April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Thrombin Affects Cyr61 Synthesis and Processing Through Two Distinct Mechanisms
Author Affiliations & Notes
  • E. A. Andreae
    Medical College of Wisconsin, Milwaukee, Wisconsin
  • S. S. Twining
    Biochemistry and Ophthalmology,
    Medical College of Wisconsin, Milwaukee, Wisconsin
  • Footnotes
    Commercial Relationships  E.A. Andreae, None; S.S. Twining, None.
  • Footnotes
    Support  RO1-EY12731
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 1976. doi:
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      E. A. Andreae, S. S. Twining; Thrombin Affects Cyr61 Synthesis and Processing Through Two Distinct Mechanisms. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1976.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Thrombin increases Cyr61 (CCN1) message and protein expression in WI-38 fibroblasts and 1321N1 astrocytoma cells. Cyr61 is an extracellular matrix-associated protein that regulates cell adhesion, migration, and proliferation. Our goal is to determine how thrombin regulates Cyr61.

Methods: : Human corneal epithelial cells (HCEC-T; ATCC) or primary donor cells were treated with thrombin, thrombin inhibitors, PAR agonists, or PAR antagonists for varying amounts of time. The overlying media fraction was collected. Protein levels in the supernatant were normalized to cell protein. Samples were probed for Cyr61 protein levels by Western blot analysis. Total mRNA was collected from HCEC-T and analyzed by RT-PCR using Cyr61-specific primers.

Results: : Treatment of cultured human corneal epithelial cells with thrombin over time increased total Cyr61 protein level and induced the expression of a smaller form of Cyr61. Cyr61 conversion from a full-length form (~40 kDa) to a smaller form (~25 kDa) occurred in a thrombin-dependent manner. PCR analysis of cDNA from thrombin-stimulated and control HCEC-T showed that Cyr61 is not alternatively spliced in these cells. Cyr61 processing with thrombin stimulation progressed independently of the cell monolayer; the conditioned media contains a proenzyme activated by thrombin which cleaves Cyr61. Protease inhibition studies revealed that this enzyme is a chymotrypsin-like enzyme. PAR-1 agonists, antagonists, and thrombin washout experiments showed thrombin cleavage of PAR-1 is required to increase the protein expression of full-length Cyr61. Extracellular Cyr61 is then cleaved to the smaller form.

Conclusions: : Human corneal epithelial cells produce and secrete full-length Cyr61. Thrombin affects Cyr61 protein in two ways: 1) it increases the transcription and translation of Cyr61 through PAR-1 signaling and 2) it activates a chymotrypsin-like serine protease that cleaves full-length Cyr61. This processed form may enhance the migration of epithelial cells upon wounding.

Keywords: cornea: epithelium • cornea: basic science • wound healing 

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