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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 promotes the acetylation of histone H2B in the TGF-β2 nucleosome.A. Western blots show the genome-wide acetylation status of histones H2A, H2B, H3, and H4. Bar-diagram (means ± SE) summarizes densitometric data normalized against β-actin. B. AtRA treatment increases the acetylation of histone H2B (lys5) on the TGF-β2 nucleosome. Bar diagram (means ± SE) shows data from ChIP assay, where the acetylated histones were pulled down and the presence of the TGF-β2 promoter region in the complex confirmed by real-time PCR. Quantification of acetyl-H2B is shown as % input. Inset: Agarose gel showing enrichment of acetyl-H2B in the TGF-β2 promoter. Data represent 3 separate experiments; * p<0.05, *** p<0.001.
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pone.0134003.g005: AtRA promotes the acetylation of histone H2B in the TGF-β2 nucleosome.A. Western blots show the genome-wide acetylation status of histones H2A, H2B, H3, and H4. Bar-diagram (means ± SE) summarizes densitometric data normalized against β-actin. B. AtRA treatment increases the acetylation of histone H2B (lys5) on the TGF-β2 nucleosome. Bar diagram (means ± SE) shows data from ChIP assay, where the acetylated histones were pulled down and the presence of the TGF-β2 promoter region in the complex confirmed by real-time PCR. Quantification of acetyl-H2B is shown as % input. Inset: Agarose gel showing enrichment of acetyl-H2B in the TGF-β2 promoter. Data represent 3 separate experiments; * p<0.05, *** p<0.001.

Mentions: We have already shown that atRA increased TGF-β2 promoter activity (Fig 1D). To support these data, we asked whether treatment of IECs with atRA resulted in histone modifications typically associated with transcriptional activation [34]. Therefore, we sought acetyl-H2AK5, acetyl-H2BK5, acetyl-H3K9, and acetyl-H4K8 first in the entire chromatin and then specifically on the nucleosome of TGF-β2. In Western blots (Fig 5A), we detected a global increase in acetyl-H2AK5 and acetyl-H2BK5 in atRA-treated IEC6 cells. To confirm the presence of these modifications specifically on the TGF-β2 nucleosome, we treated IEC6 cells with atRA and performed ChIP, where nuclear extracts of formaldehyde-fixed cells were sonicated and then subjected to immunoprecipitation using anti-acetyl-H2AK5, anti-acetyl-H2BK5, or control IgG. As depicted in Fig 5B, quantitative PCR confirmed that atRA promoted H2BK5 acetylation on the TGF-β2 nucleosome in IEC6 cells.


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 promotes the acetylation of histone H2B in the TGF-β2 nucleosome.A. Western blots show the genome-wide acetylation status of histones H2A, H2B, H3, and H4. Bar-diagram (means ± SE) summarizes densitometric data normalized against β-actin. B. AtRA treatment increases the acetylation of histone H2B (lys5) on the TGF-β2 nucleosome. Bar diagram (means ± SE) shows data from ChIP assay, where the acetylated histones were pulled down and the presence of the TGF-β2 promoter region in the complex confirmed by real-time PCR. Quantification of acetyl-H2B is shown as % input. Inset: Agarose gel showing enrichment of acetyl-H2B in the TGF-β2 promoter. Data represent 3 separate experiments; * p<0.05, *** p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4520553&req=5

pone.0134003.g005: AtRA promotes the acetylation of histone H2B in the TGF-β2 nucleosome.A. Western blots show the genome-wide acetylation status of histones H2A, H2B, H3, and H4. Bar-diagram (means ± SE) summarizes densitometric data normalized against β-actin. B. AtRA treatment increases the acetylation of histone H2B (lys5) on the TGF-β2 nucleosome. Bar diagram (means ± SE) shows data from ChIP assay, where the acetylated histones were pulled down and the presence of the TGF-β2 promoter region in the complex confirmed by real-time PCR. Quantification of acetyl-H2B is shown as % input. Inset: Agarose gel showing enrichment of acetyl-H2B in the TGF-β2 promoter. Data represent 3 separate experiments; * p<0.05, *** p<0.001.
Mentions: We have already shown that atRA increased TGF-β2 promoter activity (Fig 1D). To support these data, we asked whether treatment of IECs with atRA resulted in histone modifications typically associated with transcriptional activation [34]. Therefore, we sought acetyl-H2AK5, acetyl-H2BK5, acetyl-H3K9, and acetyl-H4K8 first in the entire chromatin and then specifically on the nucleosome of TGF-β2. In Western blots (Fig 5A), we detected a global increase in acetyl-H2AK5 and acetyl-H2BK5 in atRA-treated IEC6 cells. To confirm the presence of these modifications specifically on the TGF-β2 nucleosome, we treated IEC6 cells with atRA and performed ChIP, where nuclear extracts of formaldehyde-fixed cells were sonicated and then subjected to immunoprecipitation using anti-acetyl-H2AK5, anti-acetyl-H2BK5, or control IgG. As depicted in Fig 5B, quantitative PCR confirmed that atRA promoted H2BK5 acetylation on the TGF-β2 nucleosome in IEC6 cells.

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