺伝子プロモーターの単離
ヒトSema3A遺伝子のコード領域の既知のヌクレオチド配列に基づいて、5'-フランキング領域を、遺伝子特異的プライマー1（GSP1: 5’-TCTGATAGTTTGCTCTTGCTGTAAG-3’、配列番号３）と遺伝子特異的プライマー2（GSP2: 5’-AAGACAGACAATCCTAGTTAACCAG-3’、配列番号４)を使用して、製造業者の取扱説明書に従い、Genome Walker Kit（Clontech, Mountain View, CA）を用いて増幅した。簡単に言えば、Ex Taqpolymelaseを用いる、製造業者によって推奨されるツーステップPCRプロトコール：94℃にて25秒間、そして72℃にて4分間を7サイクル、それに続いて94℃にて25秒間、そして67℃にて4分間を32サイクル、そして最後の伸長反応は67℃にて4分間、を使用して、GSP1とアダプタープライマー（AP）1を用いた一次PCRを実施した。次いで、一次PCR混合物を希釈し、そしてGSP2とAP2を用いた二次PCR増幅の鋳型として使用した。7サイクルの代わりに5サイクルの初回サイクルと、それに続く32サイクルの代わりに22サイクルの使用を除き、二次PCRは一次PCRと同一であった。Sema3Aの5'-フランキング領域の連続的な5'-欠失体突然変異株を、表１に列挙したプライマーを使用したPCRによって産出した。増幅後に、全てのPCR産物をpGEM-T-Easyベクター（Promega Co., Madison, WI, USA）内にクローン化し、そして、ABI Prism 310 Genetic Analyzer（Applied Biosystems, Foster City, CA）を用いて配列決定した。1. Isolation of the human Sema3A gene promoter Based on the known nucleotide sequence of the coding region of the human Sema3A gene, the 5'-flanking region was replaced with gene-specific primer 1 (GSP1: 5'-TCTGATAGTTTGCTCTTGCTGTAAG-3 ', SEQ ID NO: 3). And gene-specific primer 2 (GSP2: 5'-AAGACAGACAATCCTAGTTAACCAG-3 ', SEQ ID NO: 4) according to the manufacturer's instructions and amplified using the Genome Walker Kit (Clontech, Mountain View, CA) . Briefly, the manufacturer recommended two-step PCR protocol using Ex Taqpolymelase: 94 ° C for 25 seconds, 72 ° C for 4 minutes for 7 cycles, followed by 94 ° C for 25 seconds, and Primary PCR using GSP1 and adapter primer (AP) 1 was performed using 32 cycles of 4 minutes at 67 ° C. and 4 minutes at 67 ° C. for the final extension reaction. The primary PCR mixture was then diluted and used as a template for secondary PCR amplification with GSP2 and AP2. The secondary PCR was identical to the primary PCR except that 5 cycles were used instead of 7 cycles followed by 22 cycles instead of 32 cycles. Sequential 5′-deletion mutants of the 5′-flanking region of Sema3A were generated by PCR using the primers listed in Table 1. After amplification, all PCR products were cloned into pGEM-T-Easy vector (Promega Co., Madison, Wis., USA) and sequenced using ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, Calif.) Were determined.
レポータープラスミドpGL3-1444/-1を構築するために、PCRを以下の条件下、最初の変性94℃にて5分間、94℃にて1分間、60℃にて1分間、72℃にて1分間を30サイクル、そして最後に伸長72℃にて5分間、鋳型であるpGEM-T-Sema3A-1444/-1ならびにKpnIまたはHindIIIのための制限酵素部位を含む1組の特異的なSema3Aプライマー（Sema3A-1444 KpnIおよびSema3A+1 HindIII）を使用して実施した。得られたPCR産物はKpnI及びHindIIIで消化し、そしてpGL3-Basicベクター（Promega）内にクローニングした。なお、pGL3-Basicベクターは、ホタルルシフェラーゼ遺伝子を含んでいる。構築物の全てを、QIAGEN Plasmid Maxi Ki（QIAGEN, Duesseldolf, Germany）を使用することで調製した。 To construct the reporter plasmid pGL3-1444 / -1, PCR was first denatured at 94 ° C for 5 minutes, 94 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C for 1 minute under the following conditions: A set of specific Sema3A primers containing a restriction enzyme site for the template pGEM-T-Sema3A-1444 / -1 and KpnI or HindIII for 30 minutes in minutes and finally 5 minutes at extension 72 ° C. Sema3A-1444 KpnI and Sema3A + 1 HindIII) were used. The resulting PCR product was digested with KpnI and HindIII and cloned into the pGL3-Basic vector (Promega). The pGL3-Basic vector contains a firefly luciferase gene. All of the constructs were prepared using QIAGEN Plasmid Maxi Ki (QIAGEN, Duesseldolf, Germany).
