Hypoxia reduces TGFbeta1-induced corneal keratocyte myofibroblast transformation.
OBJECTIVE
The purpose of this study was to determine whether transient hypoxia had an effect on transforming growth factor beta1 (TGFbeta1)-induced rabbit corneal keratocyte myofibroblast transformation.


METHODS
Primary isolated rabbit corneal keratocytes were cultured in a serum-free medium. The effect of transient hypoxia treatment (1% oxygen, 4 h/day) on TGFbeta1 (5 ng/ml)-induced alpha-smooth muscle actin (alpha-SM actin) expression was examined by immunofluorescence, flow cytometry, and immunocytochemistry 72 h after treatment. We found that hypoxia treatment significantly reduced the myofibroblast phenotype and alpha-SM actin expression that was induced by TGFbeta1. To explore the possible mechanism for this effect, we screened for the effects of hypoxia on several early TGFbeta-dependent signaling events including activated pSmad3, CREB (cAMP response element binding) binding protein (CBP), MAPKs (Mitogen-activated protein kinase), and RhoA by co-immunoprecipitation and western blotting.


RESULTS
Hypoxia alone increased alpha-SM actin expression and the association of pSmad3 to CBP, but it did not induce the myofibroblast phenotype. The levels of pERK (the extracellular signal-regulated protein kinase) and pSmad3 or the extent of the interaction between pSmad3 and CBP induced by TGFbeta1 were not affected by hypoxia whereas the activation of RhoA induced by TGFbeta1 was significantly reduced.


CONCLUSIONS
We conclude that hypoxia can inhibit TGFbeta1-induced corneal myofibroblast transformation and alpha-SM actin expression. Our data show that this inhibition does not occur by altering Smads or MAPK signaling but possibly by reducing the early activation of RhoA.
Xing
Dongmei
D
School of Optometry, Indiana University, Bloomington, IN 47405, USA.
Bonanno
Joseph A
JA
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects; D002461:Cell Line, Transformed; D003315:Cornea / Q000166:cytology*; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011817:Rabbits; D051900:Smad3 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D010750:Phosphoproteins; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; D048049:Extracellular Signal-Regulated MAP Kinases; D020742:rhoA GTP-Binding Protein