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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 p38 MAPK.A. Representative blots from a phospho-MAPK antibody array show increased phospho-p38α expression in IEC6 cells treated with atRA × 2h. Bar-diagram (means ± SE) summarizes densitometric data. B. Western blots show the effect of atRA on phospho-p38 (Tyr182), p38α, and β-actin expression in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data for each analyte normalized against β-actin. C. Fluorescence photomicrographs (magnification 630x) show increased phospho-p38 immunoreactivity (red) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI. D. Western blots show that pharmacological inhibition of p38 MAPK using SB203580 blocked atRA-induced TGF-β2 expression in IEC6 cells. Bar-diagram (means ± SE) summarizes densitometric data. E. IEC6 cells transduced to express a dominant-negative p38 transcript show decreased expression of TGF-β2 and phospho-p38 MAPK. We used the adenoviral vector in a multiplicity of infection of 40, which was determined to be the optimum dose in preliminary experiments. Bar-diagram (means ± SE) summarizes densitometric data. F. IEC6 cells transfected to express a MKK6-p38α fusion protein with constitutively-active p38α activity show increased TGF-β2 expression. Additional blots show increased MKK6 and phospho-p38 MAPK expression. Data represent 3 separate experiments; * p<0.05, ** p<0.01, *** p<0.001.
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pone.0134003.g003: AtRA-induced TGF-β2 expression in IECs is mediated via p38 MAPK.A. Representative blots from a phospho-MAPK antibody array show increased phospho-p38α expression in IEC6 cells treated with atRA × 2h. Bar-diagram (means ± SE) summarizes densitometric data. B. Western blots show the effect of atRA on phospho-p38 (Tyr182), p38α, and β-actin expression in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data for each analyte normalized against β-actin. C. Fluorescence photomicrographs (magnification 630x) show increased phospho-p38 immunoreactivity (red) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI. D. Western blots show that pharmacological inhibition of p38 MAPK using SB203580 blocked atRA-induced TGF-β2 expression in IEC6 cells. Bar-diagram (means ± SE) summarizes densitometric data. E. IEC6 cells transduced to express a dominant-negative p38 transcript show decreased expression of TGF-β2 and phospho-p38 MAPK. We used the adenoviral vector in a multiplicity of infection of 40, which was determined to be the optimum dose in preliminary experiments. Bar-diagram (means ± SE) summarizes densitometric data. F. IEC6 cells transfected to express a MKK6-p38α fusion protein with constitutively-active p38α activity show increased TGF-β2 expression. Additional blots show increased MKK6 and phospho-p38 MAPK expression. Data represent 3 separate experiments; * p<0.05, ** p<0.01, *** p<0.001.

Mentions: We next used a phospho-MAPK antibody array to identify MAPKs involved in atRA-induced TGF-β2 expression in IECs. Treatment with atRA significantly increased phosphorylated p38α. There was also a decrease in phospho-glycogen synthase kinase-3β and phospho-extracellular signal-regulated kinase-2 (Fig 3A). P38 activation has been previously shown to increase TGF-β2 transcription in human keloid fibroblasts [31], and therefore, we focused subsequent studies on this enzyme. We first confirmed these findings by measuring the temporal changes in phospho-p38 (Tyr182) expression in IEC6 cells following atRA treatment. As shown in Fig 3B, atRA caused a persistent increase in phospho-p38 expression starting at 1h. We also noted increased total p38α at 24h. Consistent with these findings, we also detected increased nuclear and cytoplasmic immunoreactivity of phospho-p38 following atRA treatment (Fig 3C). To determine the contribution of p38α to atRA-induced TGF-β2 expression, we used pharmacological and genetic approaches to block p38 activation in IEC6 cells. As shown in Fig 3D, SB203580, a specific inhibitor of p38, blocked atRA-induced TGF-β2 expression in a dose-dependent fashion. Similarly, cells transfected with a dominant-negative mutant of p38α failed to upregulate TGF-β2 in the presence of atRA (Fig 3E). The specific role of p38 in atRA-mediated TGF-β2 expression was confirmed in studies with pharmacological inhibitors of JNK, MEK/ERK, and the PI3K, which did not block TGF-β2 expression (not depicted). Finally, to confirm the central role of p38α in TGF-β2 expression, we transiently-transfected IEC6 cells to express a MKK6-p38α fusion protein that displays constitutively-active p38α activity. Consistent with the effects of atRA-mediated p38α activation, MKK6-p38α-expressing IEC6 cells also showed increased TGF-β2 expression (Fig 3F).


