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 LinEmail author
  • Taihao QuanEmail author
Research Article


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.


Ezrin Yap Cell size Mechanical properties 



Cysteine-rich protein 61


Connective tissue growth factor


Extracellular matrix


Yes-associated protein



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)


  1. Akamine R, Yamamoto T, Watanabe M, Yamazaki N, Kataoka M, Ishikawa M, Ooie T, Baba Y, Shinohara Y (2007) Usefulness of the 5′ region of the cDNA encoding acidic ribosomal phosphoprotein P0 conserved among rats, mice, and humans as a standard probe for gene expression analysis in different tissues and animal species. J Biochem Biophys Methods 70:481–486CrossRefPubMedGoogle Scholar
  2. Arpin M, Chirivino D, Naba A, Zwaenepoel I (2011) Emerging role for ERM proteins in cell adhesion and migration. Cell Adhes Migr 5:199–206CrossRefGoogle Scholar
  3. Bretscher A, Edwards K, Fehon RG (2002) ERM proteins and merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol 3:586–599CrossRefPubMedGoogle Scholar
  4. Butcher DT, Alliston T, Weaver VM (2009) A tense situation: forcing tumour progression. Nat Rev Cancer 9:108–122CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chan SW, Lim CJ, Chong YF, Pobbati AV, Huang C, Hong W (2011) Hippo pathway-independent restriction of TAZ and YAP by angiomotin. J Biol Chem 286:7018–7026CrossRefPubMedPubMedCentralGoogle Scholar
  6. Clucas J, Valderrama F (2014) ERM proteins in cancer progression. J Cell Sci 127:267–275CrossRefPubMedGoogle Scholar
  7. Crepaldi T, Gautreau A, Comoglio PM, Louvard D, Arpin M (1997) Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells. J Cell Biol 138:423–434CrossRefPubMedPubMedCentralGoogle Scholar
  8. Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, Zanconato F, Le Digabel J, Forcato M, Bicciato S et al (2011) Role of YAP/TAZ in mechanotransduction. Nature 474:179–183CrossRefPubMedGoogle Scholar
  9. Fehon RG, McClatchey AI, Bretscher A (2010) Organizing the cell cortex: the role of ERM proteins. Nat Rev Mol Cell Biol 11:276–287CrossRefPubMedPubMedCentralGoogle Scholar
  10. Fisher GJ, Quan T, Purohit T, Shao Y, Cho MK, He T, Varani J, Kang S, Voorhees JJ (2009) Collagen fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1 in fibroblasts in aged human skin. Am J Pathol 174:101–114CrossRefPubMedPubMedCentralGoogle Scholar
  11. Fisher GJ, Shao Y, He T, Qin Z, Perry D, Voorhees JJ, Quan T (2016) Reduction of fibroblast size/mechanical force down-regulates TGF-beta type II receptor: implications for human skin aging. Aging Cell 15:67–76CrossRefPubMedGoogle Scholar
  12. Fisher GJ, Varani J, Voorhees JJ (2008) Looking older: fibroblast collapse and therapeutic implications. Arch Dermatol 144:666–672CrossRefPubMedPubMedCentralGoogle Scholar
  13. Gautreau A, Louvard D, Arpin M (2000) Morphogenic effects of ezrin require a phosphorylation-induced transition from oligomers to monomers at the plasma membrane. J Cell Biol 150:193–203CrossRefPubMedPubMedCentralGoogle Scholar
  14. Harvey KF, Hariharan IK (2012) The hippo pathway. Cold Spring Harb Perspect Biol 4:a011288CrossRefPubMedPubMedCentralGoogle Scholar
  15. Hiscox S, Jiang WG (1999) Ezrin regulates cell-cell and cell-matrix adhesion, a possible role with E-cadherin/beta-catenin. J Cell Sci 112(Pt 18):3081–3090PubMedGoogle Scholar
  16. Hsu YY, Shi GY, Kuo CH, Liu SL, Wu CM, Ma CY, Lin FY, Yang HY, Wu HL (2012) Thrombomodulin is an ezrin-interacting protein that controls epithelial morphology and promotes collective cell migration. FASEB J 26:3440–3452CrossRefPubMedGoogle Scholar
