Thrombin Increases Expression of Urokinase Receptor by Activation of the Thrombin Receptor Zeren Yang,* Robert L. Cohen,* Ge Ming Lui,~f David A. Laxvrence* and Marc A. Shuman* Purpose. To investigate the effect of thrombin on the urokinase plasminogen activator receptor (u-PAR) in retinal pigment epithelial (RPE) cells. Methods. The authors analyzed u-PAR mRNA by Northern blot hybridization. Retinal pigment epithelial cell surface u-PAR was assayed by measuring the amount of functional urokinase plasminogen activator (u-PA) bound to cells at saturation. Retinal pigment epithelial cells were derived from fetal retinal tissue and established in primary cell culture. Results. Thrombin increased u-PAR mRNA 4-fold in RPE cells examined by Northern blot hybridization, whereas the amount of thrombin receptor mRNA was unchanged. Thrombin stimulated u-PA binding to RPE cells 2.5- to 5-fold in a time- and dose-dependent manner. Hirudin, a thrombin antagonist, completely blocked the effects of thrombin on u-PAR expres- sion in RPE cells. Phosphatidylinositol phospholipase C treatment of RPE cells resulted in the abolition of thrombin-induced u-PA binding. Recombinant soluble u-PAR competitively inhibited two-chain u-PA binding to the surface of thrombin-treated RPE cells. A thrombin receptor agonist peptide (SFLLRNPNDKYEPF) also induced a 2.5-fold increase in binding of u-PA to the surface of RPE cells. Conclusion. Thrombin increases u-PAR expression by RPE cells by a mechanism involving activation of the seven transmembrane thrombin receptor. Invest Ophthalmol Vis Sci. 1995; 36:2254-2261. In addition to initiating fibrinolysis, urokinase plas- mature, glycosylated form ranges from 40,000 to 55,000 minogen activator (u-PA) has been implicated in a daltons in different cell lines.16"20 u-PAR binds active two- variety of biologic processes, including angiogenesis, chain u-PA and its zymogen, single-chain u-PA (pro-u- tumor invasion, tissue remodeling, cellular differentia- PA).21 Phorbol ester, endothelial cell growth factor, fi- tion and proliferation, ovulation, and embryonic im- broblast growth factor, a-tumor necrosis factor, and inter- plantation.1"3 Some of the actions of u-PA appear to feron-7 increase u-PAR expression without a change in be mediated by its interaction with a specific, high- receptor affinity in U937 and endothelial cells and mono affinity membrane urokinase plasminogen activator cytes.4"21 Reuning et al22 recently reported that thrombin receptor (u-PAR).4"6 and other mitogens increased l25I-labeled u-PA binding u-PAR is expressed by many types of cells, including to vascular smooth muscle cells of the bovine aorta; how- U937 cells, endothelial cells, fibroblasts, leukocytes, tumor ever, the mechanism underlying this effect was not deter- cell lines, gastric mucosa, and ovarian tumor cells.4"l5 The mined. The serine protease thrombin, a coagulation enzyme, also has the potential to regulate fibrinolysis by limited From the * Cancer Research Institute and the ^Department of Ophthalmology, University of California at San Francisco. proteolysis and inactivation of single-chain urokinase. In Supported in part by a grant from the Joseph Drown Foundation. ZY was a addition, thrombin stimulates synthesis of types 1 and 2 Biomedical Research Trainee in the Cheng Scholar Program at the University of California at San Francisco. plasminogen activator inhibitors (PAI-1 and PAI-2) by Submitted for publication Decemljer 28, 1994; revised April 3, 1995; accepted May endothelial cells.I ":V23"25 19, 1995. Proprietary interest category: N. A significant manifestation of disordered angiogen- Reprint requests: Marc A. Shuman, Cancer Research Institute, University of esis occurs in neovascularization of the retina. Increasing California at San Francisco, 3rd Avenue and Parnassus Street, San Francisco, CA 94143-0128. evidence suggests that retinal pigment epithelial (RPE) Investigative Ophthalmology & Visual Science, October 1995, Vol. 36, No. 11 2254 Copyright © Association lor Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/30/2021 Thrombin Increases Urokinase Receptor 2255 cells play a pivotal role in the inhibition of intraocular were from Stratagene (#400701; La Jolla, CA). Im- neovascularization, a key event in the pathogenesis of a muno microliter plate (#01 1-010-3350) was from Dy- number of ophthalmic disorders, including age-related natech Laboratories (Chantilly, VA). Fast-track mRNA macular degeneration and proliferative diabetic retinopa- isolation kit, vector pCR 1000, and vector pCEP were thy.2(>"27 Regulation of plasminogen activation by RPE from Invitrogen (San Diego, CA). (#K1593-02). 