JP5773872B2 - 組成物および方法 - Google Patents
組成物および方法 Download PDFInfo
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- JP5773872B2 JP5773872B2 JP2011525630A JP2011525630A JP5773872B2 JP 5773872 B2 JP5773872 B2 JP 5773872B2 JP 2011525630 A JP2011525630 A JP 2011525630A JP 2011525630 A JP2011525630 A JP 2011525630A JP 5773872 B2 JP5773872 B2 JP 5773872B2
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Description
−試験物質を用意する工程;および
−試験物質のALK6と相互作用する能力を決定する工程
を含む。
−試験物質を用意する工程;
−Cdc42を発現する細胞を用意する工程;ならびに
−試験物質のCdc42の活性および/または発現を調節する能力を決定する工程
を含む。
メラニン転移の生物学についての研究中、本発明者らは、このプロセスにおける骨形成タンパク質(BMP)ファミリーのメンバーの潜在的な役割を研究した。本出願では、ヒト皮膚メラニン細胞からヒト皮膚ケラチン細胞へのメラニン転移を調節する新規機序について記載する。これは、このプロセスを用量依存的に刺激することができるBMP6の作用を含む。また、選択的BMP6拮抗薬(スクレロスチン)の添加は、ヒトメラニン細胞の走査電子顕微鏡(SEM)分析により証明されるように、BMP6誘導性メラノソーム転移を阻害し、(糸状仮足と呼ばれる)ナノ管のBMP6誘導性生成を阻害する。BMP6が糸状仮足形成を誘導する分子機序を分析し、BMP6がヒトメラニン細胞における(細胞分裂周期42)Cdc42の発現を用量依存的に刺激することが、逆転写分析を用いて見出された。メラニン細胞の糸状仮足形成およびメラノソーム転移におけるCdc42の動員は、トランスフェクトした構成的に活性な(CA)Cdc42および優性阻害(DN)Cdc42を有するFM55細胞のSEM分析を用いて確認された。CA−Cdc42は背側糸状仮足形成を誘導するが、DN−Cdc42は背側糸状仮足を阻害する。
細胞培養:適合する表皮ケラチン細胞および表皮メラニン細胞の分離および培養
インフォームドコンセントおよび地元の研究倫理委員会の承認とともに、選択的な美容整形手術後の、皮膚フォトタイプII(女性68y、39y、36y)および皮膚フォトタイプVI(女性42y)を有する正常で健康な白人ドナーからヒト腹部皮膚を得た。
表皮メラニン細胞(MC)および表皮ケラチン細胞(KC)を、血清を添加した完全MEMメラニン細胞およびK−SFMケラチン細胞培地で6ウェルプレート上に播種し、24時間置いた。細胞を10ng/mlおよび100ng/mlのBMP6を添加した無血清培地(いわゆる飢餓培地)に移し、24時間、48時間、および72時間置いた。細胞毒性を細胞死および細胞病理学的形態変化により評価した。
融合性T225フラスコ中のMCをトリプシン処理し、完全培地を有するT25フラスコ中に播種し、一晩接着させた。処理の24時間前に培地を飢餓培地に取り替え、24時間置いた。細胞を無菌PBSで3回洗浄し、飢餓培地のみまたは100ng/mlのBMP6を添加して異なる時間(1〜24時間)インキュベートした。異なる実験では、細胞を異なる濃度のBMP6で12時間処理した。
