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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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Binding of transactivators to COX-2 promoter is reduced in pFb vs. SF-Fb.SF-Fb and pFb were treated with or without PMA (100 nM) for 4 h. A). Binding of transactivators to COX-2 promoter was analyzed by ChIP. The precipitated promoter DNA was measured by qPCR. The results were expressed as ratio (fold) of COX-2 promoter precipitated by each transactivator antibody to input DNA. COX-2 promoter precipitated by an non-immune IgG was included as a negative control. “▪ COX-2” denotes core promoter region and “□ control” denotes negative region. The error bars denote mean ± SEM of three independent experiments (n = 3). B). Transactivator proteins in nuclear extracts were analyzed by Western blotting. C) and D). Analysis of concurrent binding of transactivators (C) and p300 (D) to a COX-2 probe (denoted “COX-2”) or control probe (denoted “C”) by streptavidin agarose pulldown assay. D). p300 proteins in nuclear extract were analyzed by Western blotting. The upper panel shows a representative blot and the lower panel, densitometry of the blot. The error bars denote mean ± SEM (n = 3). “SF” denotes SF-Fb and “P” denotes pFb.
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pone-0088507-g001: Binding of transactivators to COX-2 promoter is reduced in pFb vs. SF-Fb.SF-Fb and pFb were treated with or without PMA (100 nM) for 4 h. A). Binding of transactivators to COX-2 promoter was analyzed by ChIP. The precipitated promoter DNA was measured by qPCR. The results were expressed as ratio (fold) of COX-2 promoter precipitated by each transactivator antibody to input DNA. COX-2 promoter precipitated by an non-immune IgG was included as a negative control. “▪ COX-2” denotes core promoter region and “□ control” denotes negative region. The error bars denote mean ± SEM of three independent experiments (n = 3). B). Transactivator proteins in nuclear extracts were analyzed by Western blotting. C) and D). Analysis of concurrent binding of transactivators (C) and p300 (D) to a COX-2 probe (denoted “COX-2”) or control probe (denoted “C”) by streptavidin agarose pulldown assay. D). p300 proteins in nuclear extract were analyzed by Western blotting. The upper panel shows a representative blot and the lower panel, densitometry of the blot. The error bars denote mean ± SEM (n = 3). “SF” denotes SF-Fb and “P” denotes pFb.

Mentions: To determine whether restricted COX-2 expression in proliferative fibroblasts (pFb) as compared to serum-free quiescent fibroblasts (SF-Fb) is due to reduced binding of a specific transactivator to COX-2 promoter, we analyzed binding of several functionally essential transactivators, i.e. NF-κB (p50/P65), C/EBPβ, c-Jun, CREB-2 to a core COX-2 promoter region by ChIP assay in pFb vs. SF-Fb treated with PMA. The precipitated promoter DNA was amplified and quantified with real-time qPCR. A COX-2 promoter region (−2150 ∼ −2030) without binding motifs for any of the transactivators was selected as a negative control. The data show that binding of all the transactivators was significantly lower in pFb than that in SF-Fb (Fig. 1A) while the nuclear transactivator protein levels were not different (Fig. 1B). Analysis of concurrent binding of transactivators by streptavidin-agarose pulldown assay confirmed global reduction of transactivators binding to a 500-bp COX-2 promoter probe in pFb vs. SF-Fb (Fig. 1C). These results indicate that restrained COX-2 transactivation in pFb is not due to blocking of a specific transactivator but attributed to suppression of a master regulator. We suspected that p300 is the target and, therefore, we next determined p300 binding to COX-2 promoter probe in pFb vs. SF-Fb. p300 binding to DNA-bound transactivators on COX-2 promoter was reduced in pFb compared to that in SF-Fb while nuclear p300 protein levels were not different (Fig 1D). These results suggest that p300 co-activator is involved in regulating the differential COX-2 trans-activation in pFb vs. SF-Fb.


