For better diagnosis and management of corneal wound healing problems it is important to understand the basic mechanisms that regulate cellular proliferation and alterations in the phenotypic characteristics of the cells that determine corneal transparency during wound healing. Keratocytes, corneal stromal cells that are quiescent in the normal adult cornea, are activated during wound healing to become proliferative fibroblasts or myofibroblasts with altered phenotypic characteristics. The goal of this project is to elucidate the signaling mechanisms that control and modulate: 1) the cellular proliferation (cell cycle progression) of corneal epithelial cells and activated corneal keratocytes and 2) the phenotypic changes upon the activation of keratocytes. Our previous studies demonstrated that the small GTPase RhoA, through its downstream target ROCK, regulates cell cycle progression in both corneal epithelial cells and activated corneal stromal cells in culture. We have also determined that upon keratocyte activation, the activation of mitogen activated protein kinases (MAPKs), INK and ERK, can lead to downregulation of the expression of several proteins that are critical to the maintenance of corneal transparency.
The Specific Aims of this competing renewal proposal test specific hypotheses and predictions about the signaling mechanism in cell cycle regulation in corneal epithelial cells and activated stromal cells. In tissue culture, we will perform functional analyses of the signaling proteins by inhibiting their expression with siRNAs and by modulating their activities with constitutively active or dominant negative recombinant proteins. Using these approaches we will address the following questions:
Aim 1) Are mDia, a downstream effector of RhoA, and LIMK, a downstream effector of ROCK, involved in the RhoA/ROCK mediated changes in cell cycle regulatory proteins, cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors (GDIs)?;
Aim 2) Does connexin 43, a gap junction protein regulate cell cycle progression?;
and Aim 3) Do small GTPases RhoA, Cdc42 and Rac, and the MAPKs, INK and ERK, regulate the expression of keratan sulfate proteoglycans (keratocan and lumican), corneal crystallins (ALDH1 and DTKT) and D3(IV) collagen upon keratocyte activation? This new information will further our knowledge of corneal homeostasis and wound healing mechanisms. It will lead to better diagnoses and treatments of persistent corneal wound healing problems that are related to dysregulated cellular proliferation, and to prevention of nontransparent scar tissue formation following injury, keratoplasty or refractive surgery.
| Lathrop, Kira L; Gupta, Divya; Kagemann, Larry et al. (2012) Optical coherence tomography as a rapid, accurate, noncontact method of visualizing the palisades of Vogt. Invest Ophthalmol Vis Sci 53:1381-7 |
| Gupta, Divya; Du, Yiqin; Piluek, Jordan et al. (2012) Ethyl pyruvate ameliorates endotoxin-induced corneal inflammation. Invest Ophthalmol Vis Sci 53:6589-99 |
| Chen, Jian; Wong-Chong, Julie; SundarRaj, Nirmala (2011) FGF-2- and TGF-?1-induced downregulation of lumican and keratocan in activated corneal keratocytes by JNK signaling pathway. Invest Ophthalmol Vis Sci 52:8957-64 |
| Harvey, Stephen A K; Guerriero, Emily; Charukamnoetkanok, Nahthai et al. (2010) Responses of cultured human keratocytes and myofibroblasts to ethyl pyruvate: a microarray analysis of gene expression. Invest Ophthalmol Vis Sci 51:2917-27 |
| Chen, Jian; Guerriero, Emily; Sado, Yoshikazu et al. (2009) Rho-mediated regulation of TGF-beta1- and FGF-2-induced activation of corneal stromal keratocytes. Invest Ophthalmol Vis Sci 50:3662-70 |
| Shen, Xiang; Koga, Takahisa; Park, Bum-Chan et al. (2008) Rho GTPase and cAMP/protein kinase A signaling mediates myocilin-induced alterations in cultured human trabecular meshwork cells. J Biol Chem 283:603-12 |
| Chen, Jian; Guerriero, Emily; Lathrop, Kira et al. (2008) Rho/ROCK signaling in regulation of corneal epithelial cell cycle progression. Invest Ophthalmol Vis Sci 49:175-83 |
| Guerriero, Emily; Chen, Jian; Sado, Yoshikazu et al. (2007) Loss of alpha3(IV) collagen expression associated with corneal keratocyte activation. Invest Ophthalmol Vis Sci 48:627-35 |
| Du, Yiqin; Sundarraj, Nirmala; Funderburgh, Martha L et al. (2007) Secretion and organization of a cornea-like tissue in vitro by stem cells from human corneal stroma. Invest Ophthalmol Vis Sci 48:5038-45 |
| Chen, Jianxin; Li, Hongxia; SundarRaj, Nirmala et al. (2007) Alpha-smooth muscle actin expression enhances cell traction force. Cell Motil Cytoskeleton 64:248-57 |
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