さらに、Quikchange site-directed mutagenesis kitを用いて、Sema3Aの推定転写因子結合配列部位に部位特異的変異導入を行ない、RORα結合領域、Sox-5/9結合領域及びGABP結合領域が欠失したレポータープラスミド構築物を作製した。部位特異的変異導入のために使用したプライマーを表２に示す。 In addition, using the Quikchange site-directed mutagenesis kit, a site-directed mutagenesis site was introduced into the putative transcription factor binding sequence site of Sema3A, and the RORα binding region, Sox-5 / 9 binding region and GABP binding region were deleted. A construct was made. Table 2 shows the primers used for site-directed mutagenesis.
２．ヒトSema3A遺伝子プロモーターの特性決定
（１）NHEKを用いたSema3Aプロモーターのルシフェラーゼアッセイ
正常ヒト表皮ケラチノサイト（NHEK）はLonza社のプロトコールに従い、培養した。NHEKを12ウェル組織培養プレート内で培養し、X-treme GENE HD DNAトランスフェクション試薬（Roche Diagnostics, Basel, Switzerland）を使用して、それぞれの構築物1μgを用いて、トランスフェクションした。トランスフェクション効率を補正するために、すべての細胞にHSV-TKプロモーターの制御下にあるウミシイタケ（Renilla）ルシフェラーゼ遺伝子を含むpRK-TKベクター（Promega）を同時にトランスフェクションした。トランスフェクション後、増殖期（80%コンフルエント）もしくは、分化後（コンフルエント後、1.4mMカルシウムを添加し、さらに4日間培養を継続したもの）のNHEKに、Passive Lysis Buffer 250μlを加え、細胞を溶解させた。測定にはDual-luciferase reporter assay system（Promega Co., Madison, WI, USA）を用いた。所定の96ウェルプレートにLuciferase Assay ReagentII100μlを加え、そこにサンプル20μlを加えて、Wallac 1420 ARVOsxMultilabel counter （Perkin Elmer）を用いて測定した。その後、さらにStop&Glo reagent 100μlを加えて、同様に測定を行った。2. Characterization of human Sema3A gene promoter (1) Sema3A promoter luciferase assay using NHEK Normal human epidermal keratinocytes (NHEK) were cultured according to the protocol of Lonza. NHEKs were cultured in 12-well tissue culture plates and transfected with 1 μg of each construct using X-treme GENE HD DNA transfection reagent (Roche Diagnostics, Basel, Switzerland). To correct for transfection efficiency, all cells were co-transfected with the pRK-TK vector (Promega) containing the Renilla luciferase gene under the control of the HSV-TK promoter. After transfection, add 250 μl of Passive Lysis Buffer to NHEK in the growth phase (80% confluence) or after differentiation (adding 1.4 mM calcium after confluence and continuing the culture for 4 days) to lyse the cells. It was. A dual-luciferase reporter assay system (Promega Co., Madison, WI, USA) was used for the measurement. 100 μl of Luciferase Assay Reagent II was added to a predetermined 96-well plate, 20 μl of sample was added thereto, and measurement was performed using a Wallac 1420 ARVOsxMultilabel counter (Perkin Elmer). Thereafter, 100 μl of Stop & Glo reagent was further added and measurement was performed in the same manner.
