Thrombin alters the synthesis and processing of CYR61/CCN1 in human corneal stromal fibroblasts and myofibroblasts through multiple distinct mechanisms.

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 (TGFß-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.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 modulate corneal wound healing activities or decrease activities of the full-length Cyr61 form.

Expression of insulin-like growth factor 2 receptor in corneal keratocytes during differentiation and in response to wound healing.

PURPOSE: Insulin-like growth factor 2 receptor (IGF2R) associates with ligands that influence wound healing outcomes. However, the expression pattern of IGF2R and its role in the cornea is unknown. METHODS: Human keratocytes were isolated from donor corneas. Fibroblasts (fibroblast growth factor 2 [FGF2]-treated) or myofibroblasts (TGF-ß1-treated) were analyzed for IGF2R and α-smooth muscle actin (α-SMA) expression by Western blotting and immunolocalization. Mouse corneas were wounded in vivo and porcine corneas ex vivo. The IGF2R and α-SMA protein expression were visualized and quantified by immunohistochemistry. The IGF2R gene expression in human corneal fibroblasts was knocked-down with targeted lentiviral shRNA. RESULTS: The IGF2R is expressed in epithelial and stromal cells of normal human, mouse, and porcine corneas. The IGF2R increases (11.2 ± 0.4-fold) in the epithelial and (11.7 ± 0.9-fold) stromal layers of in vivo wounded mouse corneas. Double-staining with α-SMA- and IGF2R-specific antibodies reveals that IGF2R protein expression is increased in stromal myofibroblasts in the wounded cornea relative to keratocytes in the normal cornea (11.2 ± 0.8-fold). Human primary stromal keratocytes incubated with FGF2 or TGF-ß1 in vitro demonstrate increased expression (2.0 ± 0.4-fold) of IGF2R in myofibroblasts relative to fibroblasts. Conversion of IGF2R shRNA-lentiviral particle transduced corneal fibroblasts to myofibroblasts reveals a dependence on IGF2R expression, as only 40% ± 10% of cells transduced converted to myofibroblasts compared to 86% ± 3% in control cells. CONCLUSIONS: The IGF2R protein expression is increased during corneal wound healing and IGF2R regulates human corneal fibroblast to myofibroblast differentiation.

Maspin increases extracellular plasminogen activator activity associated with corneal fibroblasts and myofibroblasts.

Maspin, an inhibitor of cell migration and a stimulator of adhesion of cells to the ECM, is synthesized and released by corneal keratocytes into the extracellular matrix. When the cornea is wounded, the quiescent stromal keratocytes underlying the wound undergo apoptosis and cells adjacent to this apoptotic area convert to fibroblasts or myofibroblasts. This study explores the effect of extracellular maspin on the plasminogen-plasminogen activator system of corneal stromal cells following wounding. Treatment of corneal fibroblasts and myofibroblasts with r-maspin increased extracellular but not cell-associated tissue-type plasminogen activator (tPA), urinary-type plasminogen activator (uPA) or plasminogen activator inhibitor-1 (PAI-1). Despite the extracellular increase in PAI-1, the net effect of maspin treatment was an increase in plasminogen activation. At physiological levels, maspin did not alter uPA or tPA mRNA levels, in these cells. The increase in pro and active uPA was due to decreased clearance in the presence of maspin for myofibroblasts but not for fibroblasts. The clearance of pro and active tPA was normal in fibroblasts indicating different mechanisms for the increase of these homologous enzymes in the two cell types. Increased generation of plasmin by maspin treated corneal stromal fibroblasts and myofibroblasts led to conversion of plasminogen to active plasmin degradation products and angiostatin-like molecules. This study suggests that extracellular maspin increased pro and active uPA and tPA released by corneal fibroblasts and myofibroblasts on the short time scale of 1-4 h, but by 24 h there was no increase over the levels produced without maspin. This augmentation of plasminogen activator activity increases plasmin activation and angiostatin generation. It further indicates that the effect of maspin on uPA and tPA levels is cell type dependent.

Maspin, the molecular bridge between the plasminogen activator system and beta1 integrin that facilitates cell adhesion.

