Neutrophil Cathepsin G Promotes Detachment-induced Cardiomyocyte Apoptosis via a Protease-activated Receptor-independent Mechanism
2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês
10.1074/jbc.m302718200
ISSN1083-351X
AutoresAbdelkarim Sabri, Sasha G. Alcott, Hasnae Elouardighi, Elena S. Pak, Claudia K. Derian, Patricia Andrade‐Gordon, Kathleen W. Kinnally, Susan F. Steinberg,
Tópico(s)Cell Adhesion Molecules Research
ResumoCathepsin G is a neutrophil-derived serine protease that contributes to tissue damage at sites of inflammation. The actions of cathepsin G are reported to be mediated by protease-activated receptor (PAR)-4 (a thrombin receptor) in human platelets. This study provides the first evidence that cathepsin G promotes inositol 1,4,5-trisphosphate accumulation, activates ERK, p38 MAPK, and AKT, and decreases contractile function in cardiomyocytes. Because some cathepsin G responses mimic cardiomyocyte activation by thrombin, a role for PARs was considered. Cathepsin G markedly activates phospholipase C and p38 MAPK in cardiomyocytes from PAR-1–/– mice, but it fails to activate phospholipase C, ERK, p38 MAPK, or AKT in PAR-1- or PAR-4-expressing PAR-1–/– fibroblasts (which display robust responses to thrombin). These results argue that PAR-1 does not mediate the actions of cathepsin G in cardiomyocytes, and neither PAR-1 nor PAR-4 mediates the actions of cathepsin G in fibroblasts. Of note, prolonged incubation of cardiomyocytes with cathepsin G results in the activation of caspase-3, cleavage of FAK and AKT, sarcomeric disassembly, cell rounding, cell detachment from underlying matrix, and morphologic features of apoptosis. Inhibition of Src family kinases or caspases (with PP1 or benzyloxycarbonyl-VAD-fluoromethyl ketone, respectively) delays FAK and AKT cleavage and cardiomyocyte detachment from substrate. Collectively, these studies describe novel cardiac actions of cathepsin G that do not require PARs and are predicted to assume functional importance at sites of interstitial inflammation in the heart. Cathepsin G is a neutrophil-derived serine protease that contributes to tissue damage at sites of inflammation. The actions of cathepsin G are reported to be mediated by protease-activated receptor (PAR)-4 (a thrombin receptor) in human platelets. This study provides the first evidence that cathepsin G promotes inositol 1,4,5-trisphosphate accumulation, activates ERK, p38 MAPK, and AKT, and decreases contractile function in cardiomyocytes. Because some cathepsin G responses mimic cardiomyocyte activation by thrombin, a role for PARs was considered. Cathepsin G markedly activates phospholipase C and p38 MAPK in cardiomyocytes from PAR-1–/– mice, but it fails to activate phospholipase C, ERK, p38 MAPK, or AKT in PAR-1- or PAR-4-expressing PAR-1–/– fibroblasts (which display robust responses to thrombin). These results argue that PAR-1 does not mediate the actions of cathepsin G in cardiomyocytes, and neither PAR-1 nor PAR-4 mediates the actions of cathepsin G in fibroblasts. Of note, prolonged incubation of cardiomyocytes with cathepsin G results in the activation of caspase-3, cleavage of FAK and AKT, sarcomeric disassembly, cell rounding, cell detachment from underlying matrix, and morphologic features of apoptosis. Inhibition of Src family kinases or caspases (with PP1 or benzyloxycarbonyl-VAD-fluoromethyl ketone, respectively) delays FAK and AKT cleavage and cardiomyocyte detachment from substrate. Collectively, these studies describe novel cardiac actions of cathepsin G that do not require PARs and are predicted to assume functional importance at sites of interstitial inflammation in the heart. Cathepsin G is a major serine protease released by activated neutrophils at sites of vascular injury and inflammation (1Molino M. Blanchard N. Belmonte