5,5'- cells, therefore, may play an important role in these pro- dithiobis (2-nitrobenzoic acid; DTNB) was from Calbi- 520.27 cesses. ochem (LaJolla, CA). Z-lysine thiobenzyl ester (BLT) We investigated the regulation of the expression of was from Peninsula Laboratories (Belmont, CA). En- u-PAR in RPE cells by thrombin to elucidate how throm- dothelial cell growth supplement (#40006) was from bin may alter various biologic processes by modulating Collaborative Biomedical Products (Bedford, MA). cell surface PA activity.1"' We have demonstrated that u- YM-30 membrane, Centricon 10, and Centricon 30 PAR expression by RPE cells is increased by thrombin micro-concentrator were from Amicon (Lexington, through activation of a recently described seven trans- MA). membrane thrombin receptor. Recombinant Soluble u-PAR A recombinant form of u-PAR30 was produced as fol- METHODS lows: A u-PAR cDNA encoding the full-length protein was cloned by the polymerase chain reaction using Materials U937 cDNA as a template and primers based on the Two-chain u-PA was a generous gift from Jack Henkin, published u-PAR sequence.1'20 In a second round of Abbott Laboratories (Abbott Park, IL). a-thrombin polymerase chain reaction, a stop codon was intro- was a generous gift from John Fenton, Department of duced to truncate the protein after arg281. The resul- Health, New York State University (Albany, NY). The tant 957-bp fragment was cloned into pCR 1000, subse- synthetic agonist of the thrombin receptor (SFLLRN- quently excised with Kpnl and Notl, and recloned into PNDKYEPF), mimicking the new amino terminus cre- the corresponding sites of the expression vector pCEP ated by cleavage of the receptor, the control peptide to yield pCEP/Trunc. A stable cell line expressing (FSLLRNPNDKYEPF), and a thrombin receptor truncated u-PAR was obtained by transfection of 293 cDNA probe (708 bp) were kindly provided by S. R. cells with pCEP/Trunc using the calcium phosphate Coughlin, University of California at San Fran- method.1' Serum-free conditioned media were pre- cisco.282'1 The human u-PA cDNA probe used in these pared, concentrated 10-fold on a YM-30 membrane, studies has been described previously.15 Polyclonal and collected by affinity chromatography on u-PA- anti-u-PAR antiserum was generously provided by Dr. Sepharose. Bound u-PAR was eluted with acid-glycine R. F. Todd III (Ann Arbor, MI).H<) The following were as described,15 and the neutralized eluate was concen- purchased from commercial sources: rabbit polyclonal trated using a Centricon 30 micro-concentrator before antibody against u-PA (#398), plasminogen (#412), use. Sodium dodecyl sulfate-polyacrylamide gel elec- and plasmin (#411) were from American Diagnostica trophoresis followed by silver staining showed a single (Greenwich, CT).ir>32P-dCTP and Resolution were from broad band centered at 55 kDa that cross-reacted with Amersham (Arlington Heights, IL. Acrylamide, glyc- a polyclonal anti-u-PAR antiserum. erol, and nylon niters were from Bio-Rad (Richmond, CA). 3-(N-Morpholino) propane sulfonic acid, basic Cell Culture fibroblast growth factor (bFGF), collagenase, bovine Human fetal RPE cells derived from two different fetal serum albumin (BSA), N-2-hydroxythylpiperazine-N'- retinas were harvested and maintained as described 2-ethane sulfonic acid (Hepes), Tris, ethylenediamine previously.31 Tenets of the Declaration of Helsinki tetraacetic acid (EDTA) formaldehyde, Triton X-100, were followed. Informed consent for human research and Tween-20 were from Sigma (St. Louis, MO). Ko- was obtained, and institutional human experimenta- dak XAR autoradiography film was from Eastman Ko- tion committee approval was granted. Briefly, 8- to 10- dak (Rochester, NY). Fibronectin, guanidinium isothi- passage RPE cells were plated in 75 mm2 flasks coated ocynate, cesium chloride, and a random DNA labeling with fibronectin for 10 days, and basic fibroblast system were from BRL (Gaithersburg, MD). Phospha- growth factor (1 ng/ml) was added as a growth supple- tidylinositol phospholipase C (PI-PLC, #1143069) ment to cells every other day as previously reported.31 was from Boehringer Mannheim Biochemicals (India- For mRNA extraction, cells were plated on 24 X 24 napolis, IN). 293 cells (#CRL1573) and glyceralde- cm2 plates. For u-PAR functional assays, the final con- hyde-3-phosphate dehydrogenase (GAPDH, #57090) centration was approximately 2 to 2.5 X 107 cells/75 probe from PBR 322 were from American Type Cul- mm2 flask (protein concentration: 5.5 to 6 mg/ml). ture Collection (Rockville, MD). Imidazole was from J. After cells became confluent, they were treated with T. Baker Chemical Company. Nuctran push columns 0 to 10 U/ml of thrombin at 37°C for 24 hours for Downloaded from iovs.arvojournals.org on 09/30/2021 2256 Investigative Ophthalmology 8c Visual Science, October 1995, Vol. 36, No. 11 determining the effects of thrombin concentration, 5 BSA, then incubated with elution buffer at room tem- U/ml of thrombin for 0 to 48 hours at 37°C for the perature for 3 minutes with mild shaking. The eluate time course study, 5 U/ml of thrombin and/or 10 U/ was collected and 1/10 volume of 1 M Tris-HCl, pH ml of hirudin at 37°C for 24 hours for hirudin blocking 7.6, was added to the solution. Samples were concen- study,2' or 50 to 200 /xM of the thrombin receptor trated 20-fold with a Centricon 10 apparatus and ei- peptide or the same amount of the control peptide at ther assayed for u-PA activity or stored at — 70°C.