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 p38 MAPK.A. Representative blots from a phospho-MAPK antibody array show increased phospho-p38α expression in IEC6 cells treated with atRA × 2h. Bar-diagram (means ± SE) summarizes densitometric data. B. Western blots show the effect of atRA on phospho-p38 (Tyr182), p38α, and β-actin expression in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data for each analyte normalized against β-actin. C. Fluorescence photomicrographs (magnification 630x) show increased phospho-p38 immunoreactivity (red) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI. D. Western blots show that pharmacological inhibition of p38 MAPK using SB203580 blocked atRA-induced TGF-β2 expression in IEC6 cells. Bar-diagram (means ± SE) summarizes densitometric data. E. IEC6 cells transduced to express a dominant-negative p38 transcript show decreased expression of TGF-β2 and phospho-p38 MAPK. We used the adenoviral vector in a multiplicity of infection of 40, which was determined to be the optimum dose in preliminary experiments. Bar-diagram (means ± SE) summarizes densitometric data. F. IEC6 cells transfected to express a MKK6-p38α fusion protein with constitutively-active p38α activity show increased TGF-β2 expression. Additional blots show increased MKK6 and phospho-p38 MAPK expression. Data represent 3 separate experiments; * p<0.05, ** p<0.01, *** p<0.001.
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pone.0134003.g003: AtRA-induced TGF-β2 expression in IECs is mediated via p38 MAPK.A. Representative blots from a phospho-MAPK antibody array show increased phospho-p38α expression in IEC6 cells treated with atRA × 2h. Bar-diagram (means ± SE) summarizes densitometric data. B. Western blots show the effect of atRA on phospho-p38 (Tyr182), p38α, and β-actin expression in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data for each analyte normalized against β-actin. C. Fluorescence photomicrographs (magnification 630x) show increased phospho-p38 immunoreactivity (red) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI. D. Western blots show that pharmacological inhibition of p38 MAPK using SB203580 blocked atRA-induced TGF-β2 expression in IEC6 cells. Bar-diagram (means ± SE) summarizes densitometric data. E. IEC6 cells transduced to express a dominant-negative p38 transcript show decreased expression of TGF-β2 and phospho-p38 MAPK. We used the adenoviral vector in a multiplicity of infection of 40, which was determined to be the optimum dose in preliminary experiments. Bar-diagram (means ± SE) summarizes densitometric data. F. IEC6 cells transfected to express a MKK6-p38α fusion protein with constitutively-active p38α activity show increased TGF-β2 expression. Additional blots show increased MKK6 and phospho-p38 MAPK expression. Data represent 3 separate experiments; * p<0.05, ** p<0.01, *** p<0.001.
Mentions: We next used a phospho-MAPK antibody array to identify MAPKs involved in atRA-induced TGF-β2 expression in IECs. Treatment with atRA significantly increased phosphorylated p38α. There was also a decrease in phospho-glycogen synthase kinase-3β and phospho-extracellular signal-regulated kinase-2 (Fig 3A). P38 activation has been previously shown to increase TGF-β2 transcription in human keloid fibroblasts [31], and therefore, we focused subsequent studies on this enzyme. We first confirmed these findings by measuring the temporal changes in phospho-p38 (Tyr182) expression in IEC6 cells following atRA treatment. As shown in Fig 3B, atRA caused a persistent increase in phospho-p38 expression starting at 1h. We also noted increased total p38α at 24h. Consistent with these findings, we also detected increased nuclear and cytoplasmic immunoreactivity of phospho-p38 following atRA treatment (Fig 3C). To determine the contribution of p38α to atRA-induced TGF-β2 expression, we used pharmacological and genetic approaches to block p38 activation in IEC6 cells. As shown in Fig 3D, SB203580, a specific inhibitor of p38, blocked atRA-induced TGF-β2 expression in a dose-dependent fashion. Similarly, cells transfected with a dominant-negative mutant of p38α failed to upregulate TGF-β2 in the presence of atRA (Fig 3E). The specific role of p38 in atRA-mediated TGF-β2 expression was confirmed in studies with pharmacological inhibitors of JNK, MEK/ERK, and the PI3K, which did not block TGF-β2 expression (not depicted). Finally, to confirm the central role of p38α in TGF-β2 expression, we transiently-transfected IEC6 cells to express a MKK6-p38α fusion protein that displays constitutively-active p38α activity. Consistent with the effects of atRA-mediated p38α activation, MKK6-p38α-expressing IEC6 cells also showed increased TGF-β2 expression (Fig 3F).

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