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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 RhoA GTPase and ROCK1.A. Representative Western blots show increased expression of activated RhoA (RhoA-GTP) in IEC6 cells treated with atRA × 4h. Activated RhoA was pulled down from cell lysates using Rhotekin-agarose beads. Bar-diagram (means ± SE) summarizes densitometric data. Inset: Left panel: ATRA also increased the expression of total RhoA in IECs. Right panel: AtRA-treatment did not increase Rac1-GTP in IEC6 cells. B. Western blots show that atRA-induced TGF-β2 expression in IEC6 cells was reproduced by over-expression of the constitutively-active GL4V mutant of RhoA. C. Cells expressing the TN19 dominant-negative RhoA mutant did not show atRA-induced TGF-β2 expression. D. Western blots show cleaved ROCK1 in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data. Inset: Fluorescence photomicrographs (magnification 630x) show nuclear localization of ROCK1 (green) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI (blue). E. Pharmacological inhibition of ROCK1 by Y-27632 blocked atRA-induced TGF-β2 expression in IEC6 cells. Western blots show TGF-β2 and cleaved ROCK1 expression. Bar-diagram (means ± SE) summarizes densitometric data, normalized against β-actin. Data represent 3 separate experiments; * p<0.05 compared to cells cultured in media alone; # indicates p<0.05 compared to atRA-treated cells.
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pone.0134003.g002: AtRA-induced TGF-β2 expression in IECs is mediated via RhoA GTPase and ROCK1.A. Representative Western blots show increased expression of activated RhoA (RhoA-GTP) in IEC6 cells treated with atRA × 4h. Activated RhoA was pulled down from cell lysates using Rhotekin-agarose beads. Bar-diagram (means ± SE) summarizes densitometric data. Inset: Left panel: ATRA also increased the expression of total RhoA in IECs. Right panel: AtRA-treatment did not increase Rac1-GTP in IEC6 cells. B. Western blots show that atRA-induced TGF-β2 expression in IEC6 cells was reproduced by over-expression of the constitutively-active GL4V mutant of RhoA. C. Cells expressing the TN19 dominant-negative RhoA mutant did not show atRA-induced TGF-β2 expression. D. Western blots show cleaved ROCK1 in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data. Inset: Fluorescence photomicrographs (magnification 630x) show nuclear localization of ROCK1 (green) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI (blue). E. Pharmacological inhibition of ROCK1 by Y-27632 blocked atRA-induced TGF-β2 expression in IEC6 cells. Western blots show TGF-β2 and cleaved ROCK1 expression. Bar-diagram (means ± SE) summarizes densitometric data, normalized against β-actin. Data represent 3 separate experiments; * p<0.05 compared to cells cultured in media alone; # indicates p<0.05 compared to atRA-treated cells.

Mentions: Existing information indicates that atRA differentially activates the small GTPases RhoA, rac1, and cdc42 in different cell lineages [26–28]. To determine whether atRA activates one or more of these mediators in IECs, we used Rhotekin RBD- and PAK PBD-conjugated agarose beads to pull down the active forms of RhoA, rac1, and cdc42 from cell lysates and measured these GTPases in Western blots. As shown in Fig 2A, atRA increased active RhoA (RhoA-GTP) in IEC6 cells. There was no change in active Rac1, and we did not detect activated Cdc42 in atRA-treated cells. Consistent with these findings, IEC6 cells expressing the constitutively-active GL4V mutant of RhoA showed increased TGF-β2 expression similar to the effects of atRA (Fig 2B). In contrast, we did not find TGF-β2 expression in cells expressing the dominant-negative TN19 RhoA mutant, emphasizing the importance of RhoA in constitutive and atRA-induced TGF-β2 expression (Fig 2C).


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 RhoA GTPase and ROCK1.A. Representative Western blots show increased expression of activated RhoA (RhoA-GTP) in IEC6 cells treated with atRA × 4h. Activated RhoA was pulled down from cell lysates using Rhotekin-agarose beads. Bar-diagram (means ± SE) summarizes densitometric data. Inset: Left panel: ATRA also increased the expression of total RhoA in IECs. Right panel: AtRA-treatment did not increase Rac1-GTP in IEC6 cells. B. Western blots show that atRA-induced TGF-β2 expression in IEC6 cells was reproduced by over-expression of the constitutively-active GL4V mutant of RhoA. C. Cells expressing the TN19 dominant-negative RhoA mutant did not show atRA-induced TGF-β2 expression. D. Western blots show cleaved ROCK1 in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data. Inset: Fluorescence photomicrographs (magnification 630x) show nuclear localization of ROCK1 (green) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI (blue). E. Pharmacological inhibition of ROCK1 by Y-27632 blocked atRA-induced TGF-β2 expression in IEC6 cells. Western blots show TGF-β2 and cleaved ROCK1 expression. Bar-diagram (means ± SE) summarizes densitometric data, normalized against β-actin. Data represent 3 separate experiments; * p<0.05 compared to cells cultured in media alone; # indicates p<0.05 compared to atRA-treated cells.
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pone.0134003.g002: AtRA-induced TGF-β2 expression in IECs is mediated via RhoA GTPase and ROCK1.A. Representative Western blots show increased expression of activated RhoA (RhoA-GTP) in IEC6 cells treated with atRA × 4h. Activated RhoA was pulled down from cell lysates using Rhotekin-agarose beads. Bar-diagram (means ± SE) summarizes densitometric data. Inset: Left panel: ATRA also increased the expression of total RhoA in IECs. Right panel: AtRA-treatment did not increase Rac1-GTP in IEC6 cells. B. Western blots show that atRA-induced TGF-β2 expression in IEC6 cells was reproduced by over-expression of the constitutively-active GL4V mutant of RhoA. C. Cells expressing the TN19 dominant-negative RhoA mutant did not show atRA-induced TGF-β2 expression. D. Western blots show cleaved ROCK1 in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data. Inset: Fluorescence photomicrographs (magnification 630x) show nuclear localization of ROCK1 (green) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI (blue). E. Pharmacological inhibition of ROCK1 by Y-27632 blocked atRA-induced TGF-β2 expression in IEC6 cells. Western blots show TGF-β2 and cleaved ROCK1 expression. Bar-diagram (means ± SE) summarizes densitometric data, normalized against β-actin. Data represent 3 separate experiments; * p<0.05 compared to cells cultured in media alone; # indicates p<0.05 compared to atRA-treated cells.
Mentions: Existing information indicates that atRA differentially activates the small GTPases RhoA, rac1, and cdc42 in different cell lineages [26–28]. To determine whether atRA activates one or more of these mediators in IECs, we used Rhotekin RBD- and PAK PBD-conjugated agarose beads to pull down the active forms of RhoA, rac1, and cdc42 from cell lysates and measured these GTPases in Western blots. As shown in Fig 2A, atRA increased active RhoA (RhoA-GTP) in IEC6 cells. There was no change in active Rac1, and we did not detect activated Cdc42 in atRA-treated cells. Consistent with these findings, IEC6 cells expressing the constitutively-active GL4V mutant of RhoA showed increased TGF-β2 expression similar to the effects of atRA (Fig 2B). In contrast, we did not find TGF-β2 expression in cells expressing the dominant-negative TN19 RhoA mutant, emphasizing the importance of RhoA in constitutive and atRA-induced TGF-β2 expression (Fig 2C).

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