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Interaction of Cx43 with Hsc70 regulates G1/S transition through CDK inhibitor p27.

Hino H, Dai P, Yoshida T, Hatakeyama T, Harada Y, Otsuji E, Okuda T, Takamatsu T - Sci Rep (2015)

Bottom Line: Here, we report that nuclear accumulation of p27 is reduced by overexpression of Cx43, and that this reduction is restored by co-overexpression with Hsc70.We found that Cx43 competes with p27 for binding to Hsc70, and as a result, decreases the level of Hsc70 in cyclin D1-CDK4-p27 complex, leading to prevention of the nuclear translocation of the complex and the G1/S transition.Collectively, our findings suggest that, in Cx43 up-regulation, which is most likely an emergency measure, Cx43-Hsc70 interaction regulates cell cycle G1/S progression through a novel mechanism by which Cx43-Hsc70 interaction prevents the nuclear accumulation of p27 through controlling the nuclear translocation of cyclin D1-CDK4-p27 complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kyoto, Japan.

ABSTRACT
Connexin 43 (Cx43) functions as a cell growth suppressor. We have demonstrated that Cx43 interacts with heat shock cognate protein 70 (Hsc70) for regulating cell proliferation. Hsc70 interacts with CDK inhibitor p27, which regulates the assembly and subcellular localization of cyclin D1-CDK4-p27 complex. However, the involvement of p27 with Cx43-mediated cell cycle suppression is still poorly understood. Here, we report that nuclear accumulation of p27 is reduced by overexpression of Cx43, and that this reduction is restored by co-overexpression with Hsc70. We found that Cx43 competes with p27 for binding to Hsc70, and as a result, decreases the level of Hsc70 in cyclin D1-CDK4-p27 complex, leading to prevention of the nuclear translocation of the complex and the G1/S transition. Collectively, our findings suggest that, in Cx43 up-regulation, which is most likely an emergency measure, Cx43-Hsc70 interaction regulates cell cycle G1/S progression through a novel mechanism by which Cx43-Hsc70 interaction prevents the nuclear accumulation of p27 through controlling the nuclear translocation of cyclin D1-CDK4-p27 complex.

No MeSH data available.


Related in: MedlinePlus

Cx43-Hsc70 interaction decreases nuclear accumulation of p27.(a) Effect of Cx43-Hsc70 interaction on nuclear accumulation of p27. HuH-7 cells were transfected with Cx43-mRFP with or without T7-Hsc70. After 48 hr of transfection, immunofluorescence staining with anti-p27 antibodies was carried out. (b) Ratio of nuclear/cytoplasmic (N/C) fluorescence intensities in p27. Nucleocytoplasmic localization analysis in p27 was performed. At least 30 transfected cells were examined and quantified for each group, and the data were plotted. The horizontal lines represent median values. *p < 0.05.
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f2: Cx43-Hsc70 interaction decreases nuclear accumulation of p27.(a) Effect of Cx43-Hsc70 interaction on nuclear accumulation of p27. HuH-7 cells were transfected with Cx43-mRFP with or without T7-Hsc70. After 48 hr of transfection, immunofluorescence staining with anti-p27 antibodies was carried out. (b) Ratio of nuclear/cytoplasmic (N/C) fluorescence intensities in p27. Nucleocytoplasmic localization analysis in p27 was performed. At least 30 transfected cells were examined and quantified for each group, and the data were plotted. The horizontal lines represent median values. *p < 0.05.

Mentions: The Cx43-mediated cell cycle suppression can be restored by co-overexpression with Hsc70 in HuH-7 cells15. To determine whether Cx43 overexpression-decreased nuclear accumulation of p27 can be restored by co-overexpression with Hsc70, HuH-7 cells were transfected with Cx43-mRFP alone or with both Cx43-mRFP and T7-Hsc70. As expected, co-overexpression of Cx43 with Hsc70 restored the nuclear accumulation of p27 in HuH-7 cells (Fig. 2a). Moreover, the quantitative analysis showed that the Cx43-reduced ratio of nuclear to cytoplasmic intensity of p27 was abolished by co-overexpression with Hsc70 (Fig. 2b). These experimental data demonstrated that the interaction of Cx43-Hsc70 regulates the nuclear accumulation of p27 as well as cyclin D1. Similar results were also obtained in PANC-1 cells, which are derived from human pancreatic cancer cells and express endogenous Cx4329 (see Supplementary Fig. S1).


