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Emily Andreae, Debra Warejcka, Sally Twining; Thrombin regulates Cyr61 synthesis and processing in cultured human corneal stromal fibroblasts and myofibroblasts. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5248.
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Prothrombin is produced by the human cornea and is activated to thrombin. Previous research with human corneal epithelial cells indicated thrombin treatment increased Cyr61 (CCN1), an extracellular matrix associated protein related to CTGF (CCN2), at the mRNA and protein levels. In other systems, Cyr61 can regulate angiogenesis, fibrosis, and cell adhesion, migration, and apoptosis. Our goal was to characterize the role of thrombin in regulating Cyr61 mRNA and protein levels produced by human corneal stromal fibroblasts and myofibroblasts.
Donor human corneas were processed for stromal cells and put in culture. Cells were allowed to adhere for 24 hours then incubated with FGF-2 for fibroblasts or TGF-β for myofibroblasts. Cells were treated with α-thrombin and/or hirudin for varying amounts of time. Conditioned medium and cell lysates were collected and processed for mRNA analysis and SDS-PAGE. Samples were analyzed for Cyr61 mRNA by PCR and protein via immunoblot and normalized to GAPDH.
Thrombin treatment of human corneal stromal fibroblasts and myofibroblasts increased Cyr61 mRNA levels and full length 40 kDa cell-associated Cyr61 protein levels. Thrombin treatment resulted in increased levels of a 25 kDa form of extracellular Cyr61 in both cell types. Increased levels of the 40 and 25 kDa forms of Cyr61 are dependent on the proteolytic activity of thrombin. Cyr61 in non-conditioned medium was not cleaved by thrombin, indicating thrombin does not directly cleave this protein. Thrombin treatment of fibroblasts does not induce alternative splicing of Cyr61.
These results indicate thrombin regulates Cyr61 by increasing total Cyr61 mRNA and protein levels and by altering the molecular weight of secreted Cyr61 in human corneal stromal fibroblasts and myofibroblasts.
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