Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-RasV12-induced cell transformation

Hsiu Kuan Lin, Hsi Hui Lin, Yu Wei Chiou, Ching Lung Wu, Wen-Tai Chiu, Ming-Jer Tang

Research output: Contribution to journalArticle

Abstract

Caveolin-1 (Cav1) is down-regulated during MK4 (MDCK cells harbouring inducible Ha-RasV12 gene) transformation by Ha-RasV12. Cav1 overexpression abrogates the Ha-RasV12-driven transformation of MK4 cells; however, the targeted down-regulation of Cav1 is not sufficient to mimic this transformation. Cav1-silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction-related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I-CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha-RasV12-inducing MK4 cells increased exosome-like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I-CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I-CM (MK4+I-EXs). Wnt5a, a downstream product of Ha-RasV12, was markedly secreted by MK4+I-CM and MK4+I-EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha-RasV12- and MK4+I-CM-induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down-regulation, either by Ha-RasV12 or targeted shRNA, increased frizzled-2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I-EXs in MDCK cells. These data suggest that Cav1-dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha-RasV12-Wnt5a-Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha-RasV12-driven cell transformation.

Original languageEnglish
Pages (from-to)2631-2643
Number of pages13
JournalJournal of Cellular and Molecular Medicine
Volume22
Issue number5
DOIs
Publication statusPublished - 2018 May 1

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Caveolin 1
Down-Regulation
Madin Darby Canine Kidney Cells
Exosomes
Conditioned Culture Medium
Small Interfering RNA
Porcupines
Frizzled Receptors
Isopropyl Thiogalactoside
Nanoparticles
Hot Temperature
Staining and Labeling
Messenger RNA

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Cell Biology

Cite this

@article{725326c5dd5047f5bedefbc6cc1d24fb,
title = "Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-RasV12-induced cell transformation",
abstract = "Caveolin-1 (Cav1) is down-regulated during MK4 (MDCK cells harbouring inducible Ha-RasV12 gene) transformation by Ha-RasV12. Cav1 overexpression abrogates the Ha-RasV12-driven transformation of MK4 cells; however, the targeted down-regulation of Cav1 is not sufficient to mimic this transformation. Cav1-silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction-related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I-CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha-RasV12-inducing MK4 cells increased exosome-like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I-CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I-CM (MK4+I-EXs). Wnt5a, a downstream product of Ha-RasV12, was markedly secreted by MK4+I-CM and MK4+I-EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha-RasV12- and MK4+I-CM-induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down-regulation, either by Ha-RasV12 or targeted shRNA, increased frizzled-2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I-EXs in MDCK cells. These data suggest that Cav1-dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha-RasV12-Wnt5a-Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha-RasV12-driven cell transformation.",
author = "Lin, {Hsiu Kuan} and Lin, {Hsi Hui} and Chiou, {Yu Wei} and Wu, {Ching Lung} and Wen-Tai Chiu and Ming-Jer Tang",
year = "2018",
month = "5",
day = "1",
doi = "10.1111/jcmm.13531",
language = "English",
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pages = "2631--2643",
journal = "Journal of Cellular and Molecular Medicine",
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Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-RasV12-induced cell transformation. / Lin, Hsiu Kuan; Lin, Hsi Hui; Chiou, Yu Wei; Wu, Ching Lung; Chiu, Wen-Tai; Tang, Ming-Jer.

In: Journal of Cellular and Molecular Medicine, Vol. 22, No. 5, 01.05.2018, p. 2631-2643.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-RasV12-induced cell transformation

AU - Lin, Hsiu Kuan

AU - Lin, Hsi Hui

AU - Chiou, Yu Wei

AU - Wu, Ching Lung

AU - Chiu, Wen-Tai

AU - Tang, Ming-Jer

PY - 2018/5/1

Y1 - 2018/5/1

N2 - Caveolin-1 (Cav1) is down-regulated during MK4 (MDCK cells harbouring inducible Ha-RasV12 gene) transformation by Ha-RasV12. Cav1 overexpression abrogates the Ha-RasV12-driven transformation of MK4 cells; however, the targeted down-regulation of Cav1 is not sufficient to mimic this transformation. Cav1-silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction-related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I-CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha-RasV12-inducing MK4 cells increased exosome-like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I-CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I-CM (MK4+I-EXs). Wnt5a, a downstream product of Ha-RasV12, was markedly secreted by MK4+I-CM and MK4+I-EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha-RasV12- and MK4+I-CM-induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down-regulation, either by Ha-RasV12 or targeted shRNA, increased frizzled-2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I-EXs in MDCK cells. These data suggest that Cav1-dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha-RasV12-Wnt5a-Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha-RasV12-driven cell transformation.

AB - Caveolin-1 (Cav1) is down-regulated during MK4 (MDCK cells harbouring inducible Ha-RasV12 gene) transformation by Ha-RasV12. Cav1 overexpression abrogates the Ha-RasV12-driven transformation of MK4 cells; however, the targeted down-regulation of Cav1 is not sufficient to mimic this transformation. Cav1-silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction-related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I-CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha-RasV12-inducing MK4 cells increased exosome-like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I-CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I-CM (MK4+I-EXs). Wnt5a, a downstream product of Ha-RasV12, was markedly secreted by MK4+I-CM and MK4+I-EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha-RasV12- and MK4+I-CM-induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down-regulation, either by Ha-RasV12 or targeted shRNA, increased frizzled-2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I-EXs in MDCK cells. These data suggest that Cav1-dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha-RasV12-Wnt5a-Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha-RasV12-driven cell transformation.

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