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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

Nrf2 activation affects basal and TGF-β1 dependent Smad and JNK signalling pathways in premalignant and malignant pancreatic duct cells.A) HPDE or B) Colo357 cells incubated with 50 μM tBHQ, 10 μM SFN or 10 ng/mL TGF-β1 alone or in combination for the indicated periods. Then total cell lysates were analysed by westernblot (A,C) for expression of P-Smad2, P-Smad3, Smad2/3, P-JNK and JNK using Hsp90 as loading control. Representative results from three independent experiments are shown and a densitometric band intensity evaluation is provided in Figs. A and B in S5 File. B) HPDE or D) Colo357 cells were transfected with pGL3 (control) or p6SBE (SRE) together with ptkRL followed by incubation with 50 μM tBHQ and/or 10 ng/mL TGF-β1 or without. After 16h, cell lysates were analysed for firefly and renilla luciferase expression and firefly luciferase units were normalized to those of renilla luciferase. Data represent the mean ± SD of six independent experiments performed in duplicate, p<0.05 (+tBHQ versus-tBHQ). E) HPDE or F) Colo357 cells were treated with Nrf2 or control siRNA for 48h, followed by incubation with 50 μM tBHQ, 10 μM SFN and/or 10 ng/mL TGF-β1, or without. Then, total cell lysates were analysed by westernblot for expression of P-Smad2, P-Smad3, Smad2/3, P-JNK and JNK using Hsp90 as loading control. Representative results from three independent experiments are shown, and a densitometric band intensity evaluation is provided in Figs. C and D in S5 File.
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pone.0132978.g006: Nrf2 activation affects basal and TGF-β1 dependent Smad and JNK signalling pathways in premalignant and malignant pancreatic duct cells.A) HPDE or B) Colo357 cells incubated with 50 μM tBHQ, 10 μM SFN or 10 ng/mL TGF-β1 alone or in combination for the indicated periods. Then total cell lysates were analysed by westernblot (A,C) for expression of P-Smad2, P-Smad3, Smad2/3, P-JNK and JNK using Hsp90 as loading control. Representative results from three independent experiments are shown and a densitometric band intensity evaluation is provided in Figs. A and B in S5 File. B) HPDE or D) Colo357 cells were transfected with pGL3 (control) or p6SBE (SRE) together with ptkRL followed by incubation with 50 μM tBHQ and/or 10 ng/mL TGF-β1 or without. After 16h, cell lysates were analysed for firefly and renilla luciferase expression and firefly luciferase units were normalized to those of renilla luciferase. Data represent the mean ± SD of six independent experiments performed in duplicate, p<0.05 (+tBHQ versus-tBHQ). E) HPDE or F) Colo357 cells were treated with Nrf2 or control siRNA for 48h, followed by incubation with 50 μM tBHQ, 10 μM SFN and/or 10 ng/mL TGF-β1, or without. Then, total cell lysates were analysed by westernblot for expression of P-Smad2, P-Smad3, Smad2/3, P-JNK and JNK using Hsp90 as loading control. Representative results from three independent experiments are shown, and a densitometric band intensity evaluation is provided in Figs. C and D in S5 File.

Mentions: To elucidate whether the interference of tBHQ or SFN treatment with EMT marker expression in response to TGF-β1 relates to an altered Smad or MAPK signalling, westernblot analyses were conducted. As shown in Fig 6A, tBHQ or SFN treatment (16 h) decreased basal phospho-Smad3/2 level in HPDE cells and blocked the increasing effect by TGF-β1 treatment (0.5 h). Smad response element (SRE)-luciferase assays confirmed the negative interference of Nrf2 activation with the Smad-pathway in HPDE cells (Fig 6B) as shown by the attenuating effect of tBHQ treatment on TGF-β1 induced SRE-driven luciferase expression.


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)

Nrf2 activation affects basal and TGF-β1 dependent Smad and JNK signalling pathways in premalignant and malignant pancreatic duct cells.A) HPDE or B) Colo357 cells incubated with 50 μM tBHQ, 10 μM SFN or 10 ng/mL TGF-β1 alone or in combination for the indicated periods. Then total cell lysates were analysed by westernblot (A,C) for expression of P-Smad2, P-Smad3, Smad2/3, P-JNK and JNK using Hsp90 as loading control. Representative results from three independent experiments are shown and a densitometric band intensity evaluation is provided in Figs. A and B in S5 File. B) HPDE or D) Colo357 cells were transfected with pGL3 (control) or p6SBE (SRE) together with ptkRL followed by incubation with 50 μM tBHQ and/or 10 ng/mL TGF-β1 or without. After 16h, cell lysates were analysed for firefly and renilla luciferase expression and firefly luciferase units were normalized to those of renilla luciferase. Data represent the mean ± SD of six independent experiments performed in duplicate, p<0.05 (+tBHQ versus-tBHQ). E) HPDE or F) Colo357 cells were treated with Nrf2 or control siRNA for 48h, followed by incubation with 50 μM tBHQ, 10 μM SFN and/or 10 ng/mL TGF-β1, or without. Then, total cell lysates were analysed by westernblot for expression of P-Smad2, P-Smad3, Smad2/3, P-JNK and JNK using Hsp90 as loading control. Representative results from three independent experiments are shown, and a densitometric band intensity evaluation is provided in Figs. C and D in S5 File.
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Related In: Results  -  Collection

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pone.0132978.g006: Nrf2 activation affects basal and TGF-β1 dependent Smad and JNK signalling pathways in premalignant and malignant pancreatic duct cells.A) HPDE or B) Colo357 cells incubated with 50 μM tBHQ, 10 μM SFN or 10 ng/mL TGF-β1 alone or in combination for the indicated periods. Then total cell lysates were analysed by westernblot (A,C) for expression of P-Smad2, P-Smad3, Smad2/3, P-JNK and JNK using Hsp90 as loading control. Representative results from three independent experiments are shown and a densitometric band intensity evaluation is provided in Figs. A and B in S5 File. B) HPDE or D) Colo357 cells were transfected with pGL3 (control) or p6SBE (SRE) together with ptkRL followed by incubation with 50 μM tBHQ and/or 10 ng/mL TGF-β1 or without. After 16h, cell lysates were analysed for firefly and renilla luciferase expression and firefly luciferase units were normalized to those of renilla luciferase. Data represent the mean ± SD of six independent experiments performed in duplicate, p<0.05 (+tBHQ versus-tBHQ). E) HPDE or F) Colo357 cells were treated with Nrf2 or control siRNA for 48h, followed by incubation with 50 μM tBHQ, 10 μM SFN and/or 10 ng/mL TGF-β1, or without. Then, total cell lysates were analysed by westernblot for expression of P-Smad2, P-Smad3, Smad2/3, P-JNK and JNK using Hsp90 as loading control. Representative results from three independent experiments are shown, and a densitometric band intensity evaluation is provided in Figs. C and D in S5 File.
Mentions: To elucidate whether the interference of tBHQ or SFN treatment with EMT marker expression in response to TGF-β1 relates to an altered Smad or MAPK signalling, westernblot analyses were conducted. As shown in Fig 6A, tBHQ or SFN treatment (16 h) decreased basal phospho-Smad3/2 level in HPDE cells and blocked the increasing effect by TGF-β1 treatment (0.5 h). Smad response element (SRE)-luciferase assays confirmed the negative interference of Nrf2 activation with the Smad-pathway in HPDE cells (Fig 6B) as shown by the attenuating effect of tBHQ treatment on TGF-β1 induced SRE-driven luciferase expression.

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