探討TNF-alpha對人類真皮纖維母細胞中基質金屬蛋白酶(MMP-1/MMP-3)表現之機轉

Studies on the mechanism of tumor necrosis factor-alpha-induced matrix metalloproteinases(MMP-1/MMP-3)expression in human dermal fibroblasts

盧宜恩(Yi-En Lu)

指導教授 ： 陳瑩容

高雄醫學大學/藥學院/香粧品學系碩士班/碩士(2019年)

摘要

腫瘤壞死因子-alpha（TNF-alpha）會誘導基質金屬蛋白酶（Matrix metalloproteinases，MMPs）的表現，進而降解細胞外基質的膠原蛋白，此一作用與TNF-alpha誘使細胞老化機制有關。TNF-alpha透過NF-kB的轉錄活性調控其目標基因，NF-kB的蛋白質轉譯後修飾包含磷酸化（Serine 536）與乙醯化（Lysine 310），並影響其轉錄活性。p300是一種轉錄輔助因子，具有組蛋白乙醯轉移酶（Histone acetyltransferase，HAT）的活性，能調節NF-B與組蛋白（Histone）的乙醯基修飾。本研究旨在探討在人類真皮纖維母細胞CCD-966SK中TNF-alpha誘導基質金屬蛋白酶表現之分子調控機轉。實驗結果發現，TNF-alpha會誘導NF-kB產生磷酸化修飾，也會透過活化p300表現，使p300對NF-kB及組蛋白進行乙醯化修飾。當組蛋白進行乙醯化作用會改變染色質結構，促進p300與NF-kB複合體結合在MMP-1和MMP-3啟動子上，進而調控基因的表現。以冷光活性測定（Luciferase assay）與染色質免疫沉澱分析法（ChIP assay）進行檢測，結果顯示TNF-alpha誘導p300介導NF-kB蛋白質乙醯化在調控MMP-1和MMP-3表現中扮演著重要的角色。進一步實驗發現白藜蘆醇能抑制TNF-alpha活化p300介導NF-kB的乙醯化與組蛋白的乙醯化，從而減弱TNF-alpha誘導NF-kB的轉錄活性，進而達到顯著降低MMP-1和MMP-3的表現。本研究結果顯示在人類真皮纖維母細胞中TNF-alpha是透過活化p300的表現，誘導NF-kB乙醯化修飾與組蛋白乙醯化作用，進而調控MMP-1和MMP-3的作用機轉。此研究成果將有助於發展改善TNF-alpha誘導皮膚老化之策略。

關鍵字

Tumor necrosis factor-alpha ； NF-kB ； p300 ； 基質金屬蛋白酶皮膚老化

並列摘要

Tumor necrosis factor-alpha(TNF-alpha)up-regulates matrix metalloproteinases (MMPs) expression, resulting in degradation of collagen in the extracellular matrix. This event is tightly associated with TNF-alpha-induced skin aging. TNF-alpha-modulated gene expression is related to NF-kB transcriptional activity, which depends on the post-translational modification of NF-kB including phosphorylation (Serine 536) and acetylation (Lysine 310). The transcriptional coactivator p300 endows with histone acetyltransferase (HAT) activity, and regulates acetylation of NF-kB and histone. This study aims to explore the molecular mechanism responsible for the effect of TNF-alpha-induced matrix metalloproteinases expression in human dermal fibroblasts CCD-966SK. Bay11-7082 (NF-kB inhibitor), C646 (p300 inhibitor) and p300 siRNA transfection are employed to investigate the role of p300 and NF-kB in regulating TNF-alpha-stimulated MMP-1 and MMP-3 expression. Our data revealed that TNF-alpha-induced NF-kB phosphorylation and NF-kB acetylation. TNF-alpha induced p300 up-regulation promoted acetylation of NF-kB and histone. While the binding of p300 and NF-kB with MMP-1 and MMP-3 promoter region increased gene expression. The methods of Luciferase assay and chromatin immunoprecipitation assay showed that p300-induced NF-B acetylation is important for TNF-alpha-elicited up-regulation of MMP-1 and MMP-3. Treatment with resveratrol suppressed TNF-alpha-mediated p300 on NF-kB acetylation and histone acetylation, and that inhibited TNF-alpha-induced up-regulation of MMP-1 and MMP-3 expression. Our data indicate that NF-kB acetylation and histone acetylation play a crucial role in TNF-alpha-modulated up-regulation of MMP-1 and MMP-3 in human dermal fibroblasts. It may shed some light on the strategy in improving TNF-alpha-induced skin aging.

並列關鍵字

Tumor necrosis factor-alpha ； NF-kB ； p300 ； Matrix metalloproteinases ； Skin aging

參考文獻

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