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)

Binding of transactivators to COX-2 promoter is reduced in pFb vs. SF-Fb.SF-Fb and pFb were treated with or without PMA (100 nM) for 4 h. A). Binding of transactivators to COX-2 promoter was analyzed by ChIP. The precipitated promoter DNA was measured by qPCR. The results were expressed as ratio (fold) of COX-2 promoter precipitated by each transactivator antibody to input DNA. COX-2 promoter precipitated by an non-immune IgG was included as a negative control. “▪ COX-2” denotes core promoter region and “□ control” denotes negative region. The error bars denote mean ± SEM of three independent experiments (n = 3). B). Transactivator proteins in nuclear extracts were analyzed by Western blotting. C) and D). Analysis of concurrent binding of transactivators (C) and p300 (D) to a COX-2 probe (denoted “COX-2”) or control probe (denoted “C”) by streptavidin agarose pulldown assay. D). p300 proteins in nuclear extract were analyzed by Western blotting. The upper panel shows a representative blot and the lower panel, densitometry of the blot. The error bars denote mean ± SEM (n = 3). “SF” denotes SF-Fb and “P” denotes pFb.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3921189&req=5

pone-0088507-g001: Binding of transactivators to COX-2 promoter is reduced in pFb vs. SF-Fb.SF-Fb and pFb were treated with or without PMA (100 nM) for 4 h. A). Binding of transactivators to COX-2 promoter was analyzed by ChIP. The precipitated promoter DNA was measured by qPCR. The results were expressed as ratio (fold) of COX-2 promoter precipitated by each transactivator antibody to input DNA. COX-2 promoter precipitated by an non-immune IgG was included as a negative control. “▪ COX-2” denotes core promoter region and “□ control” denotes negative region. The error bars denote mean ± SEM of three independent experiments (n = 3). B). Transactivator proteins in nuclear extracts were analyzed by Western blotting. C) and D). Analysis of concurrent binding of transactivators (C) and p300 (D) to a COX-2 probe (denoted “COX-2”) or control probe (denoted “C”) by streptavidin agarose pulldown assay. D). p300 proteins in nuclear extract were analyzed by Western blotting. The upper panel shows a representative blot and the lower panel, densitometry of the blot. The error bars denote mean ± SEM (n = 3). “SF” denotes SF-Fb and “P” denotes pFb.
Mentions: To determine whether restricted COX-2 expression in proliferative fibroblasts (pFb) as compared to serum-free quiescent fibroblasts (SF-Fb) is due to reduced binding of a specific transactivator to COX-2 promoter, we analyzed binding of several functionally essential transactivators, i.e. NF-κB (p50/P65), C/EBPβ, c-Jun, CREB-2 to a core COX-2 promoter region by ChIP assay in pFb vs. SF-Fb treated with PMA. The precipitated promoter DNA was amplified and quantified with real-time qPCR. A COX-2 promoter region (−2150 ∼ −2030) without binding motifs for any of the transactivators was selected as a negative control. The data show that binding of all the transactivators was significantly lower in pFb than that in SF-Fb (Fig. 1A) while the nuclear transactivator protein levels were not different (Fig. 1B). Analysis of concurrent binding of transactivators by streptavidin-agarose pulldown assay confirmed global reduction of transactivators binding to a 500-bp COX-2 promoter probe in pFb vs. SF-Fb (Fig. 1C). These results indicate that restrained COX-2 transactivation in pFb is not due to blocking of a specific transactivator but attributed to suppression of a master regulator. We suspected that p300 is the target and, therefore, we next determined p300 binding to COX-2 promoter probe in pFb vs. SF-Fb. p300 binding to DNA-bound transactivators on COX-2 promoter was reduced in pFb compared to that in SF-Fb while nuclear p300 protein levels were not different (Fig 1D). These results suggest that p300 co-activator is involved in regulating the differential COX-2 trans-activation in pFb vs. SF-Fb.

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