p38 inhibitors prevent TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts.
OBJECTIVE
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.
Meyer-Ter-Vehn
Tobias
T
Department of Ophthalmology, University of Würzburg, Germany. meyer_t3@augenklinik.uni-wuerzburg.de
Gebhardt
Susanne
S
Sebald
Walter
W
Buttmann
Mathias
M
Grehn
Franz
F
Schlunck
Günther
G
Knaus
Petra
P
D000199:Actins / Q000235:genetics; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation; D024042:Collagen Type I / Q000235:genetics; D003239:Connective Tissue Cells / Q000166:cytology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology*; D005353:Fibronectins / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051899:Smad2 Protein / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000199:Actins; D024042:Collagen Type I; D004791:Enzyme Inhibitors; D005353:Fibronectins; D012333:RNA, Messenger; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D016212:Transforming Growth Factor beta; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases