Limits...
The Crosstalk between Nrf2 and TGF-β1 in the Epithelial-Mesenchymal Transition of Pancreatic Duct Epithelial Cells.

Arfmann-Knübel S, Struck B, Genrich G, Helm O, Sipos B, Sebens S, Schäfer H - PLoS ONE (2015)

Bottom Line: In Colo357 cells, TGF-β1 itself was capable of inducing Nrf2 whereas in HPDE cells TGF-β1 per-se did not affect Nrf2 activity, but enhanced Nrf2 induction by tBHQ.In Colo357, but not in HPDE cells, the effects of TGF-β1 on invasion were sensitive to Nrf2 knock-down.In both cell lines, E-cadherin re-expression inhibited the proinvasive effect of Nrf2.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gastroenterology, Dept. of Internal Medicine I, UKSH Campus Kiel, Arnold-Heller-Str. 3, Bldg. 6, 24105, Kiel, Germany.

ABSTRACT
Nrf2 and TGF-β1 both affect tumorigenesis in a dual fashion, either by preventing carcinogen induced carcinogenesis and suppressing tumor growth, respectively, or by conferring cytoprotection and invasiveness to tumor cells during malignant transformation. Given the involvement of Nrf2 and TGF-β1 in the adaptation of epithelial cells to persistent inflammatory stress, e.g. of the pancreatic duct epithelium during chronic pancreatitis, a crosstalk between Nrf2 and TGF-β1 can be envisaged. By using premalignant human pancreatic duct cells (HPDE) and the pancreatic ductal adenocarcinoma cell line Colo357, we could show that Nrf2 and TGF-β1 independently but additively conferred an invasive phenotype to HPDE cells, whereas acting synergistically in Colo357 cells. This was accompanied by differential regulation of EMT markers like vimentin, Slug, L1CAM and E-cadherin. Nrf2 activation suppressed E-cadherin expression through an as yet unidentified ARE related site in the E-cadherin promoter, attenuated TGF-β1 induced Smad2/3-activity and enhanced JNK-signaling. In Colo357 cells, TGF-β1 itself was capable of inducing Nrf2 whereas in HPDE cells TGF-β1 per-se did not affect Nrf2 activity, but enhanced Nrf2 induction by tBHQ. In Colo357, but not in HPDE cells, the effects of TGF-β1 on invasion were sensitive to Nrf2 knock-down. In both cell lines, E-cadherin re-expression inhibited the proinvasive effect of Nrf2. Thus, the increased invasion of both cell lines relates to the Nrf2-dependent downregulation of E-cadherin expression. In line, immunohistochemistry analysis of human pancreatic intraepithelial neoplasias in pancreatic tissues from chronic pancreatitis patients revealed strong Nrf2 activity already in premalignant epithelial duct cells, accompanied by partial loss of E-cadherin expression. Our findings indicate that Nrf2 and TGF-β1 both contribute to malignant transformation through distinct EMT related mechanisms accounting for an invasive phenotype. Provided a crosstalk between both pathways, Nrf2 and TGF-β1 mutually promote their tumorigenic potential, a condition manifesting already at an early stage during inflammation induced carcinogenesis of the pancreas.

No MeSH data available.


Related in: MedlinePlus

A Nrf2 binding site in the human E-cadherin promoter exerts transcriptional repression.HPDE (A) or Colo357 (B) cells were transfected with firefly luciferase reporter gene constructs containing the ARE like site (Ecad[–1189]) from the E-cadherin promoter, or not (Ecad[–1153]), or with the empty reporter gene vector. Cells were left untreated or were treated with tBHQ or SFN for 8h. Then, firefly luciferase activity was measured and normalized to renilla luciferase. Data represent the mean of 4 independent experiments. A scheme of the cloned E-cadherin promoter fragments is provided in Figs. A and B in S6 File.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4520686&req=5

pone.0132978.g007: A Nrf2 binding site in the human E-cadherin promoter exerts transcriptional repression.HPDE (A) or Colo357 (B) cells were transfected with firefly luciferase reporter gene constructs containing the ARE like site (Ecad[–1189]) from the E-cadherin promoter, or not (Ecad[–1153]), or with the empty reporter gene vector. Cells were left untreated or were treated with tBHQ or SFN for 8h. Then, firefly luciferase activity was measured and normalized to renilla luciferase. Data represent the mean of 4 independent experiments. A scheme of the cloned E-cadherin promoter fragments is provided in Figs. A and B in S6 File.

Mentions: Since the Nrf2 mediated downregulation of E-cadherin expression did not correlate with the effects of Nrf2 on Smad and JNK signalling, we next elucidated whether Nrf2 directly impacts on E-cadherin expression by analysing the gene promoter of human E-cadherin for potential Nrf2 binding sites (ARE). We could identify an ARE related motif at position -1153 to -1162 (Fig A in S6 File). To study the impact of this site on transcriptional activity, luciferase assays were conducted using constructs either containing (Ecad[–1189]) or lacking this site (Ecad[–1153]). As shown in Fig 7, the luciferase expression in HPDE cells transfected with Ecad[–1189] was slightly lower than in HPDE cells transfected with Ecad[–1153]. Treatment with tBHQ or SFN further reduced the luciferase activity in Ecad[–1189] transfected but not in Ecad[–1153] transfected HPDE cells. Already in the absence of tBHQ or SFN, luciferase expression was significantly lower in Colo357 cells transfected with Ecad[–1189] as compared to Ecad[–1153] transfected Colo357 cells. Again, tBHQ or SFN treatment further decreased luciferase expression in Colo357 cells transfected with Ecad[–1189] but not in Colo357 cells transfected with Ecad[–1153].