氷冷メタノール中で定着させた細胞をPBS中で洗浄した後、タンパク質発現の検出のため10%ロバ血清でブロックした。二重標識実験のため、第1の一次抗体として、BMP6(Abcam、英国ケンブリッジ)、BMPR−IB/ALK6(R&D Systems、英国オックスフォードシャー州)またはNKi/Beteb(1:30)(Monosan、オランダ、ウデン)を塗布して4℃で一晩置いた後、FITC共役二次抗体(1:100)を用いて室温で1時間インキュベートした。第2の一次抗体として、サイトケラチン(1:100)(Abcam、英国ケンブリッジ)またはβ−アクチン(1:100)(Santa Cruz、米国)を塗布して室温で1時間置いた後、TRITC共役二次抗体(1:100)(Jackson Immunoresearch Laboratories,Inc.、米国ウエストグローブ)を塗布した。DAPI(Vector Labratories、カリフォルニア州バーリンゲーム)を用いて細胞核を染色した。冷却型浜松デジタルカメラで対物100倍または40倍を用いて画像をキャプチャし、Paint Shop Pro(Jasc Software Ver.7、米国カリフォルニア州)を用いて後処理した。陰性対照は、一次抗体を省略し、二次抗体ホストからの非免疫血清で置き換え、二次抗体を含んだ。
適合するメラニン細胞−ケラチン細胞共培養を異なる用量のBMP6で刺激し、非刺激細胞と比較した。3つの独立した実験において100倍の倍率(油浸)で1ウェル当たり5つのランダムな顕微鏡視野の受容ケラチン細胞内の蛍光gp100陽性スポットを数えることにより、メラノソーム転移の評価を行った。メラニン細胞にも関連し得るメラニン顆粒を数えることを回避するため、我々はメラニン細胞と直接接触しないケラチン細胞内のgp100陽性スポットのみを数えた。
1、50、100および500ng/mlのBMP6で4時間または100ng/mlのBMP6で指定時間処理した1×106個のメラニン細胞から総RNAを得た(図4A)。いくつかの実験のため、メラニン細胞を400ng/mlのスクレロスチンの有無にかかわらず100ng/mlのBMP6の存在または非存在下でも処理した。RNA試料をデオキシリボヌクレアーゼ(QIAGEN、英国)で処理した後、濃度を測定した。次に逆転写用Superscript IIIファーストストランド合成キット(Invitrogen、カリフォルニア州)を用いて第1ストランドcDNAを合成した。その反応におけるRNAの最終濃度は、すべての試料について1μg/100μlだった。QIAGEN Fast Cycling PCRキット(QIAGEN、英国)を用いて製造業者より説明される条件下でPCRを行った。簡潔には、以下のサイクル条件を用いる:95℃で15分間(段階1);96℃で15秒間;プライマーアニーリング15秒間(Cdc42は42〜65℃、チロシナーゼは58℃、およびGAPDHは60℃);72℃で25秒間(段階2)×30サイクル。RT−PCR反応に用いるプライマーは表1に示すが、Sigma−Genosys、英国から入手した。PCR生成物を1倍TAE緩衝液中の1%アガロースゲルで分離し、臭化エチジウムで染色した。1KbのDNA Plus DNAラダー(Invitrogen、カリフォルニア州)をDNAマーカーとして用いる。
優性阻害ヒトGFP−Cdc42(15A)、構成的に活性なGFP−Cdc42(61L)およびpEGFP−C3のGFPは、Richard E.Cheney(ノースカロライナ大学、チャペルヒル)の気前の良い寄贈品だった。ベクターはジェネティシン(G418)への耐性を与えるネオマイシン遺伝子を含んでいた。約5×105個の中程度に色素沈着したFM55細胞を、トランスフェクションの前日10%FBSを添加したRPMI培地において16時間6ウェルプレートで70〜80%の密集度まで増殖させた。FM55細胞をLipofectamine 2000トランスフェクション試薬(Invitrogen、米国カリフォルニア州カールスバッド)を用いてトランスフェクトした。簡潔には、1.6mlのOpti−MEM低血清培地中で3.2μgのcDNA構成物を8μlのLipofectamine 2000トランスフェクション試薬と混合した。