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Tight junction protein 1 is regulated by transforming growth factor-β and contributes to cell motility in NSCLC cells.

Lee SH, Paek AR, Yoon K, Kim SH, Lee SY, You HJ - BMB Rep (2015)

Bottom Line: Tight junction protein 1 (TJP1), a component of tight junction, has been reported to play a role in protein networks as an adaptor protein, and TJP1 expression is altered during tumor development.SB431542, a type-I TGF-β receptor inhibitor, as well as SB203580, a p38 kinase inhibitor, significantly abrogated the effect of TGF-β on TJP1 expression.When TJP1 expression was reduced by shRNA lentiviral particles in A549 cells (A549-sh TJP1), wound healing was much lower than in cells infected with control viral particles.

View Article: PubMed Central - PubMed

Affiliation: Cancer Cell and Molecular Biology Branch, Div. of Cancer Biology, National Cancer Center, Goyang 410-769; Division of Molecular Life Sciences, Ewha Womans University, Seoul 120-750, Korea.

ABSTRACT
Tight junction protein 1 (TJP1), a component of tight junction, has been reported to play a role in protein networks as an adaptor protein, and TJP1 expression is altered during tumor development. Here, we found that TJP1 expression was increased at the RNA and protein levels in TGF-β-stimulated lung cancer cells, A549. SB431542, a type-I TGF-β receptor inhibitor, as well as SB203580, a p38 kinase inhibitor, significantly abrogated the effect of TGF-β on TJP1 expression. Diphenyleneiodonium, an NADPH oxidase inhibitor, also attenuated TJP1 expression in response to TGF-β in lung cancer cells. When TJP1 expression was reduced by shRNA lentiviral particles in A549 cells (A549-sh TJP1), wound healing was much lower than in cells infected with control viral particles. Taken together, these data suggest that TGF-β enhances TJP1 expression, which may play a role beyond structural support in tight junctions during cancer development.

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SB431542, a type I TGF-β receptor inhibitor, SB203580, a p38 kinase inhibitor, DPI, an NADPH oxidase inhibitor, and NAC, a ROS scavenger, attenuated TGF-β-mediated TJP1 expression in A549 cells. Subconfluent cells were treated with 50 pM TGF-β for 24 h in the presence of the indicated amounts of buffer, SB431542, SB203580, DPI, or NAC and evaluated for TJP1 expression by immunoblotting. All results shown are representative of at least three independent experiments.
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Figure 002: SB431542, a type I TGF-β receptor inhibitor, SB203580, a p38 kinase inhibitor, DPI, an NADPH oxidase inhibitor, and NAC, a ROS scavenger, attenuated TGF-β-mediated TJP1 expression in A549 cells. Subconfluent cells were treated with 50 pM TGF-β for 24 h in the presence of the indicated amounts of buffer, SB431542, SB203580, DPI, or NAC and evaluated for TJP1 expression by immunoblotting. All results shown are representative of at least three independent experiments.

