Korean J Ophthalmol.  2012 Feb;26(1):45-48. 10.3341/kjo.2012.26.1.45.

The Role of Focal Adhesion Kinase in the TGF-beta-Induced Myofibroblast Transdifferentiation of Human Tenon's Fibroblasts

Affiliations
  • 1Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea. shhan222@yuhs.ac

Abstract

PURPOSE
To investigate the role of focal adhesion kinase (FAK) in transforming growth factor (TGF)-beta-induced myofibroblast transdifferentiation of human Tenon's fibroblasts.
METHODS
Primary cultured human Tenon's fibroblasts were exposed to TGF-beta1 for up to 48 hours. The mRNA levels of FAK, alpha smooth muscle actin (alphaSMA), and beta-actin were determined by quantitative real time reverse transcription polymerase chain reaction. The protein levels of collagen type I, FAK, phospho-FAK, alphaSMA, and beta-actin were determined by Western immunoblots. After the small interfering RNA targeting FAK (siRNA(FAK)) molecules were delivered into the cells, the expressions of alphaSMA proteins were determined by Western immunoblots.
RESULTS
In human Tenon's fibroblasts, TGF-beta1 significantly increased the mRNA and protein expressions of alphaSMA. However, when the action of FAK was inhibited using siRNAFAK, the TGF-beta1-induced expression of alphaSMA was attenuated.
CONCLUSIONS
Our data suggest that FAK may be associated with the TGF-beta1-induced transdifferentiation of human Tenon's fibroblasts to myofibroblasts, which is the essential step of subconjunctival fibrosis.

Keyword

Fibroblast; Focal adhesion protein-tyrosine kinases; Myofibroblast; Transforming growth factors

MeSH Terms

Actins/metabolism
Analysis of Variance
Blotting, Western
Cell Transdifferentiation/drug effects
Cells, Cultured
Collagen/metabolism
Fibroblasts/cytology/drug effects/metabolism
Focal Adhesion Protein-Tyrosine Kinases/*metabolism
Humans
Myofibroblasts
RNA, Messenger/metabolism
RNA, Small Interfering/metabolism
Real-Time Polymerase Chain Reaction
Transforming Growth Factor beta/*pharmacology

Figure

  • Fig. 1 Quantitative reverse transcription polymerase chain reaction for focal adhesion kinase (FAK) and α smooth muscle actin (αSMA) after exposure to 5 ng/mL of transforming growth factor-β1 for up to 16 hours. The level of target mRNA was calculated using a relative standard curve of β-actin and expressed as the mean ± SEM. *p < 0.05.

  • Fig. 2 Representative Western immunoblots and densitometric data for collagen type I (190 kDa), focal adhesion kinase (FAK, 125 kDa), phospho-FAK (pFAK, 125 kDa), and β-actin (43 kDa) in human Tenon's fibroblasts stimulated by 5 ng/mL of transforming growth factor-β1 for up to 48 hours.

  • Fig. 3 Representative Western immunoblots and densitometric data for α smooth muscle actin (43 kDa) in human Tenon's fibroblasts stimulated by 5 ng/mL of transforming growth factor (TGF)-β1 for up to 48 hours with or without the treatment of siRNA targeting FAK (siRNAFAK) molecules.


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