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Quiescent and proliferative fibroblasts exhibit differential p300 HAT activation through control of 5-methoxytryptophan production.

Cheng HH, Wang KH, Chu LY, Chang TC, Kuo CC, Wu KK - PLoS ONE (2014)

Bottom Line: The underlying transcriptional mechanism is unclear.By ultrahigh-performance liquid chromatography coupled with a quadrupole time of flight mass spectrometer and enzyme-immunoassay, we found that production of 5-methoxytryptophan was 2-3 folds higher in proliferative fibroblasts than that in quiescent fibroblasts.Silencing of tryptophan hydroxylase-1 or hydroxyindole O-methyltransferase in proliferative fibroblasts with siRNA resulted in elevation of PMA-induced p300 histone acetyltransferase activity to the level of that in quiescent fibroblasts, which was rescued by addition of 5-hydroxytryptophan or 5-methoxytryptophan.

View Article: PubMed Central - PubMed

Affiliation: Metabolomic Medicine Research Center, China Medical University, Taichung, Taiwan ; Graduate Institute of Clinical Medicine Science, China Medical University, Taichung, Taiwan.

ABSTRACT
Quiescent fibroblasts possess unique genetic program and exhibit high metabolic activity distinct from proliferative fibroblasts. In response to inflammatory stimulation, quiescent fibroblasts are more active in expressing cyclooxygenase-2 and other proinflammatory genes than proliferative fibroblasts. The underlying transcriptional mechanism is unclear. Here we show that phorbol 12-myristate 13-acetate (PMA) and cytokines increased p300 histone acetyltransferase activity to a higher magnitude (> 2 fold) in quiescent fibroblasts than in proliferative fibroblasts. Binding of p300 to cyclooxygenase-2 promoter was reduced in proliferative fibroblasts. By ultrahigh-performance liquid chromatography coupled with a quadrupole time of flight mass spectrometer and enzyme-immunoassay, we found that production of 5-methoxytryptophan was 2-3 folds higher in proliferative fibroblasts than that in quiescent fibroblasts. Addition of 5-methoxytryptophan and its metabolic precursor, 5-hydroxytryptophan, to quiescent fibroblasts suppressed PMA-induced p300 histone acetyltransferase activity and cyclooxygenase-2 expression to the level of proliferative fibroblasts. Silencing of tryptophan hydroxylase-1 or hydroxyindole O-methyltransferase in proliferative fibroblasts with siRNA resulted in elevation of PMA-induced p300 histone acetyltransferase activity to the level of that in quiescent fibroblasts, which was rescued by addition of 5-hydroxytryptophan or 5-methoxytryptophan. Our findings indicate that robust inflammatory gene expression in quiescent fibroblasts vs. proliferative fibroblasts is attributed to uncontrolled p300 histone acetyltransferase activation due to deficiency of 5-methoxytryptophan production. 5-methoxytryptophan thus is a potential valuable lead compound for new anti-inflammatory drug development.

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p300 HAT activation is decreased in pFb vs. SF-Fb.A). PMA-induced p300 HAT activity in untransfected native cells. Upper panel shows p300 proteins analyzed by Western blotting and lower panel, p300 HAT activity. B). p300 HAT activity in p300-transfected fibroblasts. Upper panel shows p300 proteins in Fb transfected with full-length p300 vectors and lower panel PMA-induced p300 HAT activity in the p300 overexpressing cells. Nuclear histone H1 was used as a loading control. C) and D). IL-1β- and TNFα-induced p300 HAT activities in p300-transfected SF-Fb or pFb. Each error bar denotes mean ± SEM (n = 3).
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pone-0088507-g003: p300 HAT activation is decreased in pFb vs. SF-Fb.A). PMA-induced p300 HAT activity in untransfected native cells. Upper panel shows p300 proteins analyzed by Western blotting and lower panel, p300 HAT activity. B). p300 HAT activity in p300-transfected fibroblasts. Upper panel shows p300 proteins in Fb transfected with full-length p300 vectors and lower panel PMA-induced p300 HAT activity in the p300 overexpressing cells. Nuclear histone H1 was used as a loading control. C) and D). IL-1β- and TNFα-induced p300 HAT activities in p300-transfected SF-Fb or pFb. Each error bar denotes mean ± SEM (n = 3).

Mentions: To provide direct evidence for a more robust activation of p300 HAT in quiescent fibroblasts than in proliferative fibroblasts, we isolated p300 by immunoprecipitation from SF-Fb and pFb treated with and without PMA and analyzed the HAT activity. As anticipated from the autoinhibition of p300 HAT, the basal p300 HAT activity was very low in SF-Fb as well as in pFb (Fig. 3A). PMA increased p300 HAT activity to a greater extent in SF-Fb than in pFb (Fig. 3A). PMA-stimulated p300 HAT activity in SF-Fb was augmented by p300 transfection (Fig. 3B). Although PMA-induced p300 HAT activity in pFb was also augmented by p300 overexpression, the extent of increase was less than that in SF-Fb (Fig. 3B). IL-1β or TNF-α stimulated p300 HAT activity differentially in pFb vs. SF-Fb and the extent of stimulation and magnitude of difference between SF-Fb and pFb were comparable to PMA (Fig. 3C & 3D).


