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

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.

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.

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.

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.