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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 suppression in pFb is reversed by silencing of 5-MTP synthetic enzymes.5-MTP synthesis in pFb (P-Fb) is catalyzed by tryptophan hydroxylase-1 (TPH-1) and hydroxyindole o-methyltransferase (HIOMT). A) and B). pFb transfected with TPH-1 siRNA or control scRNA were treated with PMA for 4 h. A). TPH-1 proteins were analyzed by Western blotting. B). p300 proteins were isolated by IP and HAT activity was measured. siRNA but not scRNA treatment resulted in p300 HAT elevation to a level comparable to that in SF-Fb. Addition of 5-HTP (10 µM) for 30 min restored p300 HAT suppression. Error bars are mean ± SEM (n = 3). C) and D). pFb transfected with HIOMT siRNA or scRNA were treated with PMA. C). HIOMT protein analysis by Western blotting. D). p300 HAT analysis. HIOMT siRNA abrogated p300 HAT suppression which was restored by addition of 5-MTP (10 µM) for 30 min. The error bars denote mean ± SEM (n = 3).
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pone-0088507-g006: p300 HAT suppression in pFb is reversed by silencing of 5-MTP synthetic enzymes.5-MTP synthesis in pFb (P-Fb) is catalyzed by tryptophan hydroxylase-1 (TPH-1) and hydroxyindole o-methyltransferase (HIOMT). A) and B). pFb transfected with TPH-1 siRNA or control scRNA were treated with PMA for 4 h. A). TPH-1 proteins were analyzed by Western blotting. B). p300 proteins were isolated by IP and HAT activity was measured. siRNA but not scRNA treatment resulted in p300 HAT elevation to a level comparable to that in SF-Fb. Addition of 5-HTP (10 µM) for 30 min restored p300 HAT suppression. Error bars are mean ± SEM (n = 3). C) and D). pFb transfected with HIOMT siRNA or scRNA were treated with PMA. C). HIOMT protein analysis by Western blotting. D). p300 HAT analysis. HIOMT siRNA abrogated p300 HAT suppression which was restored by addition of 5-MTP (10 µM) for 30 min. The error bars denote mean ± SEM (n = 3).

Mentions: We have recently identified 5-MTP as a COX-2 suppressing cytoguardin [6]. 5-MTP production in pFb was shown to be catalyzed by tryptophan hydroxylase-1 (TPH-1) which converts L-tryptophan to 5-hydroxytryptophan (5-HTP) and hydroxyindole O-methyltransferase (HIOMT) which converts 5-HTP to 5-MTP [6]. To determine that 5-MTP production is responsible for control of p300 HAT activation, we silenced TPH-1 with siRNA (Fig. 6A) which abrogated p300 HAT suppression in pFb (Fig. 6B). PMA-induced p300 HAT activity in TPH-1 siRNA-treated pFb was increased to the level detected in SF-Fb (Fig. 6B). The control scRNA did not alter the p300 HAT in pFb (Fig. 6B). Interestingly, addition of 5-HTP restored p300 HAT suppression despite TPH-1 siRNA treatment (Fig 6B). HIOMT siRNA which inhibited HIOMT protein expression (Fig. 6C) also abrogated p300 HAT suppression in pFb and 5-MTP restored p300 HAT inhibition (Fig. 6D). These results confirm the essential role of 5-MTP production in conferring control of proinflammatory mediator-induced p300 HAT activation and COX-2 expression in proliferating fibroblasts.


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 suppression in pFb is reversed by silencing of 5-MTP synthetic enzymes.5-MTP synthesis in pFb (P-Fb) is catalyzed by tryptophan hydroxylase-1 (TPH-1) and hydroxyindole o-methyltransferase (HIOMT). A) and B). pFb transfected with TPH-1 siRNA or control scRNA were treated with PMA for 4 h. A). TPH-1 proteins were analyzed by Western blotting. B). p300 proteins were isolated by IP and HAT activity was measured. siRNA but not scRNA treatment resulted in p300 HAT elevation to a level comparable to that in SF-Fb. Addition of 5-HTP (10 µM) for 30 min restored p300 HAT suppression. Error bars are mean ± SEM (n = 3). C) and D). pFb transfected with HIOMT siRNA or scRNA were treated with PMA. C). HIOMT protein analysis by Western blotting. D). p300 HAT analysis. HIOMT siRNA abrogated p300 HAT suppression which was restored by addition of 5-MTP (10 µM) for 30 min. The error bars denote mean ± SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3921189&req=5

pone-0088507-g006: p300 HAT suppression in pFb is reversed by silencing of 5-MTP synthetic enzymes.5-MTP synthesis in pFb (P-Fb) is catalyzed by tryptophan hydroxylase-1 (TPH-1) and hydroxyindole o-methyltransferase (HIOMT). A) and B). pFb transfected with TPH-1 siRNA or control scRNA were treated with PMA for 4 h. A). TPH-1 proteins were analyzed by Western blotting. B). p300 proteins were isolated by IP and HAT activity was measured. siRNA but not scRNA treatment resulted in p300 HAT elevation to a level comparable to that in SF-Fb. Addition of 5-HTP (10 µM) for 30 min restored p300 HAT suppression. Error bars are mean ± SEM (n = 3). C) and D). pFb transfected with HIOMT siRNA or scRNA were treated with PMA. C). HIOMT protein analysis by Western blotting. D). p300 HAT analysis. HIOMT siRNA abrogated p300 HAT suppression which was restored by addition of 5-MTP (10 µM) for 30 min. The error bars denote mean ± SEM (n = 3).
Mentions: We have recently identified 5-MTP as a COX-2 suppressing cytoguardin [6]. 5-MTP production in pFb was shown to be catalyzed by tryptophan hydroxylase-1 (TPH-1) which converts L-tryptophan to 5-hydroxytryptophan (5-HTP) and hydroxyindole O-methyltransferase (HIOMT) which converts 5-HTP to 5-MTP [6]. To determine that 5-MTP production is responsible for control of p300 HAT activation, we silenced TPH-1 with siRNA (Fig. 6A) which abrogated p300 HAT suppression in pFb (Fig. 6B). PMA-induced p300 HAT activity in TPH-1 siRNA-treated pFb was increased to the level detected in SF-Fb (Fig. 6B). The control scRNA did not alter the p300 HAT in pFb (Fig. 6B). Interestingly, addition of 5-HTP restored p300 HAT suppression despite TPH-1 siRNA treatment (Fig 6B). HIOMT siRNA which inhibited HIOMT protein expression (Fig. 6C) also abrogated p300 HAT suppression in pFb and 5-MTP restored p300 HAT inhibition (Fig. 6D). These results confirm the essential role of 5-MTP production in conferring control of proinflammatory mediator-induced p300 HAT activation and COX-2 expression in proliferating fibroblasts.

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