細胞をトランスフェクション混合物で12時間インキュベートし、1倍PBSで3回洗浄し、10%FBSを含有する未使用RPMI中で48時間インキュベートした。トランスフェクト細胞を1.6mg/mlのG418を用いて15日間かけて選択した。次に1×104個の安定なトランスフェクト細胞を8ウェルLab−Tek(登録商標)チャンバースライドにおいて10%FBSを添加したRPMI培地存在下で24時間培養した。24時間後、FM55細胞の(糸状仮足を含む)形状の立体構造を保存するため、安定にトランスフェクトした細胞を0.1Mのカコジル酸ナトリウム(Agar、英国)中に緩衝させた1%グルタルアルデヒド(Sigma、英国)において37℃ですぐに定着させ、SEM分析のために処理した。8ウェルLab−Tek(登録商標)チャンバースライド上の平行な細胞も、氷冷メタノールにおいて−20℃で10分間定着させ、PBS中で洗浄した後、gp100免疫蛍光研究のため10%ロバ血清でブロックし、トランスフェクションおよび選択を確認した(補足1)。共培養研究のため、安定にトランスフェクトしたFM55細胞およびケラチン細胞(継代2)を8ウェルLab−Tek(登録商標)チャンバースライド上に1×104細胞/ウェルの細胞密度および10個のケラチン細胞に対して1個のFM55という比で播種した。共培養をK−SFMおよびMEM(共培地)の混合物中に一晩(16時間)保持し、細胞を接着させた後、培地を補充してさらに24時間置いた。次に、メラノソーム転移効率を試験するgp100およびサイトケラチンの二重免疫蛍光研究のため、細胞を氷冷メタノールにおいて−20℃で10分間定着させ、PBS中で洗浄した後、10%ロバ血清でブロックした。
400ng/mlのスクレロスチンの有無にかかわらず100ng/mlのBMP6の存在または非存在下で72時間処理した表皮メラニン細胞および安定に選択トランスフェクトされたF55細胞を0.1Mのカコジル酸ナトリウム(Agar、英国)中に緩衝させた1%グルタルアルデヒド(Sigma、英国)において37℃で定着させた。次に細胞を媒染剤として用いられる1%四酸化オスミウム(Agar、英国)および1%タンニン酸(Agar、英国)中で後定着させ、一連の20〜70%のアルコールによって脱水し、0.5%酢酸ウラニル中で染色した後、90および100%のアルコール中でさらに脱水した。へキサメチル−ジシラザン(Sigma、英国)中で最終脱水した後、空気乾燥させた。乾燥後、試料を金スパッター(EMITECH、K550)(Blazer 20mA)において金で10分間コーティングした。試料を電界放射型SEM(FEI、Quanta400)下、10keVの加速電圧で観察した。
500μg/mlの合成メラニン(Sigma、英国)を1Mの水酸化ナトリウム(NaOH)(BOH Ltd、英国)中で調製し、超音波水槽中で20分間溶解させた。この原液から、各種メラニン標準液を100μg/ml〜1μg/mlの1MのNaOH中で調製した。メラニン標準液をピペットで96ウェルプレートに取り、試験試料中のメラニン含量の評価について較正曲線を作成した。400μlの1MのNaOHを各細胞ペレットに添加し、ヒートブロック(100℃)上で15分間溶解させた。ペレットを激しく渦撹拌し、可溶化したペレットをピペットで同じ96ウェルプレートに取った。試料の光学密度をDYNEX REVELATION 4.02プログラム上495nmで読んだ。各試験試料のメラニン含量を較正曲線から読んだ。
約1×106個の表皮メラニン細胞を3つのT75フラスコ中に播種し、37℃および5%CO2で一晩インキュベートした。細胞をSDS−PAGE用に用意し、PVDF膜上にトランスブロットした。70μgの非還元タンパク質抽出物を沸騰せずにピペットで適切なウェルの8%SDS−PAGEゲルに取った。別々のタンパク質を含有するPVDF膜を1倍PBS中で1回洗浄した後、0.1Mのリン酸ナトリウム緩衝液中の5mMのL−DOPAにおいてL−DOPAを3回交換しながら室温で3時間インキュベートした。膜を蒸留水で洗浄することによりL−DOPA反応を停止し、膜を走査した。
