Abstract
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.