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The N-cadherin cytoplasmic domain confers anchorage-independent growth and the loss of contact inhibition.

Ozawa M - Sci Rep (2015)

Bottom Line: Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated.An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth.Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

ABSTRACT
Tumor growth is characterized by anchorage independence and the loss of contact inhibition. Previously, we showed that either a red fluorescent protein (DsRed)-tagged N-cadherin or E-cadherin cytoplasmic domain (DNCT or DECT) could function as a dominant negative inhibitor by blocking the cell surface localization of endogenous E-cadherin and inducing cell dissociation. Here, we show that expression of DNCT abrogated contact inhibition of proliferation and conferred anchorage-independent growth. DNCT expression induced the relocation of the tumor suppressor Merlin from the cell surface to intracellular compartments. Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated. An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth. Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

No MeSH data available.


Related in: MedlinePlus

DNCT expression induces the nuclear localization of TAZ.(a) Immunofluorescence staining of T23 MDCK cells expressing DsRed, DNCT, or DNCT and ELA (DNCT+ELA), or DNCT and ELAαC (DNCT+ELAαC). Cells were stained with anti-TAZ antibodies and DAPI. Although TAZ is excluded from the nucleus and is predominantly localized in the cytoplasm of DsRed+ cells, TAZ is distributed throughout DNCT+ cells, including a significant portion in the nucleus. Dox-induced reduction in DNCT expression caused the redistribution of TAZ from the nucleus to the cytoplasm. Expression of ELAαC in DNCT+ cells prevents nuclear localization of TAZ induced by DNCT. (b) Staining of DNCT+ cells with anti-YAP antibodies and DAPI. Cells were cultured for 2 d in the presence (+) or absence (−) of Dox. Bars, 25 μm. (c,d) Immunoblot detection of TAZ (c) and CTGF (d) revealed that the expression of DNCT does not change the amounts of these proteins. Vinculin was used as a loading control.
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f5: DNCT expression induces the nuclear localization of TAZ.(a) Immunofluorescence staining of T23 MDCK cells expressing DsRed, DNCT, or DNCT and ELA (DNCT+ELA), or DNCT and ELAαC (DNCT+ELAαC). Cells were stained with anti-TAZ antibodies and DAPI. Although TAZ is excluded from the nucleus and is predominantly localized in the cytoplasm of DsRed+ cells, TAZ is distributed throughout DNCT+ cells, including a significant portion in the nucleus. Dox-induced reduction in DNCT expression caused the redistribution of TAZ from the nucleus to the cytoplasm. Expression of ELAαC in DNCT+ cells prevents nuclear localization of TAZ induced by DNCT. (b) Staining of DNCT+ cells with anti-YAP antibodies and DAPI. Cells were cultured for 2 d in the presence (+) or absence (−) of Dox. Bars, 25 μm. (c,d) Immunoblot detection of TAZ (c) and CTGF (d) revealed that the expression of DNCT does not change the amounts of these proteins. Vinculin was used as a loading control.

Mentions: The Hippo signaling pathway plays an important role in contact inhibition of proliferation181936. In sparse cell cultures, YAP/TAZ is predominantly localized in the nucleus, but in dense cell cultures it is phosphorylated and translocated to the cytoplasm. Accordingly, we examined whether the localization of TAZ was affected by DNCT expression. Although TAZ was excluded from the nucleus and predominantly localized in the cytoplasm of DsRed+ cells, TAZ was distributed throughout DNCT+ cells, with a significant portion localized in the nucleus (Fig. 5a). Interestingly, Dox-induced reduction of DNCT expression led to the redistribution of TAZ from the nucleus to the cytoplasm. Likewise, a significant portion of YAP was localized in the nucleus (Fig. 5b). Dox-induced reduction of DNCT expression led to redistribution of YAP from the nucleus to the cytoplasm, although a portion of YAP remained in the nucleus. DNCT expression did not affect the levels of TAZ (Fig. 5c) or of connective tissue growth factor (CTGF), a well-known YAP/TAZ regulated gene product (Fig. 5d). The latter result suggests that TAZ signaling was not activated despite its nuclear localization.