Interaction of Cx43 with Hsc70 regulates G1/S transition through CDK inhibitor p27.

Hino H, Dai P, Yoshida T, Hatakeyama T, Harada Y, Otsuji E, Okuda T, Takamatsu T - Sci Rep (2015)

Cx43-Hsc70 interaction decreases nuclear accumulation of p27.(a) Effect of Cx43-Hsc70 interaction on nuclear accumulation of p27. HuH-7 cells were transfected with Cx43-mRFP with or without T7-Hsc70. After 48 hr of transfection, immunofluorescence staining with anti-p27 antibodies was carried out. (b) Ratio of nuclear/cytoplasmic (N/C) fluorescence intensities in p27. Nucleocytoplasmic localization analysis in p27 was performed. At least 30 transfected cells were examined and quantified for each group, and the data were plotted. The horizontal lines represent median values. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4612729&req=5

f2: Cx43-Hsc70 interaction decreases nuclear accumulation of p27.(a) Effect of Cx43-Hsc70 interaction on nuclear accumulation of p27. HuH-7 cells were transfected with Cx43-mRFP with or without T7-Hsc70. After 48 hr of transfection, immunofluorescence staining with anti-p27 antibodies was carried out. (b) Ratio of nuclear/cytoplasmic (N/C) fluorescence intensities in p27. Nucleocytoplasmic localization analysis in p27 was performed. At least 30 transfected cells were examined and quantified for each group, and the data were plotted. The horizontal lines represent median values. *p < 0.05.
Mentions: The Cx43-mediated cell cycle suppression can be restored by co-overexpression with Hsc70 in HuH-7 cells15. To determine whether Cx43 overexpression-decreased nuclear accumulation of p27 can be restored by co-overexpression with Hsc70, HuH-7 cells were transfected with Cx43-mRFP alone or with both Cx43-mRFP and T7-Hsc70. As expected, co-overexpression of Cx43 with Hsc70 restored the nuclear accumulation of p27 in HuH-7 cells (Fig. 2a). Moreover, the quantitative analysis showed that the Cx43-reduced ratio of nuclear to cytoplasmic intensity of p27 was abolished by co-overexpression with Hsc70 (Fig. 2b). These experimental data demonstrated that the interaction of Cx43-Hsc70 regulates the nuclear accumulation of p27 as well as cyclin D1. Similar results were also obtained in PANC-1 cells, which are derived from human pancreatic cancer cells and express endogenous Cx4329 (see Supplementary Fig. S1).

Bottom Line: Here, we report that nuclear accumulation of p27 is reduced by overexpression of Cx43, and that this reduction is restored by co-overexpression with Hsc70.We found that Cx43 competes with p27 for binding to Hsc70, and as a result, decreases the level of Hsc70 in cyclin D1-CDK4-p27 complex, leading to prevention of the nuclear translocation of the complex and the G1/S transition.Collectively, our findings suggest that, in Cx43 up-regulation, which is most likely an emergency measure, Cx43-Hsc70 interaction regulates cell cycle G1/S progression through a novel mechanism by which Cx43-Hsc70 interaction prevents the nuclear accumulation of p27 through controlling the nuclear translocation of cyclin D1-CDK4-p27 complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kyoto, Japan.

ABSTRACT
Connexin 43 (Cx43) functions as a cell growth suppressor. We have demonstrated that Cx43 interacts with heat shock cognate protein 70 (Hsc70) for regulating cell proliferation. Hsc70 interacts with CDK inhibitor p27, which regulates the assembly and subcellular localization of cyclin D1-CDK4-p27 complex. However, the involvement of p27 with Cx43-mediated cell cycle suppression is still poorly understood. Here, we report that nuclear accumulation of p27 is reduced by overexpression of Cx43, and that this reduction is restored by co-overexpression with Hsc70. We found that Cx43 competes with p27 for binding to Hsc70, and as a result, decreases the level of Hsc70 in cyclin D1-CDK4-p27 complex, leading to prevention of the nuclear translocation of the complex and the G1/S transition. Collectively, our findings suggest that, in Cx43 up-regulation, which is most likely an emergency measure, Cx43-Hsc70 interaction regulates cell cycle G1/S progression through a novel mechanism by which Cx43-Hsc70 interaction prevents the nuclear accumulation of p27 through controlling the nuclear translocation of cyclin D1-CDK4-p27 complex.

No MeSH data available.


Related in: MedlinePlus