E. Tarver A.P. Abrams C. Hoxie J.A. Cerletti C. Brass L.F. J. Biol. Chem. 1995; 270: 11168-11175Google Scholar, 2Parry M.A. Myles T. Tschopp J. Stone S.R. Biochem. J. 1996; 320: 335-341Google Scholar). Cathepsin G is a strong agonist for platelets, causing a rise in intracellular calcium, aggregation, and degranulation. Cathepsin G induces functional and morphological changes in endothelial cells that lead to increased permeability and growth factor release (1Molino M. Blanchard N. Belmonte E. Tarver A.P. Abrams C. Hoxie J.A. Cerletti C. Brass L.F. J. Biol. Chem. 1995; 270: 11168-11175Google Scholar, 3Weksler B.B. Jaffe E.A. Brower M.S. Cole O.F. Blood. 1989; 74: 1627-1634Google Scholar, 4Selak M.A. Biochem. J. 1994; 297: 269-275Google Scholar, 5Si-Tahar M. Renesto P. Falet H. Rendu F. Chignard M. Biochem. J. 1996; 313: 401-408Google Scholar). Cathepsin G also induces tissue injury and contributes to the pathogenesis of certain chronic pulmonary diseases, such as emphysema and cystic fibrosis (6Bird P.I. Immunol. Cell Biol. 1999; 77: 47-57Google Scholar). These diverse cellular actions of cathepsin G have been attributed to the cleavage and function modulation of a range of protein substrates, including clotting factors (factor V and factor VII), neutrophil chemoattractants (tumor necrosis factor α, interleukin-1, and interleukin-8), and matrix components (including collagen, fibronectin, and elastin). Recent studies have focused on cathepsin G-dependent cleavage of seven transmembrane spanning domain G protein-coupled protease-activated receptors (PARs) 1The abbreviations used are: PARs, protease-activated receptors; IP, inositol phosphate; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; PTX, pertussis toxin; DMEM, Dulbecco's modified Eagle's medium; MMPs, matrix-degrading metalloproteinases; FAK, focal adhesion kinase. as an additional potentially important mechanism whereby cathepsin G modulates coagulation and tissue remodeling at sites of injury and inflammation. PAR-1, the prototypical receptor for thrombin, is activated as a result of thrombin-dependent cleavage of its extracellular N terminus to expose a new N-terminal sequence (SFLLRN) that binds intramolecularly and serves as a tethered ligand (7Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11023-11027Google Scholar). Since the initial cloning of PAR-1, three additional structurally homologous PARs have been identified; PAR-3 and PAR-4 are activated by thrombin, whereas PAR-2 is activated by trypsin, membrane-type serine protease-1, and mast cell tryptase (but not thrombin) (8Dery O. Corvera C.U. Steinhoff M. Bunnett N.W. Am. J. Physiol. 1998; 274: C1429-C1452Google Scholar, 9Takeuchi T. Harris J.L. Huang W. Yan K.W. Coughlin S.R. Craik C.S. J. Biol. Chem. 2000; 275: 26333-26342Google Scholar). PARs are endowed with two unique regulatory features as a result of their distinctive proteolytic activation mechanism. PAR-1, PAR-3, and PAR-4 need not be entirely selective for thrombin but are predicted to be susceptible to activation by any serine protease capable of cleaving the N terminus at the site that exposes the tethered ligand sequence. Additionally, PAR cleavage at sites on the N terminus more proximal to the transmembrane domain is predicted to amputate the tethered ligand sequence and render the PAR unresponsive to subsequent proteolytic activation. Cathepsin G is reported to modulate PAR function via both mechanisms. At concentrations achieved in the vicinity of activated neutrophils, cathepsin G mimics the action of thrombin to mobilize intracellular calcium in Xenopus oocytes and fibroblast cell lines that stably overexpress human PAR-1 or human PAR-4 (but not in untransfected cells or cells that overexpress PAR-2 or PAR-3). Mutagenesis studies suggest that in each case the mechanism entails