（２）siRNAによる転写因子の発現抑制
培養NHEKを製造業者の取り扱い説明書に従って、転写因子RORα, Jun, Fos, Sox4, STAT3, NF-κB1, GABPα, Sox9, Rel (c-Rel), CREB, Pbx1, ER, JunB, JunD, Fra1, Fra2に対するsiRNA（Thermo Scientific）40nMをそれぞれLipofectamineRNAi Max（Invitrogen, Carisbad, CA）を使用してトランスフェクションした。その細胞を、抗生物質不含培地中で48時間培養し、その後、全RNAを抽出し、リアルタイムPCRで解析した。使用した各siRNAの配列（それぞれ、4種類のsiRNAの混合物を使用）を下記表３に示す。(2) Inhibition of transcription factor expression by siRNA Transcription factor RORα, Jun, Fos, Sox4, STAT3, NF-κB1, GABPα, Sox9, Rel (c-Rel), CREB, 40 nM of siRNA (Thermo Scientific) against Pbx1, ER, JunB, JunD, Fra1, and Fra2 were each transfected using LipofectamineRNAi Max (Invitrogen, Carisbad, CA). The cells were cultured in antibiotic-free medium for 48 hours, after which total RNA was extracted and analyzed by real-time PCR. The sequence of each siRNA used (each using a mixture of four siRNAs) is shown in Table 3 below.
転写因子の転写レベルを、定量リアルタイムPCRによって分析した。全RNAを、製造業者の取扱説明書に従って、RNeasy Mini Kit (QIAGEN)を用いて培養細胞から抽出し、PrimeScript RT reagent kit（Takara, Shiga, Japan）を用いて、cDNAに逆転写した。定量リアルタイムPCRは製造業者の取扱説明書に従って、SYBR Premix Ex Taq（Takara）を使用して、Applied Biosystems 7900HT Fast Real-time PCR system （Applied Biosystems）により実施した。使用したプライマーに関する情報を表４に示す。リボソームタンパク質S18（RPS18）を基準遺伝子として使用した。mRNAの量を、RPS18のmRNA量に対して標準化し、そして最終的に未処理対照のmRNAに対する比として示した。 Transcription levels of transcription factors were analyzed by quantitative real-time PCR. Total RNA was extracted from cultured cells using RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions and reverse transcribed into cDNA using PrimeScript RT reagent kit (Takara, Shiga, Japan). Quantitative real-time PCR was performed with an Applied Biosystems 7900HT Fast Real-time PCR system (Applied Biosystems) using SYBR Premix Ex Taq (Takara) according to the manufacturer's instructions. Information on the primers used is shown in Table 4. Ribosomal protein S18 (RPS18) was used as a reference gene. The amount of mRNA was normalized to the amount of RPS18 mRNA and was finally expressed as a ratio to the untreated control mRNA.
（３）電気泳動移動度シフト分析（EMSA）
一本鎖ビオチン化オリゴヌクレオチドと、これに相補的な一本鎖非標識オリゴヌクレオチドをアニーリングすることにより、二本鎖オリゴヌクレオチドプローブを調製した。プローブに用いた配列（センス鎖の配列）を表５に示す。核タンパク質の抽出とEMSAを、NE-PER Nuclear Cytoplasmic Extraction Reagents及びLightShiftChemiluminescent EMSA kit（Thermo Scientific）を使用することで実施した。核タンパク質抽出物（5μg）を、1×結合バッファー及び50ng/μlのポリ（dI・dC）、50mM KCl, 5 mM EDTA、2.5%グリセロール、0.05% NP-40、並びにSema3Aプロモーター領域の-122/-93、-77/-46、-47/-18の領域に対応するビオチン化プローブ（50 pmol/μl）と共に、室温で20分間インキュベートした。競合アッセイのために、400倍過剰の未標識プローブを、ビオチン化プローブの添加前に結合反応に加えた。これらのインキュベーション混合物を、次に0.5×TBEバッファーと共に6%ポリアクリルアミドゲル中で電気泳動し、そしてBiodyne Bナイロン膜（Pall, Port Washington, NY）に転写した。EMSAキットの中の化学発光検出試薬を使用することで、バンドを可視化した。(3) Electrophoretic mobility shift analysis (EMSA)
A double-stranded oligonucleotide probe was prepared by annealing a single-stranded biotinylated oligonucleotide and a complementary single-stranded unlabeled oligonucleotide. Table 5 shows the sequences (sense strand sequences) used for the probes. Nuclear protein extraction and EMSA were performed using NE-PER Nuclear Cytoplasmic Extraction Reagents and LightShift Chemiluminescent EMSA kit (Thermo Scientific). Nuclear protein extract (5 μg) was added to 1 × binding buffer and 50 ng / μl poly (dI · dC), 50 mM KCl, 5 mM EDTA, 2.5% glycerol, 0.05% NP-40, and −122 / of the Sema3A promoter region Incubated for 20 minutes at room temperature with biotinylated probes (50 pmol / μl) corresponding to the -93, -77 / -46, -47 / -18 regions. For competition assays, a 400-fold excess of unlabeled probe was added to the binding reaction prior to the addition of biotinylated probe. These incubation mixtures were then electrophoresed in a 6% polyacrylamide gel with 0.5 × TBE buffer and transferred to a Biodyne B nylon membrane (Pall, Port Washington, NY). Bands were visualized using the chemiluminescence detection reagent in the EMSA kit.
