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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 activity in SF-Fb is suppressed by conditioned medium (CM) and CMF2.A). SF-Fb were washed and incubated with CM from proliferative fibroblasts (pFb-CM), from quiescent fibroblasts (SF-Fb-CM) or fresh medium containing 2.5% FBS (fresh medium) for 24 h followed by treatment with PMA (100 nM) for 4 h. Each error bar denotes mean ± SEM (n = 3). B). Diluted & undiluted CMF2 prepared from pFb-CM was incubated for 24 h with washed SF-Fb transfected with p300 vectors. PMA-induced p300 HAT activity was measured. “Undil” denotes undiluted CMF2 while “1∶10” and “1∶2” denote dilution of CMF2 with medium. “0” denotes without CMF2. Each error bar denotes mean ± SEM (n = 3).
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pone-0088507-g005: p300 HAT activity in SF-Fb is suppressed by conditioned medium (CM) and CMF2.A). SF-Fb were washed and incubated with CM from proliferative fibroblasts (pFb-CM), from quiescent fibroblasts (SF-Fb-CM) or fresh medium containing 2.5% FBS (fresh medium) for 24 h followed by treatment with PMA (100 nM) for 4 h. Each error bar denotes mean ± SEM (n = 3). B). Diluted & undiluted CMF2 prepared from pFb-CM was incubated for 24 h with washed SF-Fb transfected with p300 vectors. PMA-induced p300 HAT activity was measured. “Undil” denotes undiluted CMF2 while “1∶10” and “1∶2” denote dilution of CMF2 with medium. “0” denotes without CMF2. Each error bar denotes mean ± SEM (n = 3).

Mentions: Conditioned medium of pFb (pFb-CM) was reported to contain soluble factors (named cytoguardin) which suppress COX-2 transcriptional activation by proinflammatory mediators [5]. We determined whether pFb-CM exerted an effect on p300 HAT activation. pFb-CM inhibited PMA-induced p300 HAT activation in SF-Fb by ∼50% while SF-CM did not (Fig. 5A). Nor did the control medium (Fig 5A). We prepared CMF2 fraction from pFb-CM by several purification steps and tested its ability to control p300 HAT activation in SF-Fb. Lyophilized CMF2 was serially diluted and added to washed SF-Fb. CMF2 inhibited PMA-induced p300 HAT in a concentration dependent manner (Fig. 5B) These results suggest that cytoguardins released by pFb possess inhibitory action on p300 HAT activation.


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 activity in SF-Fb is suppressed by conditioned medium (CM) and CMF2.A). SF-Fb were washed and incubated with CM from proliferative fibroblasts (pFb-CM), from quiescent fibroblasts (SF-Fb-CM) or fresh medium containing 2.5% FBS (fresh medium) for 24 h followed by treatment with PMA (100 nM) for 4 h. Each error bar denotes mean ± SEM (n = 3). B). Diluted & undiluted CMF2 prepared from pFb-CM was incubated for 24 h with washed SF-Fb transfected with p300 vectors. PMA-induced p300 HAT activity was measured. “Undil” denotes undiluted CMF2 while “1∶10” and “1∶2” denote dilution of CMF2 with medium. “0” denotes without CMF2. Each error bar denotes mean ± SEM (n = 3).
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Related In: Results  -  Collection

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pone-0088507-g005: p300 HAT activity in SF-Fb is suppressed by conditioned medium (CM) and CMF2.A). SF-Fb were washed and incubated with CM from proliferative fibroblasts (pFb-CM), from quiescent fibroblasts (SF-Fb-CM) or fresh medium containing 2.5% FBS (fresh medium) for 24 h followed by treatment with PMA (100 nM) for 4 h. Each error bar denotes mean ± SEM (n = 3). B). Diluted & undiluted CMF2 prepared from pFb-CM was incubated for 24 h with washed SF-Fb transfected with p300 vectors. PMA-induced p300 HAT activity was measured. “Undil” denotes undiluted CMF2 while “1∶10” and “1∶2” denote dilution of CMF2 with medium. “0” denotes without CMF2. Each error bar denotes mean ± SEM (n = 3).
Mentions: Conditioned medium of pFb (pFb-CM) was reported to contain soluble factors (named cytoguardin) which suppress COX-2 transcriptional activation by proinflammatory mediators [5]. We determined whether pFb-CM exerted an effect on p300 HAT activation. pFb-CM inhibited PMA-induced p300 HAT activation in SF-Fb by ∼50% while SF-CM did not (Fig. 5A). Nor did the control medium (Fig 5A). We prepared CMF2 fraction from pFb-CM by several purification steps and tested its ability to control p300 HAT activation in SF-Fb. Lyophilized CMF2 was serially diluted and added to washed SF-Fb. CMF2 inhibited PMA-induced p300 HAT in a concentration dependent manner (Fig. 5B) These results suggest that cytoguardins released by pFb possess inhibitory action on p300 HAT activation.

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