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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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PKCδ signals PMA-induced p300 HAT activation.A). SF-Fb were transfected with a PKCδ siRNA or control scRNA followed by treatment with PMA (100 nM) for 4 h. p300 HAT was analyzed. Left panel: PKCδ proteins were analyzed by Western blotting. Right panel: Analysis of p300 HAT activity in siRNA vs. scRNA transfected SF-Fb. The error bars refer to mean ± SEM (n = 3). B). SF-Fb were treated with 5-MTP at increasing concentration followed by PMA (100 nM) for 4 h. Cell lysates were collected and the level of the indicated proteins was analyzed by Western blotting. “p-PKCδ” denotes phosphorylated PKCδ. Upper panels show representative blots and lower panel, densitometry of three experiments. Error bars refer to mean ± SEM (n = 3). β-actin was included as loading control.
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pone-0088507-g010: PKCδ signals PMA-induced p300 HAT activation.A). SF-Fb were transfected with a PKCδ siRNA or control scRNA followed by treatment with PMA (100 nM) for 4 h. p300 HAT was analyzed. Left panel: PKCδ proteins were analyzed by Western blotting. Right panel: Analysis of p300 HAT activity in siRNA vs. scRNA transfected SF-Fb. The error bars refer to mean ± SEM (n = 3). B). SF-Fb were treated with 5-MTP at increasing concentration followed by PMA (100 nM) for 4 h. Cell lysates were collected and the level of the indicated proteins was analyzed by Western blotting. “p-PKCδ” denotes phosphorylated PKCδ. Upper panels show representative blots and lower panel, densitometry of three experiments. Error bars refer to mean ± SEM (n = 3). β-actin was included as loading control.

Mentions: In view of the previously reported data indicating that COX-2 transcriptional activation by PMA is mediated via PKCδ [3], we investigated the role of PKCδ in PMA-induced p300 HAT activation. We transfected SF-Fb with PKCδ siRNA or a control scRNA and analyzed PMA-induced p300 HAT activity. PKCδ siRNA abrogated the stimulatory effect of PMA while scRNA had no effect (Fig. 10A). We next determined whether PKCδ activation by PMA is suppressed by 5-MTP. 5-MTP did not inhibit PKCδ proteins (Fig. 10B), nor did it significantly alter phosphorylated PKCδ (pPKCδ) (Fig. 10B). These results support a crucial role of PKCδ in mediating PMA-induced p300 HAT activation. However, 5-MTP inhibits PMA-induced p300 activation by a mechanism independent of PKCδ.


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)

PKCδ signals PMA-induced p300 HAT activation.A). SF-Fb were transfected with a PKCδ siRNA or control scRNA followed by treatment with PMA (100 nM) for 4 h. p300 HAT was analyzed. Left panel: PKCδ proteins were analyzed by Western blotting. Right panel: Analysis of p300 HAT activity in siRNA vs. scRNA transfected SF-Fb. The error bars refer to mean ± SEM (n = 3). B). SF-Fb were treated with 5-MTP at increasing concentration followed by PMA (100 nM) for 4 h. Cell lysates were collected and the level of the indicated proteins was analyzed by Western blotting. “p-PKCδ” denotes phosphorylated PKCδ. Upper panels show representative blots and lower panel, densitometry of three experiments. Error bars refer to mean ± SEM (n = 3). β-actin was included as loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088507-g010: PKCδ signals PMA-induced p300 HAT activation.A). SF-Fb were transfected with a PKCδ siRNA or control scRNA followed by treatment with PMA (100 nM) for 4 h. p300 HAT was analyzed. Left panel: PKCδ proteins were analyzed by Western blotting. Right panel: Analysis of p300 HAT activity in siRNA vs. scRNA transfected SF-Fb. The error bars refer to mean ± SEM (n = 3). B). SF-Fb were treated with 5-MTP at increasing concentration followed by PMA (100 nM) for 4 h. Cell lysates were collected and the level of the indicated proteins was analyzed by Western blotting. “p-PKCδ” denotes phosphorylated PKCδ. Upper panels show representative blots and lower panel, densitometry of three experiments. Error bars refer to mean ± SEM (n = 3). β-actin was included as loading control.
Mentions: In view of the previously reported data indicating that COX-2 transcriptional activation by PMA is mediated via PKCδ [3], we investigated the role of PKCδ in PMA-induced p300 HAT activation. We transfected SF-Fb with PKCδ siRNA or a control scRNA and analyzed PMA-induced p300 HAT activity. PKCδ siRNA abrogated the stimulatory effect of PMA while scRNA had no effect (Fig. 10A). We next determined whether PKCδ activation by PMA is suppressed by 5-MTP. 5-MTP did not inhibit PKCδ proteins (Fig. 10B), nor did it significantly alter phosphorylated PKCδ (pPKCδ) (Fig. 10B). These results support a crucial role of PKCδ in mediating PMA-induced p300 HAT activation. However, 5-MTP inhibits PMA-induced p300 activation by a mechanism independent of PKCδ.

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