The N-cadherin cytoplasmic domain confers anchorage-independent growth and the loss of contact inhibition.

Ozawa M - Sci Rep (2015)

DNCT expression induces the nuclear localization of TAZ.(a) Immunofluorescence staining of T23 MDCK cells expressing DsRed, DNCT, or DNCT and ELA (DNCT+ELA), or DNCT and ELAαC (DNCT+ELAαC). Cells were stained with anti-TAZ antibodies and DAPI. Although TAZ is excluded from the nucleus and is predominantly localized in the cytoplasm of DsRed+ cells, TAZ is distributed throughout DNCT+ cells, including a significant portion in the nucleus. Dox-induced reduction in DNCT expression caused the redistribution of TAZ from the nucleus to the cytoplasm. Expression of ELAαC in DNCT+ cells prevents nuclear localization of TAZ induced by DNCT. (b) Staining of DNCT+ cells with anti-YAP antibodies and DAPI. Cells were cultured for 2 d in the presence (+) or absence (−) of Dox. Bars, 25 μm. (c,d) Immunoblot detection of TAZ (c) and CTGF (d) revealed that the expression of DNCT does not change the amounts of these proteins. Vinculin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5: DNCT expression induces the nuclear localization of TAZ.(a) Immunofluorescence staining of T23 MDCK cells expressing DsRed, DNCT, or DNCT and ELA (DNCT+ELA), or DNCT and ELAαC (DNCT+ELAαC). Cells were stained with anti-TAZ antibodies and DAPI. Although TAZ is excluded from the nucleus and is predominantly localized in the cytoplasm of DsRed+ cells, TAZ is distributed throughout DNCT+ cells, including a significant portion in the nucleus. Dox-induced reduction in DNCT expression caused the redistribution of TAZ from the nucleus to the cytoplasm. Expression of ELAαC in DNCT+ cells prevents nuclear localization of TAZ induced by DNCT. (b) Staining of DNCT+ cells with anti-YAP antibodies and DAPI. Cells were cultured for 2 d in the presence (+) or absence (−) of Dox. Bars, 25 μm. (c,d) Immunoblot detection of TAZ (c) and CTGF (d) revealed that the expression of DNCT does not change the amounts of these proteins. Vinculin was used as a loading control.
Mentions: The Hippo signaling pathway plays an important role in contact inhibition of proliferation181936. In sparse cell cultures, YAP/TAZ is predominantly localized in the nucleus, but in dense cell cultures it is phosphorylated and translocated to the cytoplasm. Accordingly, we examined whether the localization of TAZ was affected by DNCT expression. Although TAZ was excluded from the nucleus and predominantly localized in the cytoplasm of DsRed+ cells, TAZ was distributed throughout DNCT+ cells, with a significant portion localized in the nucleus (Fig. 5a). Interestingly, Dox-induced reduction of DNCT expression led to the redistribution of TAZ from the nucleus to the cytoplasm. Likewise, a significant portion of YAP was localized in the nucleus (Fig. 5b). Dox-induced reduction of DNCT expression led to redistribution of YAP from the nucleus to the cytoplasm, although a portion of YAP remained in the nucleus. DNCT expression did not affect the levels of TAZ (Fig. 5c) or of connective tissue growth factor (CTGF), a well-known YAP/TAZ regulated gene product (Fig. 5d). The latter result suggests that TAZ signaling was not activated despite its nuclear localization.

Bottom Line: Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated.An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth.Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

ABSTRACT
Tumor growth is characterized by anchorage independence and the loss of contact inhibition. Previously, we showed that either a red fluorescent protein (DsRed)-tagged N-cadherin or E-cadherin cytoplasmic domain (DNCT or DECT) could function as a dominant negative inhibitor by blocking the cell surface localization of endogenous E-cadherin and inducing cell dissociation. Here, we show that expression of DNCT abrogated contact inhibition of proliferation and conferred anchorage-independent growth. DNCT expression induced the relocation of the tumor suppressor Merlin from the cell surface to intracellular compartments. Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated. An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth. Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

No MeSH data available.


Related in: MedlinePlus