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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 induces TGF-β2 expression in IECs.A. Bar-diagram (means ± SE) shows fold change in mRNA expression of TGF-β1, TGF-β2, and TGF-β3 in IEC6 cells treated with atRA (10 μM) × 24h. Inset: Rat pups receiving atRA orally show increased TGF-β2 expression in the small intestine on postnatal day 7. Bar-diagram (means ± SE) shows fold change in TGF-β2 mRNA. N = 5 pups per group. B. Temporal kinetics of atRA-induced TGF-β2 expression in IEC6 cells. Line diagram (means ± SE) shows fold changes in TGF-β2 mRNA expression over cells cultured in media alone, depicted as a function of the duration of atRA treatment. C. Western blots show atRA-induced TGF-β2 protein expression in IEC6 cells. Bar-diagram (means ± SE) summarizes densitometric data. Inset: Western blots showing increased TGF-β2 expression in atRA-treated T84 and HT29 human IECs, respectively. D. AtRA activates TGF-β2 promoter in IEC6 cells. Bar-diagram (means ± SE) shows relative luciferase activity in IEC6 cells transfected with a luciferase reporter containing the TGF-β2 promoter, 8h after treatment with ATRA. Data represent 3 separate experiments; * p<0.05, ** p<0.01.
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pone.0134003.g001: AtRA induces TGF-β2 expression in IECs.A. Bar-diagram (means ± SE) shows fold change in mRNA expression of TGF-β1, TGF-β2, and TGF-β3 in IEC6 cells treated with atRA (10 μM) × 24h. Inset: Rat pups receiving atRA orally show increased TGF-β2 expression in the small intestine on postnatal day 7. Bar-diagram (means ± SE) shows fold change in TGF-β2 mRNA. N = 5 pups per group. B. Temporal kinetics of atRA-induced TGF-β2 expression in IEC6 cells. Line diagram (means ± SE) shows fold changes in TGF-β2 mRNA expression over cells cultured in media alone, depicted as a function of the duration of atRA treatment. C. Western blots show atRA-induced TGF-β2 protein expression in IEC6 cells. Bar-diagram (means ± SE) summarizes densitometric data. Inset: Western blots showing increased TGF-β2 expression in atRA-treated T84 and HT29 human IECs, respectively. D. AtRA activates TGF-β2 promoter in IEC6 cells. Bar-diagram (means ± SE) shows relative luciferase activity in IEC6 cells transfected with a luciferase reporter containing the TGF-β2 promoter, 8h after treatment with ATRA. Data represent 3 separate experiments; * p<0.05, ** p<0.01.

Mentions: To investigate retinoic acid effects on IECs, we treated IEC6 rat intestinal epithelial cells with atRA in vitro. IEC6 cells are primary IECs that were originally derived from duodenal crypt IECs from 18–24-day-old rat pups [25]. AtRA (1–10 μM) selectively induced the TGF-β2 isoform in IEC6 cells (Fig 1A). In support of these data, we detected a similar increase in TGF-β2 expression in the intestine in rat pups fed atRA (500 μg) mixed in peanut oil from postnatal day 3 through day 7 (inset).


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 induces TGF-β2 expression in IECs.A. Bar-diagram (means ± SE) shows fold change in mRNA expression of TGF-β1, TGF-β2, and TGF-β3 in IEC6 cells treated with atRA (10 μM) × 24h. Inset: Rat pups receiving atRA orally show increased TGF-β2 expression in the small intestine on postnatal day 7. Bar-diagram (means ± SE) shows fold change in TGF-β2 mRNA. N = 5 pups per group. B. Temporal kinetics of atRA-induced TGF-β2 expression in IEC6 cells. Line diagram (means ± SE) shows fold changes in TGF-β2 mRNA expression over cells cultured in media alone, depicted as a function of the duration of atRA treatment. C. Western blots show atRA-induced TGF-β2 protein expression in IEC6 cells. Bar-diagram (means ± SE) summarizes densitometric data. Inset: Western blots showing increased TGF-β2 expression in atRA-treated T84 and HT29 human IECs, respectively. D. AtRA activates TGF-β2 promoter in IEC6 cells. Bar-diagram (means ± SE) shows relative luciferase activity in IEC6 cells transfected with a luciferase reporter containing the TGF-β2 promoter, 8h after treatment with ATRA. Data represent 3 separate experiments; * p<0.05, ** p<0.01.
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pone.0134003.g001: AtRA induces TGF-β2 expression in IECs.A. Bar-diagram (means ± SE) shows fold change in mRNA expression of TGF-β1, TGF-β2, and TGF-β3 in IEC6 cells treated with atRA (10 μM) × 24h. Inset: Rat pups receiving atRA orally show increased TGF-β2 expression in the small intestine on postnatal day 7. Bar-diagram (means ± SE) shows fold change in TGF-β2 mRNA. N = 5 pups per group. B. Temporal kinetics of atRA-induced TGF-β2 expression in IEC6 cells. Line diagram (means ± SE) shows fold changes in TGF-β2 mRNA expression over cells cultured in media alone, depicted as a function of the duration of atRA treatment. C. Western blots show atRA-induced TGF-β2 protein expression in IEC6 cells. Bar-diagram (means ± SE) summarizes densitometric data. Inset: Western blots showing increased TGF-β2 expression in atRA-treated T84 and HT29 human IECs, respectively. D. AtRA activates TGF-β2 promoter in IEC6 cells. Bar-diagram (means ± SE) shows relative luciferase activity in IEC6 cells transfected with a luciferase reporter containing the TGF-β2 promoter, 8h after treatment with ATRA. Data represent 3 separate experiments; * p<0.05, ** p<0.01.
Mentions: To investigate retinoic acid effects on IECs, we treated IEC6 rat intestinal epithelial cells with atRA in vitro. IEC6 cells are primary IECs that were originally derived from duodenal crypt IECs from 18–24-day-old rat pups [25]. AtRA (1–10 μM) selectively induced the TGF-β2 isoform in IEC6 cells (Fig 1A). In support of these data, we detected a similar increase in TGF-β2 expression in the intestine in rat pups fed atRA (500 μg) mixed in peanut oil from postnatal day 3 through day 7 (inset).

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