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Ching Yuan, Heidi Roehrich, Elizabeth F. Nelson, Dale Gregerson, Deborah Ferrington; Immunoproteasome Expression In Corneal Epithelium, And Its Roles In Tissue Homeostasis And Wound Healing. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1833.
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Our previous study showed that immunoproteasome is expressed in retinal neurons and retinal pigment epithelial cells and can be up-regulated with aging or injury, suggesting roles for immunoproteasome in maintaining retinal homeostasis and in the cellular stress response (Ferrington et al., J. Neurochem. 2008). The purpose of the current study was to investigate the expression of immunoproteasome and its potential roles in homeostasis and wound healing in murine cornea.
Corneas from WT and L7M1 KO (lmp7-/-/mecl1-/-) mice were used in this study. Immunostaining specific to the immunoproteasome subunit Lmp7 was performed to investigate immunoproteasome localization within WT corneal layers. Western blotting was used to evaluate immunoproteasome expression in protein extracts from the corneal epithelium. RT-PCR for immunoproteasome subunits was conducted on total RNA extracted from corneal explant cultures. Apoptosis was determined using a fluorogenic caspase-3 substrate and TUNEL staining on corneal sections and explant cultures, respectively. Corneal epithelial debridement was performed and the progression of wound healing was evaluated using morphometric analysis, H&E staining and immunostainings.
Immunostaining revealed a strong signal for Lmp7 in the WT corneal epithelium. Expression of immunoproteasome subunits (Lmp2, Lmp7 and MECL1) was confirmed both in the corneal epithelium and corneal explant cultures by RT-PCR and Western blots. We found higher apoptosis in both corneal explant cultures and sections of L7M1 KO mice compared to WT. L7M1 KO mice also displayed extensive stromal cell activation and myofibroblast transformation 24 hours after debridement based on immunostaining with anti-alpha-SMA and MCP-1 antibodies. On average, 2.3 times more myofibroblasts were found in the stroma of L7M1 KO mice compared to the WT. CD11b staining also indicated more infiltrating inflammatory cells at 48 hours after debridement in the KO mice.
Our study revealed immunoproteasome expression in non-inflammed WT mouse corneal epithelium. The high degree of apoptosis and compromised wound healing observed in the cornea of the L7M1 KO mice suggests immunoproteasome may play an important role in maintaining corneal homeostasis.
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