p38 inhibitors prevent TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts

Invest Ophthalmol Vis Sci. 2006 Apr;47(4):1500-9. doi: 10.1167/iovs.05-0361.


Purpose: The role of mitogen-activated protein kinase (MAPK) pathways in TGF-beta-induced myofibroblast transdifferentiation of human tenon fibroblasts (HTFs) was investigated to identify potential pharmacologic targets for the inhibition of scarring after glaucoma surgery.

Methods: TGF-beta-dependent activation of Smad2, p38, and Erk-1/2 was examined by Western blot analysis. TGF-beta-induced mRNA expression of collagen Ialpha1, fibronectin, and the myofibroblast transdifferentiation marker alpha smooth muscle actin (alpha-SMA) was analyzed by real-time RT-PCR. alpha-SMA protein expression and subcellular distribution were determined by Western blot analysis and immunofluorescence cytochemistry. Fibroblast contractility was assessed in three-dimensional collagen gel contraction assays, stress fiber assembly with rhodamine-phalloidin stains, and confocal microscopy. Cell proliferation was measured with an MTT assay. Specific pharmacologic kinase inhibitors were used to characterize the involvement of MAPK-dependent pathways.

Results: TGF-beta stimulation of HTF induced a rapid and transient activation of Smad2 and Erk, whereas p38 activation was biphasic and sustained. After 24 hours of TGF-beta stimulation, increased levels of collagen Ialpha1, fibronectin, and alpha-SMA transcripts were detected. After 3 days of stimulation, HTF displayed increased alpha-SMA protein levels, enhanced contractility, and assembly of actin stress fibers. TGF-beta also induced HTF proliferation. Specific p38 inhibitors prevented all these aspects of TGF-beta-induced myofibroblastic transdifferentiation.

Conclusions: Pharmacologic inhibition of p38 abrogates TGF-beta-induced myofibroblast transdifferentiation, reduces extracellular matrix protein expression and HTF proliferation, and may therefore serve to inhibit scarring after glaucoma surgery.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / genetics
  • Blotting, Western
  • Cell Culture Techniques
  • Cell Differentiation / drug effects*
  • Cell Proliferation
  • Collagen Type I / genetics
  • Connective Tissue Cells / cytology*
  • Enzyme Inhibitors / pharmacology*
  • Fibroblasts / cytology*
  • Fibronectins / genetics
  • Humans
  • Microscopy, Fluorescence
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Smad2 Protein / metabolism
  • Transforming Growth Factor beta / pharmacology*
  • Up-Regulation
  • p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors*
  • p38 Mitogen-Activated Protein Kinases / metabolism


  • Actins
  • Collagen Type I
  • Enzyme Inhibitors
  • Fibronectins
  • RNA, Messenger
  • SMAD2 protein, human
  • Smad2 Protein
  • Transforming Growth Factor beta
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • p38 Mitogen-Activated Protein Kinases