群と処理との統計的有意性を、Prism v.4.00(GraphPad Software、米国イリノイ州シカゴ)を用いる一元配置ANOVAおよびダネット事後検定を用いて評価した。統計的に有意な差はアスタリスクで示す:**=P<0.01、***=P<0.001。
BMP6(10および100μg/ml)は正常なヒト表皮メラニン細胞およびケラチン細胞に対する細胞毒性を示さない
BMP6がメラニン細胞またはケラチン細胞に対して毒性がある可能性を排除するため、細胞を10ng/mlおよび100ng/mlのBMP6存在下で24時間、48時間および72時間保持した。BMP6の2つの用量は、メラニン細胞およびケラチン細胞の細胞増殖または形態の変化とは関係ない(図1A、1B)。
正常なヒトメラニン細胞におけるBMP6の発現を、抗BMP6(緑、Ai)と抗チロシナーゼ抗体(赤、Aii)の二重免疫標識(黄橙、Aiii)により確認した。抗BMP6(緑、Bi)および抗ALK6(赤、Bii)抗体でのメラニン細胞の二重免疫標識は、細胞全体でBMP6およびALK6の共局在化(黄、Biii)を示した。
メラニン細胞−ケラチン細胞共培養へのBMP6処理がメラニン細胞からケラチン細胞へのメラノソーム転移の刺激を用量依存的にもたらすように(図3A、B)、ALK6受容体は有効である。よって、これはメラニン細胞−ケラチン細胞共培養系におけるメラノソーム転移に対するBMP6の作用についての初めての報告である。オートクリンおよびパラクリン作用の両方が含まれ得る。
メラノソーム転移におけるBMP6の機序的役割をより良く理解するため、我々はヒトメラニン細胞におけるCdc42の発現;糸状仮足形成の主要調節因子を研究した(Scott et al.,2002)。RT−PCR分析は、未処理対照におけるCdc42 mRNAの基礎レベルの発現と比較して、Cdc42の最大誘導が100ng/mlのBMP6での処理後2〜4時間で起こった後、わずかに減少するが、最長12時間高いままであることを示した(図4A)。BMP6によるCdc42 mRNAの誘導は用量依存的に起こった(図4B)。BMP6は、正常なヒト表皮メラニン細胞におけるALK6(図4Ci)の発現の正常な基礎レベルと比較して、BMP6受容体ALK6(緑、図4Cii)の発現も刺激した(図4C)。
2種類のRhoタンパク質変異体:GTPアーゼ活性化タンパク質(GAP)が阻害されるため、構成的にGTP結合である活性化変異体;およびグアニンヌクレオチド交換因子(GEF)を滴定することにより作用する優性阻害変異体を広く用い、それらの機能を分析してきた(Feig、1999)。我々は、対照ベクターと比較すると、構成的に活性なCdc42が背側糸状仮足を誘導し、優性阻害Cdc42がF55ヒトメラノーマの背側糸状仮足を阻害することを見出した(図5A)。
スクレロスチン(選択的BMP6拮抗薬)がBMP6誘導性メラノソーム転移を阻害するかどうかを試験するため、我々はこの拮抗薬を正常なヒト表皮メラニン細胞−ケラチン細胞共培養において試験した。スクレロスチンの添加そのものは、基礎レベルのメラニン細胞からケラチン細胞へのメラノソーム転移を阻害した(図6A、B)。また、スクレロスチンはヒト表皮メラニン細胞−ケラチン細胞共培養におけるBMP6誘導性メラノソーム転移を阻害し、スクレロスチンを哺乳類の皮膚および毛髪色素沈着の新規調節因子として用いることができることを示した(図6A、B)。
我々は、メラニン細胞からケラチン細胞へのメラノソーム転移の調節因子としてのCdc42の生理学的役割について初めて示した(図5B)。スクレロスチンのBMP6誘導性メラノソーム転移を阻害する能力は、スクレロスチンがヒトメラニン細胞におけるCdc42のBMP6誘導性発現を阻害し得ることを示した。ヒトメラニン細胞のRT−PCR分析を用い、我々は実際にスクレロスチンがCdc42のBMP6誘導性発現を下方調節することを見出した(図7A)。免疫蛍光研究を用い、我々はスクレロスチンがBMP6受容体であるALK6(緑)のBMP6誘導性発現を下方調節することも見出した(図7B)。この発見は、Cdc42がBMP6シグナル伝達の考えられる増幅ループに関与することを示す。
メラニン細胞のSEM分析は、未処理対照と比較して、多数のBMP6処理による背側糸状仮足の刺激を示す(図8iii)。スクレロスチンで処理したメラニン細胞は背側糸状仮足形成の低減を示し、一方BMP6で処理した細胞はスクレロスチン存在下で糸状仮足形成を誘導しなかった(図8ii、iv)。これらのデータは、ヒトメラニン細胞における背側糸状仮足形成の刺激についてのBMP6の要件を強く裏付ける。
BMP6はヒト表皮メラニン細胞in vitro中のメラニン含量を(72時間後約32±9%)増加させた(図9A)。選択的BMP6拮抗薬スクレロスチンは、ヒト表皮メラニン細胞中のBMP6誘導性メラニン含量を(15±3%)抑制した(図9A)。チロシナーゼ活性(メラニン合成の可能性の指標)もBMP6により刺激されたが、スクレロスチンはチロシナーゼ活性のBMP6誘導性増加を抑制した(図9B)。
Claims (9)
- 対象個体の皮膚及び/又は毛髪のメラニン色素沈着を増加させるための、BMP6又はその断片若しくは変異体を含む組成物であって、
該変異体が、BMP6と少なくとも70%の配列同一性を有し、
該断片又は該変異体が、メラニン形成又はメラニン転移を調節する能力を示し、且つ配列番号1によってコードされるか又は配列番号1のアミノ酸374〜513若しくは配列番号1のアミノ酸382〜513若しくは配列番号1のアミノ酸388〜513若しくは配列番号1のアミノ酸412〜513を含む、組成物。 - 前記BMP6又はその断片若しくは変異体が、ALK6の活性を調節することによってCdc42の発現を調節する、BMP6若しくはその断片又はそれらの変異体を含む、請求項1に記載の組成物。
- 対象個体の皮膚及び/又は毛髪のメラニン色素沈着を低減するための、BMP6拮抗薬であるスクレロスチンを含む組成物。
- 皮膚及び/又は毛髪の色素沈着減少の治療に用いられる、BMP6又はその断片若しくは変異体を含む組成物であって、
該変異体が、BMP6と少なくとも70%の配列同一性を有し、
該断片又は該変異体が、メラニン形成又はメラニン転移を調節する能力を示し、且つ配列番号1によってコードされるか又は配列番号1のアミノ酸374〜513若しくは配列番号1のアミノ酸382〜513若しくは配列番号1のアミノ酸388〜513若しくは配列番号1のアミノ酸412〜513を含む、組成物。 - 白斑の治療に用いられる、BMP6又はその断片若しくは変異体を含む、請求項4に記載の組成物。
- 皮膚及び/又は毛髪の色素沈着過度の治療に用いられる、スクレロスチンを含む、組成物。
- 炎症後色素沈着過度、メラズマ、クロズマ、老年性黒子、日光黒子、又はそばかすの治療に用いられる、スクレロスチンを含む組成物。
- 皮膚及び/又は毛髪のメラニン色素沈着のレベルを調節することによって皮膚及び/又は毛髪の色を暗くするための化粧品組成物であって、
BMP6又はその断片若しくは変異体を含み、BMP6又はその断片若しくは変異体を含み、該変異体がBMP6と少なくとも70%の配列同一性を有し、該断片又は該変異体がメラニン形成又はメラニン転移を調節する能力を示し、且つ配列番号1によってコードされるか又は配列番号1のアミノ酸374〜513若しくは配列番号1のアミノ酸382〜513若しくは配列番号1のアミノ酸388〜513若しくは配列番号1のアミノ酸412〜513を含む、化粧品組成物。 - 皮膚及び/又は毛髪のメラニン色素沈着のレベルを調節することによって皮膚及び/又は毛髪の色を明るくするための化粧品組成物であって、
BMP6拮抗薬であるスクレロスチンを含む、化粧品組成物。
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