（４）転写因子の結合部位の推定
P-Match1.0 Publicプログラムによる検索で、ヒトSema3Aの5'-フランキング領域内の多数の推定転写因子結合部位が明らかになった（図１、図２）。特に転写開始部位の位置に近い-134/-1の領域の中に、RORα/ER/AP-1, Sox-4/5/9, GABPによって認識されるコンセンサス配列によく合致した配列が存在したので、これらの転写因子がSema3Aプロモーター活性の調節に関わっていることが示唆された。より厳密にSema3Aのプロモーター領域を決定するために、欠失体による分析を実施した（図３）。(4) Estimation of transcription factor binding sites
A search with the P-Match1.0 Public program revealed a number of putative transcription factor binding sites within the 5'-flanking region of human Sema3A (Figures 1 and 2). In particular, in the -134 / -1 region close to the position of the transcription start site, there was a sequence that closely matched the consensus sequence recognized by RORα / ER / AP-1, Sox-4 / 5/9, GABP. Therefore, it was suggested that these transcription factors are involved in the regulation of Sema3A promoter activity. In order to more precisely determine the promoter region of Sema3A, analysis with deletions was performed (FIG. 3).
最高レベルのルシフェラーゼ活性はpGL3-134（Sema3Aプロモーター領域内の-134/-1領域を含む）でトランスフェクションした分化型NHEKにおいて検出した。しかしながら、欠失プラスミドの相対ルシフェラーゼ活性は、欠失がpGL3-24に進むまで残存していた。構築体の中で、pGL3-1444/-1, -1244/-1, -1044/-1断片を含むプラスミドは、NHEKにおいて有意に低い活性を示し、-1444/-844領域における上流サプレッサー活性の存在が示唆された。これらの結果は、-134/-24領域がSema3A遺伝子転写のための最小プロモーターを含んでいることを実証したので、そのヌクレオチド配列を図４−１に示す。この領域にはいくつかの転写因子結合部位、たとえばRORα/ER/AP-1, Sox-4/5/9, GABPなどがこのコアプロモーター領域に存在していた。また、上流サプレッサー活性の存在が示唆された転写抑制領域（-1444/-975）の配列及び転写因子結合部位を図４−２に示す。 The highest level of luciferase activity was detected in differentiated NHEK transfected with pGL3-134 (including the -134 / -1 region within the Sema3A promoter region). However, the relative luciferase activity of the deletion plasmid remained until the deletion proceeded to pGL3-24. Among the constructs, plasmids containing the pGL3-1444 / -1, -1244 / -1, -1044 / -1 fragment showed significantly lower activity in NHEK and showed upstream suppressor activity in the -1444 / -844 region. Existence was suggested. These results demonstrated that the -134 / -24 region contains a minimal promoter for Sema3A gene transcription, so its nucleotide sequence is shown in Figure 4-1. Several transcription factor binding sites such as RORα / ER / AP-1, Sox-4 / 5/9, GABP, etc. were present in this core promoter region. In addition, the sequence of the transcription repressing region (-1444 / -975) and the transcription factor binding site, which are suggested to have upstream suppressor activity, are shown in FIG.