  17. Jin J, Jin T, Quan M, Piao Y, Lin Z (2012) Ezrin overexpression predicts the poor prognosis of gastric adenocarcinoma. Diagn Pathol 7:135CrossRefPubMedPubMedCentralGoogle Scholar
  18. Jin T, Jin J, Li X, Zhang S, Choi YH, Piao Y, Shen X, Lin Z (2014) Prognostic implications of ezrin and phosphorylated ezrin expression in non-small cell lung cancer. BMC Cancer 14:191CrossRefPubMedPubMedCentralGoogle Scholar
  19. Jung Y, McCarty JH (2012) Band 4.1 proteins regulate integrin-dependent cell spreading. Biochem Biophys Res Commun 426:578–584CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kong J, Di C, Piao J, Sun J, Han L, Chen L, Yan G, Lin Z (2016) Ezrin contributes to cervical cancer progression through induction of epithelial-mesenchymal transition. Oncotarget 7:19631–19642PubMedPubMedCentralGoogle Scholar
  21. Kong J, Li Y, Liu S, Jin H, Shang Y, Quan C, Li Y, Lin Z (2013) High expression of ezrin predicts poor prognosis in uterine cervical cancer. BMC Cancer 13:520CrossRefPubMedPubMedCentralGoogle Scholar
  22. Krieg M, Helenius J, Heisenberg CP, Muller DJ (2008) A bond for a lifetime: employing membrane nanotubes from living cells to determine receptor-ligand kinetics. Angew Chem Int Ed Engl 47:9775–9777CrossRefPubMedGoogle Scholar
  23. Larson SM, Lee HJ, Hung PH, Matthews LM, Robinson DN, Evans JP (2010) Cortical mechanics and meiosis II completion in mammalian oocytes are mediated by myosin-II and Ezrin-radixin-Moesin (ERM) proteins. Mol Biol Cell 21:3182–3192CrossRefPubMedPubMedCentralGoogle Scholar
  24. Li Q, Gao H, Xu H, Wang X, Pan Y, Hao F, Qiu X, Stoecker M, Wang E, Wang E (2012) Expression of ezrin correlates with malignant phenotype of lung cancer, and in vitro knockdown of ezrin reverses the aggressive biological behavior of lung cancer cells. Tumour Biol 33:1493–1504CrossRefPubMedGoogle Scholar
  25. Liu Y, Belkina NV, Park C, Nambiar R, Loughhead SM, Patino-Lopez G, Ben-Aissa K, Hao JJ, Kruhlak MJ, Qi H et al (2012) Constitutively active ezrin increases membrane tension, slows migration, and impedes endothelial transmigration of lymphocytes in vivo in mice. Blood 119:445–453CrossRefPubMedPubMedCentralGoogle Scholar
  26. Mammoto A, Ingber DE (2009) Cytoskeletal control of growth and cell fate switching. Curr Opin Cell Biol 21:864–870CrossRefPubMedGoogle Scholar
  27. Mammoto A, Mammoto T, Ingber DE (2012) Mechanosensitive mechanisms in transcriptional regulation. J Cell Sci 125:3061–3073CrossRefPubMedPubMedCentralGoogle Scholar
  28. Moroishi T, Hansen CG, Guan KL (2015) The emerging roles of YAP and TAZ in cancer. Nat Rev Cancer 15:73–79CrossRefPubMedPubMedCentralGoogle Scholar
  29. Naba A, Reverdy C, Louvard D, Arpin M (2008) Spatial recruitment and activation of the Fes kinase by ezrin promotes HGF-induced cell scattering. EMBO J 27:38–50CrossRefPubMedGoogle Scholar
  30. Neisch AL, Fehon RG (2011) Ezrin, radixin and Moesin: key regulators of membrane-cortex interactions and signaling. Curr Opin Cell Biol 23:377–382CrossRefPubMedPubMedCentralGoogle Scholar
  31. Ng T, Parsons M, Hughes WE, Monypenny J, Zicha D, Gautreau A, Arpin M, Gschmeissner S, Verveer PJ, Bastiaens PI et al (2001) Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility. EMBO J 20:2723–2741CrossRefPubMedPubMedCentralGoogle Scholar
  32. Piao J, Liu S, Xu Y, Wang C, Lin Z, Qin Y, Liu S (2015) Ezrin protein overexpression predicts the poor prognosis of pancreatic ductal adenocarcinomas. Exp Mol Pathol 98:1–6CrossRefPubMedGoogle Scholar
