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Originally published In Press as doi:10.1167/iovs.08-2444 on October 20, 2008
 (Investigative Ophthalmology and Visual Science. 2009;50:626-633.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-2444
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Effect of cAMP on TGFβ1-Induced Corneal Keratocyte-Myofibroblast Transformation

Dongmei Xing and Joseph A. Bonanno

From the School of Optometry, Indiana University, Bloomington, Indiana.

PURPOSE. TGFβ is the major mediator to induce myofibroblast differentiation in the corneal wound-healing process. Elevated cAMP can reduce TGFβ-induced fibrosis in other tissues. This study was conducted to determine whether elevated cAMP can inhibit TGFβ1-induced rabbit corneal keratocyte-myofibroblast transformation.

METHODS. Primary isolated rabbit corneal keratocytes were cultured in serum-free medium. The effects of the adenylate cyclase agonist forskolin (FSK; 2 µM) on TGFβ1 (5 ng/mL)-induced {alpha}-smooth muscle actin ({alpha}-SMA) expression was examined by immunofluorescence, flow cytometry, and immunochemistry 72 hours after treatment. The effects of TGFβ+FSK on activated pSmad3, CREB binding protein (CBP), MAPKs, and RhoA were determined by coimmunoprecipitation and Western blot.

RESULTS. FSK significantly reduced the myofibroblast phenotype and {alpha}-SMA expression induced by TGFβ1 in rabbit corneal keratocytes. TGFβ1 increased the phosphorylation of ERK and Smad3. TGFβ1-induced {alpha}-SMA expression was reduced by MEK inhibition (U0126); however, the levels of pERK, pSmad3, or the extent of the interaction between pSmad3 and CBP induced by TGFβ1 were not affected by FSK. TGFβ1 also activated RhoA and ROCK (Y27632) inhibition reduced {alpha}-SMA expression. Activation of RhoA was significantly reduced by FSK.

CONCLUSIONS. Raising cAMP by FSK treatment inhibits the TGFβ1-induced corneal myofibroblast transformation and {alpha}-SMA expression and thereby provides a promising method to control corneal fibrosis. The data suggest that cAMP-dependent inhibition does not occur by altering Smads or MAPK signaling, but possibly by reducing the activation of RhoA.




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A. Kim, N. Lakshman, D. Karamichos, and W. M. Petroll
Growth Factor Regulation of Corneal Keratocyte Differentiation and Migration in Compressed Collagen Matrices
Invest. Ophthalmol. Vis. Sci., February 1, 2010; 51(2): 864 - 875.
[Abstract] [Full Text] [PDF]


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