title: [Effects of focal adhesional kinase signaling on TNF-α induced gelatinases activity in cornea epithelium].
abstract: OBJECTIVE
  To investigate the effect of focal adhesional kinase (FAK) on tumor necrosis factor α (TNF-α)-induced MMP-2 and -9 activities in cornea epithelium.
  
  
  METHODS
  Experimental research. The human corneal epithelial cells (HCE) were cultured in vitro. HCEs were incubated with different concentrations of TNF-α for 24 h, including 1 µg/L (group B), 10 µg/L (group C) and 100 µg/L (group D). The control group (group A) was incubated with phosphate buffer solution. The activities of MMPs were examined by gelatin zymography and the phosphorylation of FAK was examined by western blot analysis. FAK was down regulated by FAK siRNA following lipofectamine-mediated transfection in corneal epithelial cells. Down-regulation was confirmed using western blot analysis. Cells cultured with different concentrations of TNF-α (Groups B to D) and the control group (group A) was at similar volumes of media. Then the activities of MMP-2 and -9 were examined by gelatin zymography and the phosphorylation of FAK by western blot analysis. Statistical methods adopted one-way ANOVA and Tukey's honestly significant test between each group.
  
  
  RESULTS
  Gelatin zymography: Activities of MMP-2 and -9 in TNF-α treated groups were greater than those of the control group. The activity of MMP-2 in A, B, C and D groups was 124.06 ± 4.06, 146.72 ± 5.51, 241.18 ± 5.65 and 389.95 ± 4.44, respectively with F = 2960.91, P = 0.000. The activity of MMP-9 in A, B, C and D groups was 122.78 ± 5.86, 165.70 ± 7.90, 479.49 ± 6.22 and 495.88 ± 5.03 (F = 4937.46, P = 0.000). Significant differences were found in each two groups (P = 0.000). Western blot analysis:the phosphorylation of FAK (p-FAK) in test groups (10-100 ng/ml) were significantly greater than that in control group (p-FAK of group C and D was 0.52 ± 0.03 and 0.61 ± 0.06, F = 431.03, P = 0.000). p-FAK levels in 100 ng/ml group were greater than that in 10 ng/ml group (P = 0.005). After down-regulating the protein FAK, TNF-α had no effect on the activity of MMP-2 (The data of MMP-2 were 55.13 ± 0.66, 55.67 ± 0.43, 55.49 ± 0.20 and 55.91 ± 0.37 in groups A, B, C and D, F = 2.73, P = 0.079). We detected the increasing activity of MMP-9 in group C, D and p-FAK in group D (The data of MMP-9's activity were 80.48 ± 0.39, 81.26 ± 0.62, 84.43 ± 0.47, 85.56 ± 0.61 in groups A, B, C and D, F = 105.80, P = 0.000). The activity of MMP-9 in group D was stronger than that from the group C (P = 0.019). We just only detected a small quantity of p-FAK in group D (0.47 ± 0.05), which was weaker than that before down regulating the protein FAK (t = 5.03, P = 0.001).
  
  
  CONCLUSIONS
  Our results demonstrate the critical role of FAK in TNF-α induced activity of MMP-2 and -9 in human corneal epithelium cells. Blocking the FAK signaling pathway can reduce the activity of MMP-2 and -9 which may play an important role in prevention and treatment of corneal diseases.
authors.affiliation: Research Institute of Ophthalmology, Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.forename: Yan-ning
authors.initials: YN
authors.lastname: Yang
authors.forename: Yi-qiao
authors.initials: YQ
authors.lastname: Xing
authors.forename: Jing
authors.initials: J
authors.lastname: Yuan
authors.forename: Xuan
authors.initials: X
authors.lastname: Xiao
authors.forename: Ruo-xi
authors.initials: RX
authors.lastname: Wu
authors.forename: Wan-ju
authors.initials: WJ
authors.lastname: Yang
mesh_terms: D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D019573:Epithelium, Corneal / Q000187:drug effects / Q000201:enzymology*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation
chemical_list: D014409:Tumor Necrosis Factor-alpha; D051416:Focal Adhesion Protein-Tyrosine Kinases; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9