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All-Trans Retinoic Acid Induces TGF-β2 in Intestinal Epithelial Cells via RhoA- and p38α MAPK-Mediated Activation of the Transcription Factor ATF2.

Namachivayam K, MohanKumar K, Arbach D, Jagadeeswaran R, Jain SK, Natarajan V, Mehta D, Jankov RP, Maheshwari A - PLoS ONE (2015)

Bottom Line: AtRA effects on intestinal epithelium were investigated using IEC6 cells.AtRA effects were mediated via RhoA GTPase, Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), p38α MAPK, and activating transcription factor (ATF)-2.AtRA increased phospho-ATF2 binding to the TGF-β2 promoter and increased histone H2B acetylation in the TGF-β2 nucleosome, which is typically associated with transcriptional activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois, United States of America; Department of Pediatrics, Morsani College of Medicine, University of South Florida, Tampa, Florida, United States of America.

ABSTRACT

Objective: We have shown previously that preterm infants are at risk of necrotizing enterocolitis (NEC), an inflammatory bowel necrosis typically seen in infants born prior to 32 weeks' gestation, because of the developmental deficiency of transforming growth factor (TGF)-β2 in the intestine. The present study was designed to investigate all-trans retinoic acid (atRA) as an inducer of TGF-β2 in intestinal epithelial cells (IECs) and to elucidate the involved signaling mechanisms.

Methods: AtRA effects on intestinal epithelium were investigated using IEC6 cells. TGF-β2 expression was measured using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Western blots. Signaling pathways were investigated using Western blots, transiently-transfected/transduced cells, kinase arrays, chromatin immunoprecipitation, and selective small molecule inhibitors.

Results: AtRA-treatment of IEC6 cells selectively increased TGF-β2 mRNA and protein expression in a time- and dose-dependent fashion, and increased the activity of the TGF-β2 promoter. AtRA effects were mediated via RhoA GTPase, Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), p38α MAPK, and activating transcription factor (ATF)-2. AtRA increased phospho-ATF2 binding to the TGF-β2 promoter and increased histone H2B acetylation in the TGF-β2 nucleosome, which is typically associated with transcriptional activation.

Conclusions: AtRA induces TGF-β2 expression in IECs via RhoA- and p38α MAPK-mediated activation of the transcription factor ATF2. Further studies are needed to investigate the role of atRA as a protective/therapeutic agent in gut mucosal inflammation.

No MeSH data available.


Related in: MedlinePlus

AtRA-induced TGF-β2 expression in IECs is mediated via ATF2.A. Western blots show phospho- and total ATF2 in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data normalized against β-actin. B. Fluorescence photomicrographs (magnification 630x) show increased nuclear localization of phospho-ATF2 (red) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI. C. AtRA treatment increases phospho-ATF2 binding to the TGF-β2 promoter. Bar diagram (means ± SE) shows data from chromatin immunoprecipitation (ChIP) assay of phospho-ATF2 binding to the TGF-β2 promoter region. Quantification of phospho-ATF2 binding was performed using real-time PCR and is shown as % input. To calculate % input, ChIP results for the specific antibody were determined using a standard curve of input DNA from the same cells. Control IgG ChIP results were subtracted from specific antibody ChIP results. Inset: Agarose gel showing enrichment of phospho-ATF2 on the TGF-β2 promoter. Data represent 3 separate experiments; * p<0.05, ** p<0.01.
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pone.0134003.g004: AtRA-induced TGF-β2 expression in IECs is mediated via ATF2.A. Western blots show phospho- and total ATF2 in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data normalized against β-actin. B. Fluorescence photomicrographs (magnification 630x) show increased nuclear localization of phospho-ATF2 (red) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI. C. AtRA treatment increases phospho-ATF2 binding to the TGF-β2 promoter. Bar diagram (means ± SE) shows data from chromatin immunoprecipitation (ChIP) assay of phospho-ATF2 binding to the TGF-β2 promoter region. Quantification of phospho-ATF2 binding was performed using real-time PCR and is shown as % input. To calculate % input, ChIP results for the specific antibody were determined using a standard curve of input DNA from the same cells. Control IgG ChIP results were subtracted from specific antibody ChIP results. Inset: Agarose gel showing enrichment of phospho-ATF2 on the TGF-β2 promoter. Data represent 3 separate experiments; * p<0.05, ** p<0.01.

Mentions: Existing evidence indicates that the transcriptional factor ATF2, which is known to be activated by atRA [32], is a key regulator of TGF-β2 expression [33]. Therefore, we next asked whether atRA activates ATF2 in IECs. As shown in Fig 4A, treatment of IEC6 cells with atRA promoted ATF2 phosphorylation (Thr71), starting within 15 min and lasting for nearly 96h with some oscillations. In support of these findings, we also detected increased nuclear immunoreactivity of phospho-ATF2 in cultured IEC6 cells (Fig 4B). Phospho-ATF2 binding to the TGF-β2 promoter in atRA-treated cells was confirmed by ChIP (Fig 4C).