Maspin is a non-inhibitory serine protease inhibitor (serpin) that influences many cellular functions including adhesion, migration, and invasion. The underlying molecular mechanisms that facilitate these actions are still being elucidated. In this study we determined the mechanism by which maspin mediates increased MCF10A cell adhesion. Utilizing competition peptides and mutation analyses, we discovered two unique regions (amino acid residues 190-202 and 260-275) involved in facilitating the increased adhesion function of maspin. In addition, we demonstrate that the urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) complex is required for the localization and adhesion function of maspin. Finally, we showed that maspin, uPAR, and ß1 integrin co-immunoprecipitate, suggesting a novel maspin-uPA-uPAR-ß1 integrin mega-complex that regulates mammary epithelial cell adhesion.

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts.

Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down-regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation is involved in the mechanism of down-regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-beta1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for Western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-beta1 were hypermethylated. Down-regulation of maspin synthesis was also associated with histone H3 dimethylation at lysine 9. Both maspin mRNA and protein were re-expressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down-regulation of maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation.

Urokinase receptor cleavage: a crucial step in fibroblast-to-myofibroblast differentiation.

Fibroblasts migrate into and repopulate connective tissue wounds. At the wound edge, fibroblasts differentiate into myofibroblasts, and they promote wound closure. Regulated fibroblast-to-myofibroblast differentiation is critical for regenerative healing. Previous studies have focused on the role in fibroblasts of urokinase plasmingen activator/urokinase plasmingen activator receptor (uPA/uPAR), an extracellular protease system that promotes matrix remodeling, growth factor activation, and cell migration. Whereas fibroblasts have substantial uPA activity and uPAR expression, we discovered that cultured myofibroblasts eventually lost cell surface uPA/uPAR. This led us to investigate the relevance of uPA/uPAR activity to myofibroblast differentiation. We found that fibroblasts expressed increased amounts of full-length cell surface uPAR (D1D2D3) compared with myofibroblasts, which had reduced expression of D1D2D3 but increased expression of the truncated form of uPAR (D2D3) on their cell surface. Retaining full-length uPAR was found to be essential for regulating myofibroblast differentiation, because 1) protease inhibitors that prevented uPAR cleavage also prevented myofibroblast differentiation, and 2) overexpression of cDNA for a noncleavable form of uPAR inhibited myofibroblast differentiation. These data support a novel hypothesis that maintaining full-length uPAR on the cell surface regulates the fibroblast to myofibroblast transition and that down-regulation of uPAR is necessary for myofibroblast differentiation.

Corneal activation of prothrombin to form thrombin, independent of vascular injury.

PURPOSE: Two major functions of thrombin observed in the cornea are activation of thrombin-sensitive, proteinase-activated receptors and cleavage of fibrinogen to fibrin. The purpose of this study was to determine whether the normal human cornea itself is competent to convert prothrombin to thrombin and synthesizes the mRNA for the proteins required. METHODS: Human corneas were processed for immunolocalization studies or separated into epithelial, stromal, and endothelial layers for proteins and RNA isolation. The protein extracts were used for Western blots, prothrombin time, and activated partial thromboplastin time assays and fibrinopeptide A generation tests. RNA was used for RT-PCR. Apoptosis of cultured human corneal cells was induced with sodium nitroprusside or camptothecin and activation of prothrombin tested. RESULTS: Prothrombin and its mRNA were present in all three layers of human donor cornea. It was found to be associated with the cells and the extracellular matrix at similar levels across the cornea. With corneal stromal extracts, activation of either the intrinsic or extrinsic coagulation pathways resulted in thrombin activation and fibrin formation with fibrinopeptide A release. Detection of key components of the coagulation cascades confirmed noninjured human corneas contain factors required for prothrombin activation. In addition, mRNAs for representative factors and inhibitors were detected by RT-PCR and confirmed by sequencing. Apoptotic corneal stromal cells provide a surface for prothrombin activation. CONCLUSIONS: These studies suggest that the normal avascular human cornea contains and synthesizes the components required for thrombin generation and that this process does not depend on a breech in the limbal vascular endothelium.

Differential conversion of plasminogen to angiostatin by human corneal cell populations.

