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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

Maintained expression of E-cadherin affects the inducing effect of Nrf2 activation on the invasion of premalignant and malignant pancreatic duct cells.HPDE (A) or Colo357 (B) cells were transfected with a constitutive E-cadherin expression vector or an empty pcDNA3.1 vector (mock). Afterwards, cells were incubated with 50 μM tBHQ or 10 ng/mL TGF-β1 for 24h, or were left untreated. Then, cells were submitted to the modified Boyden assay on collagen-I coated transwells (A,B, right panels). In parallel, total cell lysates were analysed by E-cadherin western blots (A,B, left panels) using Hsp90 as loading control. A densitometric band intensity evaluation is provided in Figs. A and B in S7 File. The westernblots show representative results from four independent experiments. The invasion data (A,B, right panels) represent the mean ± SD of four independent experiments performed in duplicate, *p<0.05 (treated versus untreated).
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pone.0132978.g008: Maintained expression of E-cadherin affects the inducing effect of Nrf2 activation on the invasion of premalignant and malignant pancreatic duct cells.HPDE (A) or Colo357 (B) cells were transfected with a constitutive E-cadherin expression vector or an empty pcDNA3.1 vector (mock). Afterwards, cells were incubated with 50 μM tBHQ or 10 ng/mL TGF-β1 for 24h, or were left untreated. Then, cells were submitted to the modified Boyden assay on collagen-I coated transwells (A,B, right panels). In parallel, total cell lysates were analysed by E-cadherin western blots (A,B, left panels) using Hsp90 as loading control. A densitometric band intensity evaluation is provided in Figs. A and B in S7 File. The westernblots show representative results from four independent experiments. The invasion data (A,B, right panels) represent the mean ± SD of four independent experiments performed in duplicate, *p<0.05 (treated versus untreated).

Mentions: The observation that Nrf2 activation directly decreases the expression of E-cadherin but does not upregulate mesenchymal markers like vimentin prompted us to look whether the proinvasive effect of Nrf2 depends on the decline of E-cadherin expression. For this purpose, HPDE and Colo357 cells were transfected with E-cadherin to ensure its sustained expression during treatment with tBHQ or TGF-β1 for 24h. Boyden chamber analyses (Fig 8A) revealed that the inducing effect of tBHQ on HPDE cell invasion was significantly decreased by E-cadherin overexpression. In mock-transfected HPDE cells the basal invasion rate (42.3 ± 5.3%) increased 1.67 fold after tBHQ treatment (72.2 ± 12.1%) whereas in E-cadherin transfected HPDE cells the invasion rate increased only 1.25-fold from 39.7 ± 10.2% to 50.1 ± 13.7%. Interestingly, TGF-β1 induced migration was only slightly affected by E-cadherin re-expression (64.7 ± 11.5% versus 68.4 ± 12.4%).


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)

Maintained expression of E-cadherin affects the inducing effect of Nrf2 activation on the invasion of premalignant and malignant pancreatic duct cells.HPDE (A) or Colo357 (B) cells were transfected with a constitutive E-cadherin expression vector or an empty pcDNA3.1 vector (mock). Afterwards, cells were incubated with 50 μM tBHQ or 10 ng/mL TGF-β1 for 24h, or were left untreated. Then, cells were submitted to the modified Boyden assay on collagen-I coated transwells (A,B, right panels). In parallel, total cell lysates were analysed by E-cadherin western blots (A,B, left panels) using Hsp90 as loading control. A densitometric band intensity evaluation is provided in Figs. A and B in S7 File. The westernblots show representative results from four independent experiments. The invasion data (A,B, right panels) represent the mean ± SD of four independent experiments performed in duplicate, *p<0.05 (treated versus untreated).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132978.g008: Maintained expression of E-cadherin affects the inducing effect of Nrf2 activation on the invasion of premalignant and malignant pancreatic duct cells.HPDE (A) or Colo357 (B) cells were transfected with a constitutive E-cadherin expression vector or an empty pcDNA3.1 vector (mock). Afterwards, cells were incubated with 50 μM tBHQ or 10 ng/mL TGF-β1 for 24h, or were left untreated. Then, cells were submitted to the modified Boyden assay on collagen-I coated transwells (A,B, right panels). In parallel, total cell lysates were analysed by E-cadherin western blots (A,B, left panels) using Hsp90 as loading control. A densitometric band intensity evaluation is provided in Figs. A and B in S7 File. The westernblots show representative results from four independent experiments. The invasion data (A,B, right panels) represent the mean ± SD of four independent experiments performed in duplicate, *p<0.05 (treated versus untreated).
Mentions: The observation that Nrf2 activation directly decreases the expression of E-cadherin but does not upregulate mesenchymal markers like vimentin prompted us to look whether the proinvasive effect of Nrf2 depends on the decline of E-cadherin expression. For this purpose, HPDE and Colo357 cells were transfected with E-cadherin to ensure its sustained expression during treatment with tBHQ or TGF-β1 for 24h. Boyden chamber analyses (Fig 8A) revealed that the inducing effect of tBHQ on HPDE cell invasion was significantly decreased by E-cadherin overexpression. In mock-transfected HPDE cells the basal invasion rate (42.3 ± 5.3%) increased 1.67 fold after tBHQ treatment (72.2 ± 12.1%) whereas in E-cadherin transfected HPDE cells the invasion rate increased only 1.25-fold from 39.7 ± 10.2% to 50.1 ± 13.7%. Interestingly, TGF-β1 induced migration was only slightly affected by E-cadherin re-expression (64.7 ± 11.5% versus 68.4 ± 12.4%).

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