cleavage of the N terminus (at the Arg41–Ser42 bond in PAR-1 and the Arg47–Gly48 bond in PAR-4) to expose the tethered ligand (1Molino M. Blanchard N. Belmonte E. Tarver A.P. Abrams C. Hoxie J.A. Cerletti C. Brass L.F. J. Biol. Chem. 1995; 270: 11168-11175Google Scholar, 10Sambrano G.R. Huang W. Faruqi T. Mahrus S. Craik C. Coughlin S.R. J. Biol. Chem. 2000; 275: 6819-6823Google Scholar). These results suggest that cathepsin G should display generalized agonist actions in cells that express either PAR-1 or PAR-4. However, for unclear reasons, agonist actions of cathepsin G are consistently identified in human platelets but not other thrombin-responsive cell types, such as human umbilical vein endothelial cells and human lung fibroblasts (1Molino M. Blanchard N. Belmonte E. Tarver A.P. Abrams C. Hoxie J.A. Cerletti C. Brass L.F. J. Biol. Chem. 1995; 270: 11168-11175Google Scholar, 3Weksler B.B. Jaffe E.A. Brower M.S. Cole O.F. Blood. 1989; 74: 1627-1634Google Scholar, 4Selak M.A. Biochem. J. 1994; 297: 269-275Google Scholar, 11LeRoy E.C. Ager A. Gordon J.L. J. Clin. Invest. 1984; 84: 1003-1010Google Scholar, 12Molino M. Woolkalis M. Reavey-Cantwell J. Pratico D. Andrade-Gordon P. Barnathan E.S. Brass L.F. J. Biol. Chem. 1997; 272: 11133-11141Google Scholar). Rather, in cells that express PAR-1 but do not display stimulatory responses to cathepsin G, the predominant effect of cathepsin G is to non-productively cleave PAR-1 at a site close to the first transmembrane domain (Phe55–Trp56). This amputates the tethered ligand domain and renders PAR-1 unresponsive to subsequent stimulation by thrombin (although cathepsin G-cleaved PAR-1 is otherwise structurally intact and fully responsive to SFLLRN) (1Molino M. Blanchard N. Belmonte E. Tarver A.P. Abrams C. Hoxie J.A. Cerletti C. Brass L.F. J. Biol. Chem. 1995; 270: 11168-11175Google Scholar). Recent studies (13Cumashi A. Ansuini H. Celli N. De Blasi A. O'Brien P.J. Brass L.F. Molino M. Thromb. Haemostasis. 2001; 85: 533-538Google Scholar) indicate that human and murine PAR-3 also can be disabled by cathepsin G (although via distinct mechanisms) and that certain cathepsin G actions on PARs may be species-dependent. The cardiovascular signaling properties of PARs generally have been explored in platelets (where the actions of thrombin are critical for normal hemostasis and arterial thrombosis) and the vessel wall (where thrombin promotes changes in endothelial cell morphology, which lead to altered monolayer permeability, and induces proliferation of vascular smooth muscle cells). However, recent studies identify cardiomyocytes as an additional cardiovascular target for thrombin and related proteases. Cardiomyocytes cultured from neonatal rat ventricles co-express PAR-1, PAR-2, and PAR-4 (mRNA for PAR-3 is not detected) (14Jiang T. Kuznetsov V. Pak E. Zhang H.L. Robinson R.B. Steinberg S.F. Circ. Res. 1996; 78: 553-563Google Scholar, 15Glembotski C.C. Irons C.E. Krown K.A. Murray S.F. Sprenkle A.B. Sei C.A. J. Biol. Chem. 1993; 268: 20646-20652Google Scholar, 16Yasutake M. Haworth R.S. King A. Avkiran M. Circ. Res. 1996; 79: 705-715Google Scholar, 17Landau E. Tirosh R. Pinson A. Banai S. Even-Ram S. Maoz M. Katzav S. Bar-Shavit R. J. Biol. Chem. 2000; 275: 2281-2287Google Scholar, 18Sabri A. Muske G. Zhang H. Pak E. Darrow A. Andrade-Gordon P. Steinberg S.F. Circ. Res. 2000; 86: 1054-1061Google Scholar, 19Sabri A. Guo J. Elouardighi H. Darrow A.L. Andrade-Gordon P. Steinberg S.F. J. Biol. Chem. 2003; 278: 11714-11720Google Scholar). PAR-1 and PAR-2 agonists induce similar increases in phosphoinositide hydrolysis and intracellular calcium; both receptors link to the activation of ERK, stimulation of p38 MAPK, and cardiomyocyte hypertrophy (18Sabri A. Muske G. Zhang H. Pak E. Darrow A. Andrade-Gordon P. Steinberg S.F. Circ. Res. 2000; 