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Journal of Cell Communication and Signaling

, Volume 12, Issue 3, pp 549–560 | Cite as

Ezrin regulates skin fibroblast size/mechanical properties and YAP-dependent proliferation

  • Chunji Quan
  • Yan Yan
  • Zhaoping Qin
  • Zhenhua Lin
  • Taihao Quan
Research Article

Abstract

Ezrin acts as a dynamic linkage between plasma membrane and cytoskeleton, and thus involved in many fundamental cellular functions. Yet, its potential role in human skin is virtually unknown. Here we investigate the role of Ezrin in primary skin fibroblasts, the major cells responsible extracellular matrix (ECM) production. We report that Ezrin play an important role in the maintenance of skin fibroblast size/mechanical properties and proliferation. siRNA-mediated Ezrin knockdown decreased fibroblast size and mechanical properties, and thus impaired the nuclear translocation of YAP, a protein commonly response to cell size and mechanical force. Functionally, depletion of Ezrin significantly inhibited YAP target gene expression and fibroblast proliferation. Conversely, restoration of YAP nuclear translocation by overexpression of constitutively active YAP reversed YAP target genes expression and rescued proliferation in Ezrin knockdown cells. These data reveal a novel role for Ezrin in maintenance of fibroblast size/mechanical force and regulating YAP-mediated proliferation.

Keywords

Ezrin Yap Cell size Mechanical properties 

Abbreviations

CCN1

Cysteine-rich protein 61

CCN2

Connective tissue growth factor

ECM

Extracellular matrix

YAP

Yes-associated protein

Notes

Acknowledgements

Y Yan is supported by Milstein Medical Asian American Partnership Foundation (2015 Fellowship Award in Skin Disease).

Funding information

This work was supported by a grant from the NIH (AG019364 to T Quan).

Supplementary material

12079_2017_406_MOESM1_ESM.pdf (59 kb)
Supplementary Figure 1 Ezrin siRNA #2 reduces cell size/mechanical properties and impairs YAP-dependent proliferation in primary human skin fibroblasts. Primary human skin fibroblasts were transfected with non-specific control siRNA or Ezrin siRNA #2 (20 nM) for 48 h. (A) Cell size was reduced by Ezrin siRNA #2. Cells were stained with CellTracker® fluorescent dye. Red fluorescence delineates cell cytoplasm; blue fluorescence delineates nuclei. The relative cell surface areas were quantified by ImageJ. Bars = 50 μm. N = 3. (B) Cell traction force (nN) was reduced by Ezrin siRNA #2. N = 3. (C) Cell tensile strength (Pa) was reduced by Ezrin siRNA #2. N = 3. (D) Cell deformation was increased by Ezrin siRNA #2. N = 3. Mechanical properties were determined by atomic force microscopy (AFM) PeakForce Quantitative NanoMechanics mode and analyzed by Nanoscope Analysis software. (E) Impaired YAP nuclear translocation was determined by immunostaining. Images represent three independent experiments. Blue fluorescence delineates nuclei. Bar = 50 μm. (F) Restoration of YAP nuclear translocation reversed YAP target gene expression. Cells were transfected with non-specific control siRNA or Ezrin siRNAs or Ezrin siRNAs plus constitutively active YAP for two days. CCN1 and CCN2 protein levels were determined by Western blots. Protein levels were normalized by β-actin as a loading control. Insets show representative Western blots. N = 3. (G) Ezrin knockdown inhibits fibroblasts proliferation via impaired YAP activity. Cells were transfected with non-specific control siRNA or Ezrin siRNAs or Ezrin siRNAs plus constitutively active YAP for two days. Cells were harvested two days after transfection and 2.5 × 105 cells were cultured in 60 mm plates. Cells were harvested at indicated days and counted. Data are expressed as mean±SEM, *p < 0.05 vs control. (PDF 59 kb)

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Copyright information

© The International CCN Society 2017

Authors and Affiliations

  1. 1.Department of PathologyAffiliated Hospital of Yanbian University Medical CollegeJilinPeople’s Republic of China
  2. 2.Department of Dermatology, Plastic Surgery HospitalChinese Academy of Medical Sciences & Peking Union Medical CollegeBeijingChina
  3. 3.Department of DermatologyUniversity of Michigan Medical SchoolAnn ArborUSA

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