（５）Sema3A遺伝子調節に関与するcisエレメントの同定
次に、これらの転写因子が推定結合部位のそれぞれに実際に結合できるかどうかを解析した。このために、電気泳動移動度シフトアッセイ（EMSA）を培養NHEKの核タンパク質抽出物、及びSema3Aプロモーター領域の-122/-93、-77/-46、-47/-18の領域に対応するビオチン化二本鎖オリゴヌクレオチドプローブを用いて実施した。図５に示されているように、RORα/ER/AP-1結合部位を含む-122/-93、Sox 5/9結合部位を含む-77/-46、GABP結合部位を含む-47/-18の各プローブの標的配列には転写因子が結合し、シフトしたバンドが検出された。これらの結果は-122/-18 bp領域のこれらの結合部位が、Sema3A転写のためのシスエレメントとして不可欠であることを示している。(5) Identification of cis element involved in Sema3A gene regulation Next, it was analyzed whether or not these transcription factors can actually bind to each of the putative binding sites. For this purpose, electrophoretic mobility shift assay (EMSA) was performed on the nucleoprotein extract of cultured NHEK and biotin corresponding to the -122 / -93, -77 / -46, and -47 / -18 regions of the Sema3A promoter region. This was carried out using a double-stranded oligonucleotide probe. As shown in FIG. 5, -122 / -93 containing the RORα / ER / AP-1 binding site, -77 / -46 containing the Sox 5/9 binding site, -47 /-containing the GABP binding site. A transcription factor was bound to the target sequence of each of the 18 probes, and a shifted band was detected. These results indicate that these binding sites in the -122 / -18 bp region are essential as cis elements for Sema3A transcription.
Sema3A発現に重要な転写因子結合配列を同定するために、各転写因子結合配列を部位特異的に欠失後、増殖期のNHEKを用いてプロモーターアッセイを行った。正常ヒト表皮角化細胞（NHEK）における各変異体のルシフェラーゼアッセイの結果を図６に示す。RORα結合配列が存在する-108/-98領域を欠失した変異体でルシフェラーゼ活性の著しい抑制が認められ、この領域にSema3A発現を上方制御する転写因子が結合することが明らかとなった。 In order to identify transcription factor binding sequences important for Sema3A expression, each transcription factor binding sequence was deleted in a site-specific manner, and then a promoter assay was performed using NHEK in the growth phase. FIG. 6 shows the results of luciferase assay of each mutant in normal human epidermal keratinocytes (NHEK). Mutants lacking the -108 / -98 region where the RORα binding sequence was present showed marked suppression of luciferase activity, and it was revealed that transcription factors that upregulate Sema3A expression bind to this region.
さらに、RORα, Jun, Fos, Sox4, STAT3, NF-κB, GABPα, Sox9, Rel (c-Rel), CREB, Pbx1, ER, JunB, JunD, Fra1, Fra2がSema3A発現に関与していることを確認するために、低分子干渉RNA（siRNA）を使用して転写因子の発現を抑制し、リアルタイムPCRを用いて、各転写因子及びSema3Aの遺伝子発現を解析した。 Furthermore, RORα, Jun, Fos, Sox4, STAT3, NF-κB, GABPα, Sox9, Rel (c-Rel), CREB, Pbx1, ER, JunB, JunD, Fra1, Fra2 are involved in Sema3A expression. To confirm, transcription factor expression was suppressed using small interfering RNA (siRNA), and gene expression of each transcription factor and Sema3A was analyzed using real-time PCR.
RORα, Jun, Fos, Sox4, STAT3, NF-κB1のノックダウンについての結果を図７に示す。RORα、Jun、Fos，Sox4、STAT3の各転写因子に対するsiRNAでトランスフェクションしたNHEKでは、Sema3A発現は有意に抑制された。一方、NF-κB1のsiRNAでトランスフェクションしたNHEKでは、Sema3A発現は有意に増強した（約8倍）。これらの結果は、RORα, Jun, Fos, Sox4, STAT3がSema3A発現に不可欠であり、NF-κB1がSema3Aの発現抑制因子であることを強く示唆している。 FIG. 7 shows the results of knockdown of RORα, Jun, Fos, Sox4, STAT3, and NF-κB1. In NHEK transfected with siRNA against RORα, Jun, Fos, Sox4 and STAT3 transcription factors, Sema3A expression was significantly suppressed. On the other hand, in NHEK transfected with NF-κB1 siRNA, Sema3A expression was significantly enhanced (about 8 times). These results strongly suggest that RORα, Jun, Fos, Sox4, and STAT3 are indispensable for Sema3A expression, and that NF-κB1 is a suppressor of Sema3A expression.