86: 1054-1061Google Scholar). Recent studies (19Sabri A. Guo J. Elouardighi H. Darrow A.L. Andrade-Gordon P. Steinberg S.F. J. Biol. Chem. 2003; 278: 11714-11720Google Scholar) indicate that PAR-4 also effectively activates p38 MAPK in cardiomyocytes; PAR-4 stimulates phosphoinositide hydrolysis, activates ERK, and induces hypertrophy but more weakly. The predominant natural activator(s) of endogenous cardiomyocyte PARs remains uncertain. Although cardiomyocyte PAR-1 may be activated by thrombin in the setting of hemorrhagic infarction (where the endothelial barrier is broken and cardiomyocytes come into direct contact with blood-borne substances), most myocardial events are not accompanied by hemorrhage into the myocardium. Hence, other potential mechanisms for PAR activation must be considered. This study tests whether cathepsin G elaborated by neutrophils at sites of injury and/or inflammation (myocarditis, the border zone of a myocardial infarction) exerts a primary agonist action in cardiomyocytes and whether cathepsin G responses can be attributed to activation of a known PAR. Materials—Chemicals were purchased commercially from the following sources: thrombin (Calbiochem); fura 2-AM (Molecular Probes); [3H]myoinositol (24.4 Ci/mmol, PerkinElmer Life Sciences); pertussis toxin (PTX, List Biologicals). Cathepsin G was obtained from Calbiochem for most experiments; selected experiments were performed with cathepsin G from Sigma and yielded equivalent results. All other chemicals were reagent-grade and obtained from standard chemical suppliers. SFLLRN and AYPGKF were synthesized at the State University of New York, Stony Brook, as C-terminal amides, purified by high pressure liquid chromatography, and characterized by mass spectroscopy. Peptide solutions were made fresh from powder for all experiments. Cardiomyocyte Cultures—Cardiac myocytes were dissociated from the ventricles of Wistar rats (postnatal day two) or PAR-1–/– or background strain C57BL/6 mice (embryonic day 18–20) by a trypsin digestion protocol which incorporates a differential attachment procedure to enrich for cardiac myocytes (14Jiang T. Kuznetsov V. Pak E. Zhang H.L. Robinson R.B. Steinberg S.F. Circ. Res. 1996; 78: 553-563Google Scholar). Although the preplating step effectively decreases fibroblast contamination, myocytes were subjected to 30 gray of X-rays on day 1 of culture to halt the proliferative potential of any residual contaminating fibroblasts (14Jiang T. Kuznetsov V. Pak E. Zhang H.L. Robinson R.B. Steinberg S.F. Circ. Res. 1996; 78: 553-563Google Scholar). Cardiomyocytes were plated at a density of 0.5 × 106 cells per ml (2 ml per 35-mm dish) and were cultured in DMEM supplemented with 10% fetal calf serum. For assays of ERK, p38 MAPK, or AKT activation, cells were serum-starved in 1:1 DMEM/F-12 medium for 24 h. Cell Lines—Immortalized murine lung myofibroblasts derived from PAR-1-deficient mice (20Darrow A.L. Fung-Leung W.P. Ye R.D. Santulli R.J. Cheung W.M. Derian C.K. Burns C.L. Damiano B.P. Zhou L. Keenan C.M. Peterson P.A. Andrade-Gordon P. Thromb. Haemostasis. 1996; 76: 860-866Google Scholar, 21Andrade-Gordon P. Maryanoff B.E. Derian C.K. Zhang H.C. Addo M.F. Darrow A.L. Eckardt A.J. Hoekstra W.J. McComsey D.F. Oksenberg D. Reynolds E.E. Santulli R.J. Scarborough R.M. Smith C.E. White K.