The Crosstalk between Nrf2 and TGF-β1 in the Epithelial-Mesenchymal Transition of Pancreatic Duct Epithelial Cells.

Arfmann-Knübel S, Struck B, Genrich G, Helm O, Sipos B, Sebens S, Schäfer H - PLoS ONE (2015)

A Nrf2 binding site in the human E-cadherin promoter exerts transcriptional repression.HPDE (A) or Colo357 (B) cells were transfected with firefly luciferase reporter gene constructs containing the ARE like site (Ecad[–1189]) from the E-cadherin promoter, or not (Ecad[–1153]), or with the empty reporter gene vector. Cells were left untreated or were treated with tBHQ or SFN for 8h. Then, firefly luciferase activity was measured and normalized to renilla luciferase. Data represent the mean of 4 independent experiments. A scheme of the cloned E-cadherin promoter fragments is provided in Figs. A and B in S6 File.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520686&req=5

pone.0132978.g007: A Nrf2 binding site in the human E-cadherin promoter exerts transcriptional repression.HPDE (A) or Colo357 (B) cells were transfected with firefly luciferase reporter gene constructs containing the ARE like site (Ecad[–1189]) from the E-cadherin promoter, or not (Ecad[–1153]), or with the empty reporter gene vector. Cells were left untreated or were treated with tBHQ or SFN for 8h. Then, firefly luciferase activity was measured and normalized to renilla luciferase. Data represent the mean of 4 independent experiments. A scheme of the cloned E-cadherin promoter fragments is provided in Figs. A and B in S6 File.
Mentions: Since the Nrf2 mediated downregulation of E-cadherin expression did not correlate with the effects of Nrf2 on Smad and JNK signalling, we next elucidated whether Nrf2 directly impacts on E-cadherin expression by analysing the gene promoter of human E-cadherin for potential Nrf2 binding sites (ARE). We could identify an ARE related motif at position -1153 to -1162 (Fig A in S6 File). To study the impact of this site on transcriptional activity, luciferase assays were conducted using constructs either containing (Ecad[–1189]) or lacking this site (Ecad[–1153]). As shown in Fig 7, the luciferase expression in HPDE cells transfected with Ecad[–1189] was slightly lower than in HPDE cells transfected with Ecad[–1153]. Treatment with tBHQ or SFN further reduced the luciferase activity in Ecad[–1189] transfected but not in Ecad[–1153] transfected HPDE cells. Already in the absence of tBHQ or SFN, luciferase expression was significantly lower in Colo357 cells transfected with Ecad[–1189] as compared to Ecad[–1153] transfected Colo357 cells. Again, tBHQ or SFN treatment further decreased luciferase expression in Colo357 cells transfected with Ecad[–1189] but not in Colo357 cells transfected with Ecad[–1153].

Bottom Line: In Colo357 cells, TGF-β1 itself was capable of inducing Nrf2 whereas in HPDE cells TGF-β1 per-se did not affect Nrf2 activity, but enhanced Nrf2 induction by tBHQ.In Colo357, but not in HPDE cells, the effects of TGF-β1 on invasion were sensitive to Nrf2 knock-down.In both cell lines, E-cadherin re-expression inhibited the proinvasive effect of Nrf2.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gastroenterology, Dept. of Internal Medicine I, UKSH Campus Kiel, Arnold-Heller-Str. 3, Bldg. 6, 24105, Kiel, Germany.

ABSTRACT
Nrf2 and TGF-β1 both affect tumorigenesis in a dual fashion, either by preventing carcinogen induced carcinogenesis and suppressing tumor growth, respectively, or by conferring cytoprotection and invasiveness to tumor cells during malignant transformation. Given the involvement of Nrf2 and TGF-β1 in the adaptation of epithelial cells to persistent inflammatory stress, e.g. of the pancreatic duct epithelium during chronic pancreatitis, a crosstalk between Nrf2 and TGF-β1 can be envisaged. By using premalignant human pancreatic duct cells (HPDE) and the pancreatic ductal adenocarcinoma cell line Colo357, we could show that Nrf2 and TGF-β1 independently but additively conferred an invasive phenotype to HPDE cells, whereas acting synergistically in Colo357 cells. This was accompanied by differential regulation of EMT markers like vimentin, Slug, L1CAM and E-cadherin. Nrf2 activation suppressed E-cadherin expression through an as yet unidentified ARE related site in the E-cadherin promoter, attenuated TGF-β1 induced Smad2/3-activity and enhanced JNK-signaling. In Colo357 cells, TGF-β1 itself was capable of inducing Nrf2 whereas in HPDE cells TGF-β1 per-se did not affect Nrf2 activity, but enhanced Nrf2 induction by tBHQ. In Colo357, but not in HPDE cells, the effects of TGF-β1 on invasion were sensitive to Nrf2 knock-down. In both cell lines, E-cadherin re-expression inhibited the proinvasive effect of Nrf2. Thus, the increased invasion of both cell lines relates to the Nrf2-dependent downregulation of E-cadherin expression. In line, immunohistochemistry analysis of human pancreatic intraepithelial neoplasias in pancreatic tissues from chronic pancreatitis patients revealed strong Nrf2 activity already in premalignant epithelial duct cells, accompanied by partial loss of E-cadherin expression. Our findings indicate that Nrf2 and TGF-β1 both contribute to malignant transformation through distinct EMT related mechanisms accounting for an invasive phenotype. Provided a crosstalk between both pathways, Nrf2 and TGF-β1 mutually promote their tumorigenic potential, a condition manifesting already at an early stage during inflammation induced carcinogenesis of the pancreas.

No MeSH data available.


Related in: MedlinePlus