Limits...
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.

Show MeSH

Related in: MedlinePlus

5-MTP blocks PMA-induced p300 HAT activity in SF-Fb.A). SF-Fb were pretreated with 5-MTP at indicated concentrations for 30 min followed by PMA for 4 h. p300 HAT activity was measured. The error bars show mean ± SEM (n = 3). B). SF-Fb were treated with 5-MTP (10 µM) in the absence of PMA (left panel) or presence of PMA (right panel). Right panel also shows treatment of pFb with or without 5-MTP followed by PMA. Each error bar refers to mean ± SEM (n = 3). “NS” denotes statistically non-significant. C). SF-Fb were pretreated with 5-MTP (10 µM) for 30 min followed by PMA (100 nM) for 4 h. COX-2 proteins were analyzed with Western blotting. This Western blot is representative of two experiments with similar results. D). SF-Fb or pFb were treated with or without PMA (100 nM) for 4 h. p300 proteins were isolated by IP, washed and treated with 5-MTP (10 µM) for 30 min. HAT activity was measured. The error bars refer to mean ± SEM (n = 3). “NS” denotes statistically non-significant.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3921189&req=5

pone-0088507-g009: 5-MTP blocks PMA-induced p300 HAT activity in SF-Fb.A). SF-Fb were pretreated with 5-MTP at indicated concentrations for 30 min followed by PMA for 4 h. p300 HAT activity was measured. The error bars show mean ± SEM (n = 3). B). SF-Fb were treated with 5-MTP (10 µM) in the absence of PMA (left panel) or presence of PMA (right panel). Right panel also shows treatment of pFb with or without 5-MTP followed by PMA. Each error bar refers to mean ± SEM (n = 3). “NS” denotes statistically non-significant. C). SF-Fb were pretreated with 5-MTP (10 µM) for 30 min followed by PMA (100 nM) for 4 h. COX-2 proteins were analyzed with Western blotting. This Western blot is representative of two experiments with similar results. D). SF-Fb or pFb were treated with or without PMA (100 nM) for 4 h. p300 proteins were isolated by IP, washed and treated with 5-MTP (10 µM) for 30 min. HAT activity was measured. The error bars refer to mean ± SEM (n = 3). “NS” denotes statistically non-significant.

Mentions: To provide further evidence for the control of p300 HAT by 5-MTP, we evaluated the effect of 5-MTP on PMA-induced p300 HAT activity. 5-MTP inhibited PMA-induced p300 HAT activity in SF-Fb in a concentration-dependent manner (Fig. 9A) and at 10 µM it reduced p300 HAT activity by >50% without influencing the basal activity (Fig. 9B). By contrast, 5-MTP did not inhibit PMA-induced p300 HAT activity in pFb (Fig. 9B). Consistent with our previous report [6], 5-MTP inhibited PMA-induced COX-2 protein expression in a manner correlated with its inhibition of p300 HAT activation (Fig. 9C). It is worthy noting that 5-HTP inhibits p300 HAT activation and COX-2 expression at concentrations comparable to 5-MTP (Fig. 8B and C vs. Fig. 9A-C), consistent with the interpretation that 5-HTP exerts its effect via 5-MTP. Taken together, these findings confirm that robust p300 HAT activation and COX-2 expression in SF-Fb is attributed to deficiency of 5-MTP production whereas restrained p300 HAT activation and COX-2 expression in pFb is contributed by abundant 5-MTP production.


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)

5-MTP blocks PMA-induced p300 HAT activity in SF-Fb.A). SF-Fb were pretreated with 5-MTP at indicated concentrations for 30 min followed by PMA for 4 h. p300 HAT activity was measured. The error bars show mean ± SEM (n = 3). B). SF-Fb were treated with 5-MTP (10 µM) in the absence of PMA (left panel) or presence of PMA (right panel). Right panel also shows treatment of pFb with or without 5-MTP followed by PMA. Each error bar refers to mean ± SEM (n = 3). “NS” denotes statistically non-significant. C). SF-Fb were pretreated with 5-MTP (10 µM) for 30 min followed by PMA (100 nM) for 4 h. COX-2 proteins were analyzed with Western blotting. This Western blot is representative of two experiments with similar results. D). SF-Fb or pFb were treated with or without PMA (100 nM) for 4 h. p300 proteins were isolated by IP, washed and treated with 5-MTP (10 µM) for 30 min. HAT activity was measured. The error bars refer to mean ± SEM (n = 3). “NS” denotes statistically non-significant.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088507-g009: 5-MTP blocks PMA-induced p300 HAT activity in SF-Fb.A). SF-Fb were pretreated with 5-MTP at indicated concentrations for 30 min followed by PMA for 4 h. p300 HAT activity was measured. The error bars show mean ± SEM (n = 3). B). SF-Fb were treated with 5-MTP (10 µM) in the absence of PMA (left panel) or presence of PMA (right panel). Right panel also shows treatment of pFb with or without 5-MTP followed by PMA. Each error bar refers to mean ± SEM (n = 3). “NS” denotes statistically non-significant. C). SF-Fb were pretreated with 5-MTP (10 µM) for 30 min followed by PMA (100 nM) for 4 h. COX-2 proteins were analyzed with Western blotting. This Western blot is representative of two experiments with similar results. D). SF-Fb or pFb were treated with or without PMA (100 nM) for 4 h. p300 proteins were isolated by IP, washed and treated with 5-MTP (10 µM) for 30 min. HAT activity was measured. The error bars refer to mean ± SEM (n = 3). “NS” denotes statistically non-significant.
Mentions: To provide further evidence for the control of p300 HAT by 5-MTP, we evaluated the effect of 5-MTP on PMA-induced p300 HAT activity. 5-MTP inhibited PMA-induced p300 HAT activity in SF-Fb in a concentration-dependent manner (Fig. 9A) and at 10 µM it reduced p300 HAT activity by >50% without influencing the basal activity (Fig. 9B). By contrast, 5-MTP did not inhibit PMA-induced p300 HAT activity in pFb (Fig. 9B). Consistent with our previous report [6], 5-MTP inhibited PMA-induced COX-2 protein expression in a manner correlated with its inhibition of p300 HAT activation (Fig. 9C). It is worthy noting that 5-HTP inhibits p300 HAT activation and COX-2 expression at concentrations comparable to 5-MTP (Fig. 8B and C vs. Fig. 9A-C), consistent with the interpretation that 5-HTP exerts its effect via 5-MTP. Taken together, these findings confirm that robust p300 HAT activation and COX-2 expression in SF-Fb is attributed to deficiency of 5-MTP production whereas restrained p300 HAT activation and COX-2 expression in pFb is contributed by abundant 5-MTP production.

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