GABPα, Sox9, Rel, CREB, Pbx1, ER, JunB, JunD, Fra1, Fra2のノックダウンについての結果を図８に示す。GABPα, Sox9, CREB, JunB, JunD, Fra2の発現抑制により、Sema3A発現は有意に（*, p＜0.05; ***, p＜0.001）抑制された。特に、CREBとJunBの発現を抑制した際のSema3A発現の抑制が顕著であった。 FIG. 8 shows the results of knockdown of GABPα, Sox9, Rel, CREB, Pbx1, ER, JunB, JunD, Fra1, and Fra2. Sema3A expression was significantly suppressed (*, p <0.05; ***, p <0.001) by suppressing the expression of GABPα, Sox9, CREB, JunB, JunD, and Fra2. In particular, Sema3A expression was significantly suppressed when CREB and JunB expression was suppressed.
（６）NHEKにおけるSema3A発現に対するRORα作動薬及び拮抗薬の作用
siRNA及び部位特異的変異導入構築体のプロモーターアッセイの結果より、レチノイド関連オーファン受容体α（RORα）がSema3A発現の上方制御に重要な転写因子の一つであることが判明した。そこで、NHEKにRORα作動薬または拮抗薬を添加後、一定時間経過後（SR1078とSR1001は48時間後、コレステロール硫酸は96時間後）にtotal RNAを回収し、定量リアルタイムPCRでSema3A発現量を解析した。(6) Effects of RORα agonists and antagonists on Sema3A expression in NHEK
From the results of promoter assays of siRNA and site-directed mutagenesis constructs, retinoid-related orphan receptor α (RORα) was found to be one of the transcription factors important for up-regulation of Sema3A expression. Therefore, after adding RORα agonist or antagonist to NHEK, total RNA was collected after a certain period of time (48 hours for SR1078 and SR1001, 96 hours for cholesterol sulfate), and Sema3A expression was analyzed by quantitative real-time PCR did.
結果を図９に示す。AがRORα作動薬SR1078、BがRORα作動薬のコレステロール硫酸、CがRORα拮抗薬SR1001の結果である。RORα作動薬のSR1078及びコレステロール硫酸を添加したNHEKでSema3A発現は濃度依存的に有意に促進された。一方、RORα拮抗薬のSR1001を添加した時にSema3A発現が濃度依存的に有意に抑制された。いずれの化合物も分子量は表皮角層に浸透しやすい500Da以下であることから、外用薬として応用できる可能性がある。 The results are shown in FIG. A is the result of the RORα agonist SR1078, B is the result of the RORα agonist cholesterol sulfate, and C is the result of the RORα antagonist SR1001. Sema3A expression was significantly enhanced in NHEK supplemented with RORα agonist SR1078 and cholesterol sulfate in a concentration-dependent manner. On the other hand, when RORα antagonist SR1001 was added, Sema3A expression was significantly suppressed in a concentration-dependent manner. Since any compound has a molecular weight of 500 Da or less that easily penetrates into the epidermal stratum corneum, it may be applicable as a topical drug.
