title: Down-regulation of 14-3-3 Zeta Inhibits TGF-β1-Induced Actomyosin Contraction in Human Trabecular Meshwork Cells Through RhoA Signaling Pathway.
abstract: OBJECTIVE
  The aim of this study was to describe the expression and distribution of 14-3-3 zeta in trabecular meshwork (TM) cells and its regulatory role in the actomyosin system.
  
  
  METHODS
  The expression of 14-3-3 zeta was detected using Western blot analysis, RT-PCR, and immunofluorescence staining. TGF-β1 was used to induce cell contraction. Changes in the levels of 14-3-3 zeta, total RhoA, and the phosphorylation of myosin light chain (MLC) and cofilin were determined using Western blot analysis. The effects of 14-3-3 zeta knockdown on the actin cytoskeleton and focal adhesion were determined using immunofluorescence. The mRNA levels of fibronectin and collagen I and III were examined using quantitative RT-PCR. The contraction of TM cells was detected using collagen gel contraction (CGC) assays. The activation of the RhoA pathway was analyzed using a specific kit.
  
  
  RESULTS
  The 14-3-3 zeta protein was highly expressed in TM cells. Down-regulation of 14-3-3 zeta resulted in the following: a decrease in the phosphorylation of both MLC and cofilin, a decrease in the formation of stress fibers and focal adhesion, alteration of the mRNA composition of the extracellular matrix (ECM), and the inhibition of TGF-β1-induced cell contraction. In addition, silencing of 14-3-3 zeta directly decreased total RhoA levels in TM cells.
  
  
  CONCLUSIONS
  Collectively, our data suggest that 14-3-3 zeta plays a crucial role in regulating cytoskeletal structures, ECM homeostasis, and TGF-β1-induced contraction in TM cells by acting through the RhoA signaling pathway.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Ye
authors.forename: Yangfan
authors.initials: Y
authors.lastname: Yang
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Cai
authors.forename: Liling
authors.initials: L
authors.lastname: Liu
authors.forename: Kaili
authors.initials: K
authors.lastname: Wu
authors.forename: Minbin
authors.initials: M
authors.lastname: Yu
mesh_terms: D048948:14-3-3 Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism*; D001082:Aqueous Humor / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D051338:Cofilin 1 / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D015536:Down-Regulation; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D000199:Actins; C495810:CFL1 protein, human; D051338:Cofilin 1; D005353:Fibronectins; D018994:Myosin Light Chains; D053773:Transforming Growth Factor beta1; C572235:YWHAZ protein, human; D003094:Collagen; D000205:Actomyosin; D020742:rhoA GTP-Binding Protein