Quiescent and proliferative fibroblasts exhibit differential p300 HAT activation through control of 5-methoxytryptophan production.

Cheng HH, Wang KH, Chu LY, Chang TC, Kuo CC, Wu KK - PLoS ONE (2014)

p300 HAT activation is decreased in pFb vs. SF-Fb.A). PMA-induced p300 HAT activity in untransfected native cells. Upper panel shows p300 proteins analyzed by Western blotting and lower panel, p300 HAT activity. B). p300 HAT activity in p300-transfected fibroblasts. Upper panel shows p300 proteins in Fb transfected with full-length p300 vectors and lower panel PMA-induced p300 HAT activity in the p300 overexpressing cells. Nuclear histone H1 was used as a loading control. C) and D). IL-1β- and TNFα-induced p300 HAT activities in p300-transfected SF-Fb or pFb. Each error bar denotes mean ± SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921189&req=5

pone-0088507-g003: p300 HAT activation is decreased in pFb vs. SF-Fb.A). PMA-induced p300 HAT activity in untransfected native cells. Upper panel shows p300 proteins analyzed by Western blotting and lower panel, p300 HAT activity. B). p300 HAT activity in p300-transfected fibroblasts. Upper panel shows p300 proteins in Fb transfected with full-length p300 vectors and lower panel PMA-induced p300 HAT activity in the p300 overexpressing cells. Nuclear histone H1 was used as a loading control. C) and D). IL-1β- and TNFα-induced p300 HAT activities in p300-transfected SF-Fb or pFb. Each error bar denotes mean ± SEM (n = 3).
Mentions: To provide direct evidence for a more robust activation of p300 HAT in quiescent fibroblasts than in proliferative fibroblasts, we isolated p300 by immunoprecipitation from SF-Fb and pFb treated with and without PMA and analyzed the HAT activity. As anticipated from the autoinhibition of p300 HAT, the basal p300 HAT activity was very low in SF-Fb as well as in pFb (Fig. 3A). PMA increased p300 HAT activity to a greater extent in SF-Fb than in pFb (Fig. 3A). PMA-stimulated p300 HAT activity in SF-Fb was augmented by p300 transfection (Fig. 3B). Although PMA-induced p300 HAT activity in pFb was also augmented by p300 overexpression, the extent of increase was less than that in SF-Fb (Fig. 3B). IL-1β or TNF-α stimulated p300 HAT activity differentially in pFb vs. SF-Fb and the extent of stimulation and magnitude of difference between SF-Fb and pFb were comparable to PMA (Fig. 3C & 3D).

Bottom Line: The underlying transcriptional mechanism is unclear.By ultrahigh-performance liquid chromatography coupled with a quadrupole time of flight mass spectrometer and enzyme-immunoassay, we found that production of 5-methoxytryptophan was 2-3 folds higher in proliferative fibroblasts than that in quiescent fibroblasts.Silencing of tryptophan hydroxylase-1 or hydroxyindole O-methyltransferase in proliferative fibroblasts with siRNA resulted in elevation of PMA-induced p300 histone acetyltransferase activity to the level of that in quiescent fibroblasts, which was rescued by addition of 5-hydroxytryptophan or 5-methoxytryptophan.

View Article: PubMed Central - PubMed

Affiliation: Metabolomic Medicine Research Center, China Medical University, Taichung, Taiwan ; Graduate Institute of Clinical Medicine Science, China Medical University, Taichung, Taiwan.

ABSTRACT
Quiescent fibroblasts possess unique genetic program and exhibit high metabolic activity distinct from proliferative fibroblasts. In response to inflammatory stimulation, quiescent fibroblasts are more active in expressing cyclooxygenase-2 and other proinflammatory genes than proliferative fibroblasts. The underlying transcriptional mechanism is unclear. Here we show that phorbol 12-myristate 13-acetate (PMA) and cytokines increased p300 histone acetyltransferase activity to a higher magnitude (> 2 fold) in quiescent fibroblasts than in proliferative fibroblasts. Binding of p300 to cyclooxygenase-2 promoter was reduced in proliferative fibroblasts. By ultrahigh-performance liquid chromatography coupled with a quadrupole time of flight mass spectrometer and enzyme-immunoassay, we found that production of 5-methoxytryptophan was 2-3 folds higher in proliferative fibroblasts than that in quiescent fibroblasts. Addition of 5-methoxytryptophan and its metabolic precursor, 5-hydroxytryptophan, to quiescent fibroblasts suppressed PMA-induced p300 histone acetyltransferase activity and cyclooxygenase-2 expression to the level of proliferative fibroblasts. Silencing of tryptophan hydroxylase-1 or hydroxyindole O-methyltransferase in proliferative fibroblasts with siRNA resulted in elevation of PMA-induced p300 histone acetyltransferase activity to the level of that in quiescent fibroblasts, which was rescued by addition of 5-hydroxytryptophan or 5-methoxytryptophan. Our findings indicate that robust inflammatory gene expression in quiescent fibroblasts vs. proliferative fibroblasts is attributed to uncontrolled p300 histone acetyltransferase activation due to deficiency of 5-methoxytryptophan production. 5-methoxytryptophan thus is a potential valuable lead compound for new anti-inflammatory drug development.

Show MeSH
Related in: MedlinePlus