（７）アトピー性皮膚炎モデルNC/Ngaマウスの病変部に対するRORα作動薬の作用
アトピー性皮膚炎（AD）モデルNC/Ngaマウスの病変部にRORα作動薬のコレステロール硫酸を外用する実験を行った。AD素因を有するNC/Ngaマウスの剃毛した背部にダニ抗原を配合したビオスタAD軟膏を週2回3週間外用し、ADを発症させた。皮膚炎を発症したマウスを抽出し、(i)基剤のジメチルスルホキシドのみ、(ii)1mMコレステロール硫酸の２群（各4匹）に分類後、マウスの病変部に基剤又はコレステロール硫酸溶液を9日間連続で塗布した。外用終了後に皮膚を摘出し、0.02％トリプシン/EDTA溶液に一晩浸漬後、表皮を剥離して、RNAを抽出した。定量リアルタイムPCRによる表皮のSema3A発現量を図１０に示す。基剤を外用した群と比較し、1mMコレステロール硫酸を外用した群でSema3A発現は有意に（**, p＜0.01）促進された。(7) Effects of RORα agonists on lesions of atopic dermatitis model NC / Nga mice An experiment was carried out in which the topical RORα agonist cholesterol sulfate was applied to lesions of atopic dermatitis (AD) model NC / Nga mice. . Biosta AD ointment containing mite antigen on the shaved back of NC / Nga mice with AD predisposition was applied twice a week for 3 weeks to develop AD. Mice that developed dermatitis were extracted and classified into 2 groups (4 animals each) of (i) base dimethyl sulfoxide only, (ii) 1 mM cholesterol sulfate, and then the base or cholesterol sulfate solution was applied to the affected area of the mice. It was applied for 9 consecutive days. After the external application was completed, the skin was removed, immersed in a 0.02% trypsin / EDTA solution overnight, the epidermis was peeled off, and RNA was extracted. The expression level of Sema3A in the epidermis by quantitative real-time PCR is shown in FIG. Sema3A expression was significantly (**, p <0.01) promoted in the group externally applied with 1 mM cholesterol sulfate compared with the group externally applied with the base.
Claims (24)
(1) 配列番号１に示す塩基配列。
(2) 配列番号２に示す塩基配列。
(3) (1)又は(2)と９０％以上の同一性を有する塩基配列。The method according to claim 18, wherein the semaphorin 3A promoter region comprises any one of the following base sequences (1) to (3):
(1) The base sequence shown in SEQ ID NO: 1.
(2) The base sequence shown in SEQ ID NO: 2.
(3) A nucleotide sequence having 90% or more identity with (1) or (2).
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008297243A (en) * | 2007-05-31 | 2008-12-11 | Yokohama City Univ | Therapeutic or prophylactic agent for pruritic dermatosis |
| JP4975231B2 (en) * | 2000-07-28 | 2012-07-11 | 大日本住友製薬株式会社 | Nerve regeneration promoter containing semaphorin inhibitor as active ingredient |
| WO2013005603A1 (en) * | 2011-07-01 | 2013-01-10 | 公立大学法人横浜市立大学 | Agent for prevention and/or treatment of allergic inflammation in conjunctiva |
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| JP2008297243A (en) * | 2007-05-31 | 2008-12-11 | Yokohama City Univ | Therapeutic or prophylactic agent for pruritic dermatosis |
| WO2013005603A1 (en) * | 2011-07-01 | 2013-01-10 | 公立大学法人横浜市立大学 | Agent for prevention and/or treatment of allergic inflammation in conjunctiva |
Non-Patent Citations (6)
| Title |
|---|
| ACS CHEM BIOL., 2010, VOL.5 NO.11, P.1029-1034, JPN6014030545, ISSN: 0003755552 * |
| BIOCHEM BIOPHYS RES COMMUN., 2012, VOL.428 NO.1, P.99-104, JPN6014030546, ISSN: 0003646835 * |
| J INVESTIGATION DERMATOLOGY., 2009, VOL.129 SUPPL.1, P.S48（ABSTRACT NO.288）, JPN6018008777, ISSN: 0003755551 * |
| MOL MED, 2002, VOL.39 NO.3 P.286-292, JPN6014030548, ISSN: 0003646836 * |
| 実験医学, 2013, VOL.31,NO.4, P.488-494, JPN6014030552, ISSN: 0003646838 * |
| 生体の科学, 2013.4.15, VOL.64 NO.2 P.122-131, JPN6014030550, ISSN: 0003646837 * |
Cited By (2)
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| CN114848653A (en) * | 2021-02-03 | 2022-08-05 | 四川大学 | Application of RORa protein and agonist thereof in preparation of acute DNA injury repair agent |
| CN114848653B (en) * | 2021-02-03 | 2023-05-30 | 四川大学 | Application of RORa protein and agonist thereof in preparation of acute DNA damage repair agent |
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