All-Trans Retinoic Acid Induces TGF-β2 in Intestinal Epithelial Cells via RhoA- and p38α MAPK-Mediated Activation of the Transcription Factor ATF2.

Namachivayam K, MohanKumar K, Arbach D, Jagadeeswaran R, Jain SK, Natarajan V, Mehta D, Jankov RP, Maheshwari A - PLoS ONE (2015)

AtRA-induced TGF-β2 expression in IECs is mediated via ATF2.A. Western blots show phospho- and total ATF2 in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data normalized against β-actin. B. Fluorescence photomicrographs (magnification 630x) show increased nuclear localization of phospho-ATF2 (red) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI. C. AtRA treatment increases phospho-ATF2 binding to the TGF-β2 promoter. Bar diagram (means ± SE) shows data from chromatin immunoprecipitation (ChIP) assay of phospho-ATF2 binding to the TGF-β2 promoter region. Quantification of phospho-ATF2 binding was performed using real-time PCR and is shown as % input. To calculate % input, ChIP results for the specific antibody were determined using a standard curve of input DNA from the same cells. Control IgG ChIP results were subtracted from specific antibody ChIP results. Inset: Agarose gel showing enrichment of phospho-ATF2 on the TGF-β2 promoter. Data represent 3 separate experiments; * p<0.05, ** p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4520553&req=5

pone.0134003.g004: AtRA-induced TGF-β2 expression in IECs is mediated via ATF2.A. Western blots show phospho- and total ATF2 in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data normalized against β-actin. B. Fluorescence photomicrographs (magnification 630x) show increased nuclear localization of phospho-ATF2 (red) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI. C. AtRA treatment increases phospho-ATF2 binding to the TGF-β2 promoter. Bar diagram (means ± SE) shows data from chromatin immunoprecipitation (ChIP) assay of phospho-ATF2 binding to the TGF-β2 promoter region. Quantification of phospho-ATF2 binding was performed using real-time PCR and is shown as % input. To calculate % input, ChIP results for the specific antibody were determined using a standard curve of input DNA from the same cells. Control IgG ChIP results were subtracted from specific antibody ChIP results. Inset: Agarose gel showing enrichment of phospho-ATF2 on the TGF-β2 promoter. Data represent 3 separate experiments; * p<0.05, ** p<0.01.
Mentions: Existing evidence indicates that the transcriptional factor ATF2, which is known to be activated by atRA [32], is a key regulator of TGF-β2 expression [33]. Therefore, we next asked whether atRA activates ATF2 in IECs. As shown in Fig 4A, treatment of IEC6 cells with atRA promoted ATF2 phosphorylation (Thr71), starting within 15 min and lasting for nearly 96h with some oscillations. In support of these findings, we also detected increased nuclear immunoreactivity of phospho-ATF2 in cultured IEC6 cells (Fig 4B). Phospho-ATF2 binding to the TGF-β2 promoter in atRA-treated cells was confirmed by ChIP (Fig 4C).

Bottom Line: AtRA effects on intestinal epithelium were investigated using IEC6 cells.AtRA effects were mediated via RhoA GTPase, Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), p38α MAPK, and activating transcription factor (ATF)-2.AtRA increased phospho-ATF2 binding to the TGF-β2 promoter and increased histone H2B acetylation in the TGF-β2 nucleosome, which is typically associated with transcriptional activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois, United States of America; Department of Pediatrics, Morsani College of Medicine, University of South Florida, Tampa, Florida, United States of America.

ABSTRACT

Objective: We have shown previously that preterm infants are at risk of necrotizing enterocolitis (NEC), an inflammatory bowel necrosis typically seen in infants born prior to 32 weeks' gestation, because of the developmental deficiency of transforming growth factor (TGF)-β2 in the intestine. The present study was designed to investigate all-trans retinoic acid (atRA) as an inducer of TGF-β2 in intestinal epithelial cells (IECs) and to elucidate the involved signaling mechanisms.

Methods: AtRA effects on intestinal epithelium were investigated using IEC6 cells. TGF-β2 expression was measured using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Western blots. Signaling pathways were investigated using Western blots, transiently-transfected/transduced cells, kinase arrays, chromatin immunoprecipitation, and selective small molecule inhibitors.

Results: AtRA-treatment of IEC6 cells selectively increased TGF-β2 mRNA and protein expression in a time- and dose-dependent fashion, and increased the activity of the TGF-β2 promoter. AtRA effects were mediated via RhoA GTPase, Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), p38α MAPK, and activating transcription factor (ATF)-2. AtRA increased phospho-ATF2 binding to the TGF-β2 promoter and increased histone H2B acetylation in the TGF-β2 nucleosome, which is typically associated with transcriptional activation.

Conclusions: AtRA induces TGF-β2 expression in IECs via RhoA- and p38α MAPK-mediated activation of the transcription factor ATF2. Further studies are needed to investigate the role of atRA as a protective/therapeutic agent in gut mucosal inflammation.

No MeSH data available.


Related in: MedlinePlus