Thrombin alters the synthesis and processing of CYR61/CCN1 in human corneal stromal fibroblasts and myofibroblasts through multiple distinct mechanisms. Mol Vis 2020;26:540-562
Date
08/21/2020Pubmed ID
32818017Pubmed Central ID
PMC7406864Scopus ID
2-s2.0-85088944673 (requires institutional sign-in at Scopus site)Abstract
PURPOSE: Previous research in our laboratory indicated that prothrombin and other coagulation enzymes required to activate prothrombin to thrombin are synthesized by the cornea and that apoptotic human corneal stromal cells can provide a surface for prothrombin activation through the intrinsic and extrinsic coagulation pathways. The purpose of the work reported here is to study the role of thrombin activity in the regulation of matricellular protein Cyr61 (CCN1) produced by wounded phenotype human corneal stromal fibroblasts and myofibroblasts.
METHODS: Stromal cells from human donor corneas were converted to defined wounded phenotype fibroblasts and myofibroblasts with fetal bovine serum, followed by basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGFβ-1), respectively, and stimulated with varying concentrations (0-10.0 units (U)/ml) of thrombin from 1-7 h. Cyr61 transcript levels were determined using reverse transcriptase-PCR (RT-PCR) and quantitative PCR (qPCR) while protein forms were analyzed using western blot data. Protease activities were characterized via protease class-specific inhibitors and western blot analysis. Thrombin activity was quantified using the fluorogenic peptide Phe-Pro-Arg-AFC. Protease-activated receptor (PAR) agonist peptides-1 and -4 were used to determine whether cells increased Cyr61 through PAR signaling pathways. The PAR-1 antagonist SCH 79797 was used to block the thrombin cleavage of the receptor. PCR data were analyzed using MxPro software and western blot data were analyzed using Image Lab™ and Image J software. Student t test and one- and two-way ANOVA (with or without ranking, depending on sample distribution), together with Dunnett's test or Tukey comparison tests for post-hoc analysis, were used to determine statistical significance.
UNLABELLED: Results: Full-length Cyr61 is expressed by human corneal stromal fibroblasts and myofibroblasts and is significantly upregulated by active thrombin stimulation at the message (p<0.03) and protein (p<0.03) levels for fibroblasts and myofibroblasts. Inhibition by the allosteric thrombin-specific inhibitor hirudin prevented the thrombin-associated increase in the Cyr61 protein expression, indicating that the proteolytic activity of thrombin is required for the increase of the Cyr61 protein level. PAR-1 agonist stimulation of fibroblasts and myofibroblasts significantly increased cell-associated Cyr61 protein levels (p<0.04), and PAR-1 antagonist SCH 79797 significantly inhibited the thrombin stimulated increase of Cyr61 in fibroblasts but not in myofibroblasts. In the fibroblast and myofibroblast conditioned media, Cyr61 was detected as the full-length 40 kDa protein in the absence of thrombin, and mainly at 24 kDa in the presence of thrombin at ≥0.5 U/ml, using an antibody directed toward the internal linker region between the von Willebrand factor type C and thrombospondin type-1 domains. Although known to undergo alternative splicing, Cyr61 that is synthesized by corneal fibroblasts and myofibroblasts is not alternatively spliced in response to thrombin stimulation nor is Cyr61 directly cleaved by thrombin to generate its 24 kDa form; instead, Cyr61 is proteolytically processed into 24 kDa N- and 16 kDa C-terminal fragments by a thrombin activated leupeptin-sensitive protease present in conditioned media with activity distinct from the proteolytic activity of thrombin.
CONCLUSIONS: In cultured human corneal stromal fibroblasts and myofibroblasts, thrombin regulates Cyr61 through two mechanisms: 1) thrombin increases the Cyr61 expression at the message and protein levels, and 2) thrombin increases the activation of a leupeptin-sensitive protease that stimulates the cleavage of Cyr61 into N- and C-terminal domain populations in or near the thrombospondin type-1 domain. Generation of Cyr61 peptides during corneal injury stimulation may reveal additional functions of the protein, which
Author List
Andreae EA, Warejcka DJ, Twining SSAuthor
Sally S. Twining PhD Assistant Dean, Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Alternative SplicingCell Differentiation
Corneal Stroma
Culture Media, Conditioned
Cysteine-Rich Protein 61
Fibroblast Growth Factor 2
Fibroblasts
Hirudins
Humans
Leupeptins
Myofibroblasts
Primary Cell Culture
Proteolysis
Pyrroles
Quinazolines
RNA, Messenger
Receptors, Proteinase-Activated
Signal Transduction
Stromal Cells
Thrombin
Transforming Growth Factor beta1
