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Prolonged overexpression of Wnt10b induces epidermal keratinocyte transformation through activating EGF pathway

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Abstract

Wnt10b is a signaling protein regulating skin development and homeostasis, and the expression of Wnt10b is restricted to epidermal keratinocytes in embryonic and postnatal skin. Recent studies indicate an elevated expression of Wnt10b in skin tumors. However, how Wnt10b regulates skin tumorigenesis remains largely unknown. Here we report that continuous expression of Wnt10b mediates transformation of epidermal keratinocytes through activating genes involved in EGF/MAPK signaling pathways. We first established a prolonged Wnt10b overexpression system in JB6P− cells to represent the elevated Wnt10b expression level in skin keratinocytes. Through expression assays and observations under phase-contrast microscopy, prolonged expression of Wnt10b activated Wnt/β-catenin pathway and induced morphological changes of cells showing longer protrusions and multilayer growth, indicating early-stage cell transformation. Wnt10b also increased cellular proliferation and migration according to BrdU incorporation and cell mobility assays. Furthermore, multi-doses of AdWnt10b treatment to JB6P− cells induced colony formation, stronger invasive ability in transwell system, and anchorage-independent growth in agar gel. In molecular level, AdWnt10b treatment induced increased transcriptional expressions of Egf, downstream Mapk pathway factors, and MMPs. Administration of Wnt antagonist DKK1 blocked the tumor promotion process induced by Wnt10b. Taken together, these findings clearly demonstrate that Wnt10b promotes epidermal keratinocyte transformation through induced Egf pathway.

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Abbreviations

AdGFP:

Control adenoviruses

AdWnt10b:

Wnt10b adenoviruses

AP-1:

Activator protein

BCC:

Basal cell carcinoma

DKK1:

Dickkopf 1

ECM:

Extracellular matrix

EGF:

Epidermal growth factor

NF-KB:

Nuclear factor kappa B

PAGE:

Polyacrylamide gel electrophoresis

PBS:

Phosphate-buffered saline

PVDF:

Polyvinylidene fluoride

RT-PCR:

Reverse transcription-polymerase chain reaction

SCC:

Squamous cell carcinomas

SDS:

Sodium dodecyl sulfate

TNF-a:

Tumor necrosis factor-a

TPA:

12-O-tetradecanoylphorbol-13-acetate

Wnt10b:

Wingless-type MMTV integration site family, member 10B

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Acknowledgments

This study was supported by Grants 81171515 from National Nature Science Foundation of China and CSTC; Innovation and Attracting Talents Program for College and University (‘111’ Project) (B06023), China; and the “BaYu” Scholars Project in Chongqing for Dr. Cheng-Ming Chuong. We thank Dr. Pin-Chi Tang (National Chung Hsing University) and Dr. Ya-Chen Liang (University of Southern California) for carefully revising the manuscript.

Conflict of interest

The authors declare no conflict of interest.

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    Authors and Affiliations

    1. Department of Cell Biology, the Third Military Medical University, Chongqing, 400038, People’s Republic of China

      Mingxing Lei, Xiangdong Lai, Xiufeng Bai, Weiming Qiu, Tian Yang & Xiaohua Lian

    2. Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing, 400044, People’s Republic of China

      Li Yang & Julia Li Zhong

    3. ‘111’ Project Laboratory of Biomechanics and Tissue Repair, Bioengineering College, Chongqing University, Chongqing, 400044, People’s Republic of China

      Li Yang & Julia Li Zhong

    4. Institute of Biomedical Engineering, Chongqing University of Science and Technology, Chongqing, 401331, People’s Republic of China

      Xiaoling Liao

    5. Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

      Cheng-Ming Chuong

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    1. Mingxing Lei
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    2. Xiangdong Lai
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    Correspondence to Xiaohua Lian or Julia Li Zhong.

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    M. Lei and X. Lai have contributed equally to this work.

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    Lei, M., Lai, X., Bai, X. et al. Prolonged overexpression of Wnt10b induces epidermal keratinocyte transformation through activating EGF pathway. Histochem Cell Biol 144, 209–221 (2015). https://doi.org/10.1007/s00418-015-1330-6

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