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12257-12262Google Scholar), which lack functional PAR-1, PAR-2, and PAR-4, were transfected with human PAR-1 or PAR-4 in a modified pCDNA3 construct encoding a hygromycin resistance gene. Stable transfectants were selected in 250 mg/ml of hygromycin B and screened by specific agonist-induced calcium mobilization. Cultures were maintained in DMEM containing 10% calf serum and 250 μg/ml hygromycin; for assays of MAPK or AKT activation, cultures were serum-starved overnight. Phosphoinositide Hydrolysis—Cells were incubated for 72 (cardiomyocytes) or 24 h (cell lines) with 3 μCi/ml [3H]myoinositol, washed, preincubated with 10 mm LiCl for 20 min, and then stimulated with agonists for the indicated intervals at room temperature. Inositol phosphates (IPs) were extracted and eluted sequentially by ion-exchange chromatography on Dowex columns according to methods published previously (14Jiang T. Kuznetsov V. Pak E. Zhang H.L. Robinson R.B. Steinberg S.F. Circ. Res. 1996; 78: 553-563Google Scholar). Immunoblotting—Immunoblot analysis was used to compare levels of total and phosphorylated ERK, p38 MAPK, and AKT and to track the accumulation of caspase-3 and FAK cleavage products. Following exposure to test agents as indicated in individual experimental protocols, cells were washed three times with ice-cold calcium/magnesium-free phosphate-buffered saline (pH 7.4), scraped into hot SDS-PAGE sample buffer, sonicated, and then centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was diluted in SDS-PAGE sample buffer, boiled for 5 min, and stored at –70 °C. Western blot analysis was performed according to manufacturer's instructions, with bands detected by enhanced chemiluminescence and quantified by laser scanning densitometry. For each panel in each figure, the results are from a single gel exposed for a uniform duration. Measurement of Cytosolic Free Calcium and Cell Shortening—Intracellular calcium and shortening was simultaneously measured photometrically in fura-2-loaded cultured neonatal rat ventricular myocytes according to methods published previously (14Jiang T. Kuznetsov V. Pak E. Zhang H.L. Robinson R.B. Steinberg S.F. Circ. Res. 1996; 78: 553-563Google Scholar). In brief, myocytes cultured on coverslips were loaded with fura-2 by incubation in Tyrode's solution containing 3 μm of the acetoxymethyl ester form of fura-2 (fura-2/AM) and 1.5 μl/ml of 25% (w/v in dimethyl sulfoxide) Pluronic F-127 for 20 min at 37 °C. Cardiomyocytes were rinsed with fresh Tyrode's solution and maintained for at least 15 min at room temperature to allow for de-esterification of the dye. Cardiomyocytes were superfused with room temperature Tyrode's solution gassed with 95% O2, 5% CO2 at a rate of 1 ml/min and paced by electrical field stimulation at 1 Hz throughout the experimental protocol (to avoid changes in cell calcium due to potential chronotropic actions of agonists). Drugs were introduced as a bolus into the prechamber of a three compartment superfusion chamber described previously (14Jiang T. Kuznetsov V. Pak E. Zhang H.L. Robinson R.B. Steinberg S.F. Circ. Res. 1996; 78: 553-563Google Scholar). Intracellular fura-2 fluorescence was monitored (at a sampling rate of 100 Hz) with a device that alternately illuminates the cells with 340 and 380 nm light while measuring emission at 520 nm (Photon Technologies, Inc., Princeton, NJ). Intracellular calcium is reported as the fura-2 fluorescence ratio (due to the uncertainties inherent in any attempt to calibrate these signals, as discussed in detail by us and others elsewhere (14Jiang T. Kuznetsov V. Pak E. Zhang H.L. Robinson R.B. Steinberg S.F. Circ. Res. 1996; 78: 553-563Google Scholar)). Glass microspheres (2.1 ± 0.5 μm) were added to the monolayer cultures to provide high contrast spots for tracking cell motion. The cell image was directed to a video optical system (Crescent Electronics), and a glass bead attached to the cardiomyocyte surface was tracked along a raster line segment during electrically stimulated contractions. The lack of a single axis of myofibrillar alignment renders measurements of cell length during electrically stimulated contractions meaningless in this preparation. Therefore, relative cell motion rather than actual cell length is reported. The analog