Limits...
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

Both ROCK1 and p38α MAPK are required for atRA-mediated phosphorylation of ATF2 in IECs.A. Western blots show the effect of the ROCK inhibitor Y-27632 on the expression of cleaved ROCK1, phospho-p38, phospho-MAPKAPK2, and phospho-ATF2. Β-actin was used as the loading control. B. Effect of the p38 inhibitor SB203580 on the expression of cleaved ROCK1, phospho-p38, phospho-MAPKAPK2, and phospho-ATF2. Data represent 3 separate experiments. C. Bar-diagrams (means ± SE) summarize ROCK activity in IEC6 cells treated with atRA (10 μM, top panel), with Y-27632 (10 μM) followed by atRA (10 μM; middle panel), and SB203580 (10 μM) followed by atRA (10 μM; bottom panel). Data represent 3 separate experiments; * p<0.05, ** p<0.01, *** p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4520553&req=5

pone.0134003.g006: Both ROCK1 and p38α MAPK are required for atRA-mediated phosphorylation of ATF2 in IECs.A. Western blots show the effect of the ROCK inhibitor Y-27632 on the expression of cleaved ROCK1, phospho-p38, phospho-MAPKAPK2, and phospho-ATF2. Β-actin was used as the loading control. B. Effect of the p38 inhibitor SB203580 on the expression of cleaved ROCK1, phospho-p38, phospho-MAPKAPK2, and phospho-ATF2. Data represent 3 separate experiments. C. Bar-diagrams (means ± SE) summarize ROCK activity in IEC6 cells treated with atRA (10 μM, top panel), with Y-27632 (10 μM) followed by atRA (10 μM; middle panel), and SB203580 (10 μM) followed by atRA (10 μM; bottom panel). Data represent 3 separate experiments; * p<0.05, ** p<0.01, *** p<0.001.

Mentions: To determine whether RhoA/ROCK1 and p38α signals are both required for atRA-mediated phosphorylation of ATF2 and TGF-β2 expression in IECs, we added Y-27632 and SB203580 to inhibit ROCK and p38 activity, respectively, and measured cleaved ROCK1, phospho-p38, phospho-MAPKAPK2, and phospho-ATF2. Y-27632 suppressed atRA-induced expression of the cleaved ROCK1 fragment, phosphorylation of p38 MAPK and its downstream MAPKAPK2, and phosphorylation of ATF2 (Fig 6A). Similarly, SB203580 blocked atRA-mediated ATF2 phosphorylation (Fig 6B). Interestingly, SB203580 inhibited atRA-induced expression of cleaved ROCK1, indicating that p38α contributes to atRA-mediated activation of ROCK1. These findings were confirmed in IEC6 cells expressing the DN variant of p38. The inhibitory effect of SB203580 on atRA-activated RhoA signaling was confirmed in direct measurements of ROCK activity in IEC6 cells (Fig 6C).


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)

Both ROCK1 and p38α MAPK are required for atRA-mediated phosphorylation of ATF2 in IECs.A. Western blots show the effect of the ROCK inhibitor Y-27632 on the expression of cleaved ROCK1, phospho-p38, phospho-MAPKAPK2, and phospho-ATF2. Β-actin was used as the loading control. B. Effect of the p38 inhibitor SB203580 on the expression of cleaved ROCK1, phospho-p38, phospho-MAPKAPK2, and phospho-ATF2. Data represent 3 separate experiments. C. Bar-diagrams (means ± SE) summarize ROCK activity in IEC6 cells treated with atRA (10 μM, top panel), with Y-27632 (10 μM) followed by atRA (10 μM; middle panel), and SB203580 (10 μM) followed by atRA (10 μM; bottom panel). Data represent 3 separate experiments; * p<0.05, ** p<0.01, *** p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134003.g006: Both ROCK1 and p38α MAPK are required for atRA-mediated phosphorylation of ATF2 in IECs.A. Western blots show the effect of the ROCK inhibitor Y-27632 on the expression of cleaved ROCK1, phospho-p38, phospho-MAPKAPK2, and phospho-ATF2. Β-actin was used as the loading control. B. Effect of the p38 inhibitor SB203580 on the expression of cleaved ROCK1, phospho-p38, phospho-MAPKAPK2, and phospho-ATF2. Data represent 3 separate experiments. C. Bar-diagrams (means ± SE) summarize ROCK activity in IEC6 cells treated with atRA (10 μM, top panel), with Y-27632 (10 μM) followed by atRA (10 μM; middle panel), and SB203580 (10 μM) followed by atRA (10 μM; bottom panel). Data represent 3 separate experiments; * p<0.05, ** p<0.01, *** p<0.001.
Mentions: To determine whether RhoA/ROCK1 and p38α signals are both required for atRA-mediated phosphorylation of ATF2 and TGF-β2 expression in IECs, we added Y-27632 and SB203580 to inhibit ROCK and p38 activity, respectively, and measured cleaved ROCK1, phospho-p38, phospho-MAPKAPK2, and phospho-ATF2. Y-27632 suppressed atRA-induced expression of the cleaved ROCK1 fragment, phosphorylation of p38 MAPK and its downstream MAPKAPK2, and phosphorylation of ATF2 (Fig 6A). Similarly, SB203580 blocked atRA-mediated ATF2 phosphorylation (Fig 6B). Interestingly, SB203580 inhibited atRA-induced expression of cleaved ROCK1, indicating that p38α contributes to atRA-mediated activation of ROCK1. These findings were confirmed in IEC6 cells expressing the DN variant of p38. The inhibitory effect of SB203580 on atRA-activated RhoA signaling was confirmed in direct measurements of ROCK activity in IEC6 cells (Fig 6C).

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