Mentions: To assess the regulatory mechanism for TJP1 expression in response to TGF-β in A549 human lung carcinoma cells, cells were treated with TGF-β in the presence of SB431542, a type I TGF-β receptor inhibitor, SB203580, a p38 kinase inhibitor, PB98059, a MEK1 inhibitor, SP600126, a c-Jun amino-terminal kinase (JNK) inhibitor, diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, N-acetyl cysteine (NAC), a reactive oxygen species scavenger, wortmannin, a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, and dimethyl sulfoxide (DMSO) for 24 h. Inhibitors were added 30 min prior to TGF-β addition. SB431542, SB203580, NAC, and DPI attenuated TJP1 expression in A549 lung carcinoma cells 3 (Fig. 2) while PD98059, SP600125, and wortmannin did not (data not shown). These results suggest that the type I TGF-β receptor, p38 kinase, and ROS may be involved in TJP1 expression in response to TGF-β. To further confirm the role of the type I TGF-β receptor, p38 kinase and ROS in TGF-β signaling-mediated TJP1 expression, we examined downstream signaling activation in TGF-β-stimulated A549 cells (Fig. 3A). As expected, Smad2 was phosphorylated after 10 min and reached a maximum at 60 min in response to TGF-β in A549 lung cancer cells. p38 kinase was also phosphorylated in response to TGF-β, occurring at 30 min and lasting for 3 h. We did not observe any significant change in the phosphorylation status of extracellular signal-regulated kinase (ERK) or JNK in TGF-β-stimulated A549 lung cancer cells (data not shown). To examine any crosstalk between downstream signaling pathways, cells were treated with TGF-β in the presence of SB431542, SB203580, DPI, NAC, or vehicles (DMSO or H2O) for 10 min and then harvested for immunoblotting. SB431542 inhibited Smad2 phosphorylation markedly in response to TGF-β, but it did not affect p38 kinase phosphorylation by TGF-β in A549 lung cancer cells (Fig. 3B). SB203580, a p38 kinase inhibitor, did not affect the phosphorylation of Smad2, indicating that Smad2, as well as p38 kinase, may be involved independently in TGF-β-mediated TJP1 expression in lung cancer cells. Recently, TGF-β was reported to increase cellular ROS with the help of NADPH oxidase 4, contributing to cellular differentiation (25). Here, we observed ROS generation in response to TGF-β in A549 lung cancer cells. Moreover, TGF-β-induced ROS generation was inhibited by DPI, an NADPH oxidase inhibitor and NAC, a ROS scavenger (Fig. 3E, F). Additionally, neither DPI nor NAC altered the phosphorylation status of Smad2 (Fig. 3C), while ROS generation by TGF-β was attenuated by SB431542 and SB203580 (Fig. 3E), indicating that ROS may contribute to TJP1 expression after activation of Smad 2 and p38 kinase in TGF-β-stimulated A549 lung cancer cells.


Tight junction protein 1 is regulated by transforming growth factor-β and contributes to cell motility in NSCLC cells.

Lee SH, Paek AR, Yoon K, Kim SH, Lee SY, You HJ - BMB Rep (2015)

SB431542, a type I TGF-β receptor inhibitor, SB203580, a p38 kinase inhibitor, DPI, an NADPH oxidase inhibitor, and NAC, a ROS scavenger, attenuated TGF-β-mediated TJP1 expression in A549 cells. Subconfluent cells were treated with 50 pM TGF-β for 24 h in the presence of the indicated amounts of buffer, SB431542, SB203580, DPI, or NAC and evaluated for TJP1 expression by immunoblotting. All results shown are representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4352613&req=5

