Abstract
Purpose:
Embryonic corneas heal scar free and show a transient expression of alpha smooth muscle actin (αSMA). We sought to elucidate the mechanistic regulation of the myofibroblast phenotype transition during scar free corneal regeneration.
Methods:
Differential expression of BMPs was screened during various stages of embryonic cornea regeneration via RT-PCR. Spatiotemporal expression of TGFβ-2 and BMP-3 mRNA was identified in sections of regenerating embryonic corneas by in situ hybridization. The temporal response of keratocytes to TGF-β and BMP signaling in regenerating corneas were determined by immunostaining for pSMAD2 and pSMAD1/5/8 respectively. Primary cultures of embryonic keratocytes were treated with BMP-3; a novel antagonist to TGFβ mediated myofibroblast differentiation. To determine the effect of BMP-3 on TGFβ in embryonic keratocytes, the number of αSMA-positive cells was analyzed in primary cultures treated with BMP-3 and/or TGFβ-2. The activity of TGFβ in the presence BMP-3 was assayed by measuring the transcript levels of downstream target genes of TGFβ via qPCR.
Results:
Robust expression of TGFβ-2 and BMP-3 was identified in regenerating embryonic corneal stroma and epithelium, respectively. Nuclear localization of pSMAD2 and pSMAD1/5/8 respectively correlated with the expression and down regulation of αSMA. In vitro, BMP-3 was identified as a potent antagonist to the induction of myofibroblast phenotype in primary embryonic keratocytes. Interestingly, transcript analysis indicated that BMP-3 does not inhibit TGFβ mediated expression of αSMA. However, cells treated with BMP-3 expressed low levels of focal adhesion components and assumed a spherical morphology.
Conclusions:
These data build a mechanistic model for embryonic cornea regeneration, by which TGFβ mediated myofibroblast differentiation is negatively regulated by BMP-3. While BMP-3 does not directly inhibit TGFβ-2 activity, focal adhesion appears to be abrogated by BMP-3, which may prevent myofibroblast maturation.