Abstract
Purpose:
After corneal injury or surgery, corneal keratocytes transform from a quiescent phenotype with a dendritic morphology to active fibroblast and/or myofibroblast phenotypes that generate and transmit contractile forces to the surrounding matrix. We have previously shown that the mechanical activity of these cells is mediated by growth factor induced activation of the cytoskeletal regulatory proteins Rho and Rac of the Rho GTPase family. In addition to growth factors, cytokines, and extracellular matrix (ECM) interactions, microRNAs also have the potential to control key signaling pathways during wound healing.
Methods:
Specific microRNAs were previously found by others to be differentially expressed during corneal wound healing after laser ablation in mice. Several of these microRNAs have been reported to modulate cell motility and tissue fibrosis through regulating members of the Rho GTPase superfamily in other tissues. Using qRT-PCR, we assayed the expression of these microRNAs in 2-D cultures of both human corneal fibroblasts (HTK cells) and rabbit corneal keratocytes (NRK cells). MicroRNA expression was also assayed before and after stimulation by TGF-β1(10 ng/ml) and treatment with histone deacetylase inhibitor Trichostatin A (50nM).
Results:
Endogenous expression of MiR-21 was abundant in both corneal fibroblasts and keratocytes. Furthermore, miR-21 was induced by TGF-β1 stimulation in both cell types. This induction was blocked by Trichostatin A. With regard to miR-205 expression, there was significantly higher endogenous expression in rabbit corneal keratocytes as compared to human corneal fibroblasts (HTK cells). Treatment with TGF-β1 increased miR-205 expression in HTK cells. However, corneal keratocytes, despite their higher endogenous expression, did not show a significant change in miR-205 expression after TGF-β1 treatment.
Conclusions:
Upregulation of miR-21 by TGF-β1 stimulation coincides temporally with the transformation of cells into myofibroblasts, and is consistent with previous reports of the pro-fibrotic role of this microRNA in TGF-β signaling. The reduction in miR-21 induced by Trichostatin A suggests that epigenetic modifications influence miR-21 gene expression. miR-205 does not appear to mediate TGF-β1 induced myofibroblast transformation of corneal keratocytes. Based on studies in other cell types, we hypothesize it instead may regulate cell spreading and migration induced by Rac activation.