Figure 002: SB431542, a type I TGF-β receptor inhibitor, SB203580, a p38 kinase inhibitor, DPI, an NADPH oxidase inhibitor, and NAC, a ROS scavenger, attenuated TGF-β-mediated TJP1 expression in A549 cells. Subconfluent cells were treated with 50 pM TGF-β for 24 h in the presence of the indicated amounts of buffer, SB431542, SB203580, DPI, or NAC and evaluated for TJP1 expression by immunoblotting. All results shown are representative of at least three independent experiments.
Mentions: To assess the regulatory mechanism for TJP1 expression in response to TGF-β in A549 human lung carcinoma cells, cells were treated with TGF-β in the presence of SB431542, a type I TGF-β receptor inhibitor, SB203580, a p38 kinase inhibitor, PB98059, a MEK1 inhibitor, SP600126, a c-Jun amino-terminal kinase (JNK) inhibitor, diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, N-acetyl cysteine (NAC), a reactive oxygen species scavenger, wortmannin, a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, and dimethyl sulfoxide (DMSO) for 24 h. Inhibitors were added 30 min prior to TGF-β addition. SB431542, SB203580, NAC, and DPI attenuated TJP1 expression in A549 lung carcinoma cells 3 (Fig. 2) while PD98059, SP600125, and wortmannin did not (data not shown). These results suggest that the type I TGF-β receptor, p38 kinase, and ROS may be involved in TJP1 expression in response to TGF-β. To further confirm the role of the type I TGF-β receptor, p38 kinase and ROS in TGF-β signaling-mediated TJP1 expression, we examined downstream signaling activation in TGF-β-stimulated A549 cells (Fig. 3A). As expected, Smad2 was phosphorylated after 10 min and reached a maximum at 60 min in response to TGF-β in A549 lung cancer cells. p38 kinase was also phosphorylated in response to TGF-β, occurring at 30 min and lasting for 3 h. We did not observe any significant change in the phosphorylation status of extracellular signal-regulated kinase (ERK) or JNK in TGF-β-stimulated A549 lung cancer cells (data not shown). To examine any crosstalk between downstream signaling pathways, cells were treated with TGF-β in the presence of SB431542, SB203580, DPI, NAC, or vehicles (DMSO or H2O) for 10 min and then harvested for immunoblotting. SB431542 inhibited Smad2 phosphorylation markedly in response to TGF-β, but it did not affect p38 kinase phosphorylation by TGF-β in A549 lung cancer cells (Fig. 3B). SB203580, a p38 kinase inhibitor, did not affect the phosphorylation of Smad2, indicating that Smad2, as well as p38 kinase, may be involved independently in TGF-β-mediated TJP1 expression in lung cancer cells. Recently, TGF-β was reported to increase cellular ROS with the help of NADPH oxidase 4, contributing to cellular differentiation (25). Here, we observed ROS generation in response to TGF-β in A549 lung cancer cells. Moreover, TGF-β-induced ROS generation was inhibited by DPI, an NADPH oxidase inhibitor and NAC, a ROS scavenger (Fig. 3E, F). Additionally, neither DPI nor NAC altered the phosphorylation status of Smad2 (Fig. 3C), while ROS generation by TGF-β was attenuated by SB431542 and SB203580 (Fig. 3E), indicating that ROS may contribute to TJP1 expression after activation of Smad 2 and p38 kinase in TGF-β-stimulated A549 lung cancer cells.

Bottom Line: Tight junction protein 1 (TJP1), a component of tight junction, has been reported to play a role in protein networks as an adaptor protein, and TJP1 expression is altered during tumor development.SB431542, a type-I TGF-β receptor inhibitor, as well as SB203580, a p38 kinase inhibitor, significantly abrogated the effect of TGF-β on TJP1 expression.When TJP1 expression was reduced by shRNA lentiviral particles in A549 cells (A549-sh TJP1), wound healing was much lower than in cells infected with control viral particles.

View Article: PubMed Central - PubMed

Affiliation: Cancer Cell and Molecular Biology Branch, Div. of Cancer Biology, National Cancer Center, Goyang 410-769; Division of Molecular Life Sciences, Ewha Womans University, Seoul 120-750, Korea.

ABSTRACT
Tight junction protein 1 (TJP1), a component of tight junction, has been reported to play a role in protein networks as an adaptor protein, and TJP1 expression is altered during tumor development. Here, we found that TJP1 expression was increased at the RNA and protein levels in TGF-β-stimulated lung cancer cells, A549. SB431542, a type-I TGF-β receptor inhibitor, as well as SB203580, a p38 kinase inhibitor, significantly abrogated the effect of TGF-β on TJP1 expression. Diphenyleneiodonium, an NADPH oxidase inhibitor, also attenuated TJP1 expression in response to TGF-β in lung cancer cells. When TJP1 expression was reduced by shRNA lentiviral particles in A549 cells (A549-sh TJP1), wound healing was much lower than in cells infected with control viral particles. Taken together, these data suggest that TGF-β enhances TJP1 expression, which may play a role beyond structural support in tight junctions during cancer development.

Show MeSH
Related in: MedlinePlus