靜止期纖維母細胞（quiescent fibroblast；SF-Fb）具有特定的基因表現，並與增殖期纖維母細胞（proliferative fibroblast；pFb）擁有不同的代謝活性。先前的研究指出，對於免疫反應的刺激，SF-Fb在表達環氧酵素-2 （Cyclooxygenase-2；COX-2）以及其它發炎基因的表現量會比pFb的表現量更多。此相關的轉錄機制尚不清楚。在本研究中，我們發現SF-Fb會比pFb表現出更大量的促發炎基因，原因為兩者的p300組蛋白乙酰轉移酶（Histone acetyltransferase；HAT）有不同程度的活性。當SF-Fb利用佛波醇-12-十四烷酸酯-13-乙酸酯 （Phorbol 12-myristate 13-acetate；PMA）和細胞激素刺激後會使pFb有較高的p300 HAT活性（> 2倍）。實驗室先前的研究發現pFb中的5-甲氧基色胺酸（5-methoxytryptophan；5-MTP） 是控制COX-2轉錄的關鍵因子，因此我們推測，當細胞中5-MTP 的量下降, 會使Fb表現出更大量的p300 HAT活性，進而導致COX-2表現量上升。利用超高性能液相色譜外加四極棒暨飛行時間串聯式質譜儀（ultrahigh-performance liquid chromatography coupled with a quardrupole time of flight mass spectrometer） 以及酵素免疫分析 （enzyme immunoassay；EIA）進行5-MTP的測量, 結果顯示pFb的5-MTP的產量是SF-Fb 的2-3倍。而在SF-Fb中外加5-MTP則可降低PMA所促進的p300 HAT活性和COX -2的表現。此外我們將色胺酸代謝中的酵素：第一色胺酸氫氧基化酶（Tryptophan hydroxylase-1；TPH-1）或5-羥氧吲哚-O-甲基轉移酶（Hydroxyindole O-methyltransferase；HIOMT） 的小干擾核糖核酸 (Small interfering RNA；siRNA) 送入pFb中，結果發現以siRNA干擾的pFb一旦受到PMA刺激，其p300 HAT活性會大量升高，甚至與SF-Fb相同，而受到siRNA影響的pFb外加5-MTP後，其p300 HAT的活性會降到與未處理過的pFb相似。在色胺酸的代謝過程中，5-羥基色胺酸（5-hydroxytryptophan；5-HTP）是藉由HIOMT轉化成為5-MTP。在實驗中發現5-HTP會影響p300 HAT活性。在SF-Fb中外加5-HTP可抑制p300 HAT活性和COX- 2的表現。從以上的實驗結果證明，由於 SF-Fb中的5-MTP表現量不足，使得p300 HAT活性不受控制，進而影響許多免疫反應的基因表現。

Quiescent fibroblasts (SF-Fb) feature unique genetic program and exhibit high metabolic activity different from proliferative fibroblasts (pFb). We recently reported that in response to inflammatory stimulation, SF-Fb is more active in expressing cyclooxygenase-2 (COX-2) and other proinflammatory genes than pFb. The underlying transcriptional mechanism is unclear. Here we show that more robust proinflammatory gene expression in SF-Fb vs. pFb is attributed to differential activation of p300 histone acetyltransferase (HAT). Phorbol 12-myristate 13-acetate (PMA) and cytokines increased p300 HAT activity to a higher magnitude (> 2 fold) in SF-Fb than in pFb. 5-methoxytryptophan production was recently reported to be pivotal in controlling COX-2 transcription in pFb. We determined whether more robust p300 HAT activation in SF-Fb vs. pFb may be due to underproduction of 5-methoxytryptophan (5-MTP). 5-MTP production was analyzed by ultrahigh-performance liquid chromatography coupled with a quardrupole time of flight mass spectrometer and enzyme-immunoassay. 5-MTP produced by pFb was 2-3 fold higher than that by SF-Fb. Addition of 5-MTP to SF-Fb reduced PMA-induced p300 HAT activity and COX-2 expression to the level of pFb. Silencing of 5-MTP synthetic enzymes, tryptophan hydroxylase-1 (TPH-1) or hydroxyindole O-methyltransferase (HIOMT) in pFb with siRNA transfection resulted in elevation of PMA-induced p300 HAT activity to the level in SF-Fb, which was rescued by addition of 5-MTP. 5-hydroxytryptophan (5-HTP) is an intermediate metabolite in 5-MTP production. We determined whether 5-HTP influences p300 HAT activation. Addition of 5-HTP resulted in suppression of p300 HAT activation and COX-2 expression. Our findings indicate that robust inflammatory gene expressions in SF-Fb vs. pFb are attributed to uncontrolled p300 HAT activation due to deficiency of 5-MTP production.

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