  33. Plouffe SW, Hong AW, Guan KL (2015) Disease implications of the hippo/YAP pathway. Trends Mol Med 21:212–222CrossRefPubMedPubMedCentralGoogle Scholar
  34. Pujuguet P, Del Maestro L, Gautreau A, Louvard D, Arpin M (2003) Ezrin regulates E-cadherin-dependent adherens junction assembly through Rac1 activation. Mol Biol Cell 14:2181–2191CrossRefPubMedPubMedCentralGoogle Scholar
  35. Qin Z, Voorhees JJ, Fisher GJ, Quan T (2014) Age-associated reduction of cellular spreading/mechanical force up-regulates matrix metalloproteinase-1 expression and collagen fibril fragmentation via c-Jun/AP-1 in human dermal fibroblasts. Aging Cell 13:1028–1037CrossRefPubMedPubMedCentralGoogle Scholar
  36. Quan C, Cho MK, Perry D, Quan T (2015) Age-associated reduction of cell spreading induces mitochondrial DNA common deletion by oxidative stress in human skin dermal fibroblasts: implication for human skin connective tissue aging. J Biomed Sci 22:62CrossRefPubMedPubMedCentralGoogle Scholar
  37. Quan T, Fisher GJ (2015) Role of age-associated alterations of the dermal extracellular matrix microenvironment in human skin aging: a mini-review. Gerontology 61:427–434CrossRefPubMedPubMedCentralGoogle Scholar
  38. Quan T, Little E, Quan H, Qin Z, Voorhees JJ, Fisher GJ (2013a) Elevated matrix metalloproteinases and collagen fragmentation in photodamaged human skin: impact of altered extracellular matrix microenvironment on dermal fibroblast function. J Invest Dermatol 133:1362–1366CrossRefPubMedPubMedCentralGoogle Scholar
  39. Quan T, Qin Z, Voorhees JJ, Fisher GJ (2012) Cysteine-rich protein 61 (CCN1) mediates replicative senescence-associated aberrant collagen homeostasis in human skin fibroblasts. J Cell Biochem 113:3011–3018CrossRefPubMedGoogle Scholar
  40. Quan T, Wang F, Shao Y, Rittie L, Xia W, Orringer JS, Voorhees JJ, Fisher GJ (2013b) Enhancing structural support of the dermal microenvironment activates fibroblasts, endothelial cells, and keratinocytes in aged human skin in vivo. J Invest Dermatol 133:658–667CrossRefPubMedGoogle Scholar
  41. Rouven Bruckner B, Pietuch A, Nehls S, Rother J, Janshoff A (2015) Ezrin is a major regulator of membrane tension in epithelial cells. Sci Rep 5:14700CrossRefPubMedPubMedCentralGoogle Scholar
  42. Sato N, Funayama N, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S (1992) A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites. J Cell Sci 103(Pt 1):131–143PubMedGoogle Scholar
  43. Takeuchi K, Sato N, Kasahara H, Funayama N, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S (1994) Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J Cell Biol 125:1371–1384CrossRefPubMedGoogle Scholar
  44. Varani J, Schuger L, Dame MK, Leonard C, Fligiel SE, Kang S, Fisher GJ, Voorhees JJ (2004) Reduced fibroblast interaction with intact collagen as a mechanism for depressed collagen synthesis in photodamaged skin. J Invest Dermatol 122:1471–1479CrossRefPubMedGoogle Scholar
  45. Wang F, Garza LA, Kang S, Varani J, Orringer JS, Fisher GJ, Voorhees JJ (2007) In vivo stimulation of de novo collagen production caused by cross-linked hyaluronic acid dermal filler injections in photodamaged human skin. Arch Dermatol 143:155–163PubMedGoogle Scholar
  46. Zhao B, Tumaneng K, Guan KL (2011) The hippo pathway in organ size control, tissue regeneration and stem cell self-renewal. Nat Cell Biol 13:877–883CrossRefPubMedPubMedCentralGoogle Scholar

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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