PURPOSE: Maintenance of avascularity of the normal cornea and control of neovascularization during wound healing depend on a balance of angiogenic and antiangiogenic factors. The purpose of this paper is to determine the ability of corneal cells to convert plasminogen to angiostatins and to compare these products with those made by intact corneas. METHODS: RT-PCR was performed using plasminogen specific primers and the generated cDNA was sequenced. The proteins in corneal extracts, cornea conditioned medium, and medium from corneal epithelial cells, stromal fibroblasts, and myofibroblasts incubated with plasminogen were separated by SDS-PAGE and electroblotted. Western blots used monoclonal antibodies to kringles 1-3 to detect plasminogen and angiostatins. Angiostatins were isolated and tested for activity in a vascular endothelial cell proliferation inhibition assay. RESULTS: Plasminogen, its mRNA and angiostatins were found in human corneal tissue extracts from the epithelial, stromal, and endothelial layers and from cornea conditioned medium, but not in medium from cultured epithelial cells, stromal fibroblasts, or myofibroblasts. However, cultures of corneal epithelial cells and stromal fibroblasts were able to convert exogenously added plasminogen to angiostatins, whereas cultured myofibroblasts did not. Angiostatins of 38 and 34 kDa were found under all angiostatin generating conditions; however other angiostatins differed in size. Further, the angiostatins isolated from fibroblast culture supernatants inhibited vascular endothelial cell proliferation. CONCLUSIONS: Conversion of plasminogen to angiostatin is cell-type dependent. Because corneal cells generate angiostatins, use of human angiostatins may be a means of treating abnormal corneal neovascularization without the risk of side effects.

Specific conformational changes of plasminogen induced by chloride ions, 6-aminohexanoic acid and benzamidine, but not the overall openness of plasminogen regulate, production of biologically active angiostatins.

The overall conformation of plasminogen depends upon the presence of anions and molecules such as AHA (6-aminohexanoic acid) and BZ (benzamidine). The purpose of the present study was to determine the effect of conformation on the initial and secondary cleavages of plasminogen to generate active angiostatins. Plasminogen was digested with the physiologically relevant neutrophil elastase in one of the four Tris/acetate buffers: buffer alone or buffer plus NaCl, AHA or BZ. The initial cleavage of Glu1-plasminogen was much slower in the tight NaCl-induced alpha-conformation, fastest in the intermediate BZ-induced beta-conformation and intermediate both in the control and in the AHA-induced open gamma-conformation. Although the buffer system determined the relative amounts of the initial cleavage products, the same four cleavage sites were utilized under all conditions. A fifth major initial cleavage within the protease domain was observed in the presence of BZ. N-terminal peptide cleavage required for angiostatin formation occurred as either the initial or the secondary cleavage. Angiostatins were generated fastest in the presence of BZ and slowest in the presence of NaCl. Both the initial and secondary cleavages were affected by the modifying agents, indicating that they influence the conformation of both Glu-plasminogen and the initial cleavage products. The angiostatins produced under the different conditions inhibited proliferation of human umbilical-vein endothelial cells. These results suggest that plasminogen conversion into active angiostatins is dependent more on the specific conformation changes induced by the various modifying reagents rather than on the overall openness of the molecule.

Sufficiency of the reactive site loop of maspin for induction of cell-matrix adhesion and inhibition of cell invasion. Conversion of ovalbumin to a maspin-like molecule.

Maspin, an ov-serpin, inhibits tumor invasion and induces cell adhesion to extracellular matrix molecules. Here, we use maspin/ovalbumin chimeric proteins and the maspin reactive site loop (RSL) peptide to characterize the role of the RSL in maspin-mediated functions. Replacement of the RSL plus the C-terminal region or the RSL alone of maspin with that of ovalbumin resulted in the loss of the stimulatory effect on adhesion of corneal stromal cells to type I collagen, fibronectin, and laminin and of mammary carcinoma MDA-MB-231 cells to fibronectin. Maspin with ovalbumin as the C-terminal region retained activity, suggesting the maspin C-terminal polypeptide is not required. An R340Q mutant retained full maspin activity; however, an R340A mutant lost activity. This indicates the arginine side chain at the putative P1 site forms a hydrogen bond and not an ionic bond. The RSL peptide (P10-P5', amino acids 330-345) alone induced cell-matrix adhesion of mammary carcinoma cells and corneal stromal cells and inhibited invasion of the carcinoma cells. Substitution of the RSL of ovalbumin with that of maspin converted inactive ovalbumin into a fully active molecule. Maspin bound specifically to the surface of the mammary carcinoma cells with a kd of 367 +/- 67 nM and 32.0 +/- 2.2 x 10(6) binding sites/cell. The maspin RSL peptide inhibited binding, suggesting the RSL is involved in maspin binding to cells. Sufficiency of the maspin RSL for activity suggests the mechanism by which maspin regulates cell-matrix adhesion and tumor cell invasion does not involve the serpin mechanism of protease inhibition.