voltage output from the motion detector was calibrated to convert to micrometers of movement. The motion signal, obtained at a rate of 60 Hz, was digitized and stored using Deltascan software (Photon Technology Int.). Time-lapse Video and Fluorescence Microscopy—Cells were grown on 25-mm slides that were mounted in sealed rose chambers maintained at 37 °C, in the presence of media with or without cathepsin G. Images were taken every 5 min for ∼24 h with a Spot RT Monochrome CCD camera (Diagnostics Instruments) on a Nikon Eclipse TE300 Phase contrast/differential interference contrast microscope. Some slides were stained with Hoechst 33342 or 33258 and imaged through a UV-2A filter cube. The Uniblitz Shutter (model VMMD1) was controlled by Spot RT software or Scion Imaging (National Institutes of Health). Pictures were captured and then stacked by Spot software, and montages were prepared through Photoshop software. Cathepsin G Promotes Phosphoinositide Hydrolysis and Activates ERK, p38 MAPK, and AKT in Cardiomyocytes; Distinct Signaling by Cathepsin G and PAR Agonists—Fig. 1 shows that cathepsin G markedly increases IP accumulation in neonatal rat cardiomyocytes. Responses are detected at relatively low concentrations of cathepsin G, typical of those reported to be generated at sites of inflammation. The characteristics of IP accumulation in response to conventional PAR-1 agonists (thrombin, SFLLRN) versus cathepsin G are quite distinct. Consistent with previous results, PAR-1 activation by maximally stimulatory concentrations of thrombin (1 unit/ml) or the PAR-1 agonist peptide SFLLRN (300 μm) results in transient elevations of IP2/IP3, followed by a more sustained accumulation of IP1 (22Sabri A. Pak E. Alcott S.A. Wilson B.A. Steinberg S.F. Circ. Res. 2000; 86: 1047-1053Google Scholar). In contrast, cathepsin G promotes the progressive accumulation of IP2 and IP3 (to levels as high or higher than the levels transiently observed in cells stimulated with thrombin or SFLLRN); the accompanying cathepsin G-induced rise in IP1 levels is trivial in comparison. Because we recently identified PAR-4 as an additional cellular receptor for thrombin in cardiomyocytes (19Sabri A. Guo J. Elouardighi H. Darrow A.L. Andrade-Gordon P. Steinberg S.F. J. Biol. Chem. 2003; 278: 11714-11720Google Scholar), we also compared responses to cathepsin G and AYPGKF (a derivative of the PAR-4 tethered ligand sequence that is highly specific for PAR-4 and acts at ∼10-fold lower concentrations than the tethered ligand sequence GYPGKF itself) (23Faruqi T.R. Weiss E.J. Shapiro M.J. Huang W. Coughlin S.R. J. Biol. Chem. 2000; 275: 19728-19734Google Scholar). Table I shows that AYPGKF induces a rise in IP1 levels, but the magnitude of this response is relatively modest compared with the rise in IP1 levels elicited by 1 unit/ml thrombin (a concentration that maximally activates phosphoinositide hydrolysis in cardiomyocytes). AYPGKF actions cannot be attributed to robust PAR-4 signaling in a minor contaminating fibroblast population, because cardiac fibroblasts display brisk/pronounced increases in IP accumulation in response to thrombin and SFLLRN, but cardiac fibroblasts do not respond to AYPGKF (24Sabri A. Short J. Guo J. Steinberg S.F. Circ. Res. 2002; 91: 532-539Google Scholar). Of note, stimulation with AYPGKF results in the sustained accumulation of IP1, with only a relatively minor associated increase in IP2/IP3 levels. This is quite distinct from the effect of cathepsin G, which promotes the preferential accumulation of IP2/IP3. The actions of cathepsin G and thrombin are distinguished further based upon their sensitivities to the inhibitory effects of PTX. Previous studies established that the effect of thrombin to activate phospholipase C in cardiomyocyte cultures is severely curtailed by pretreatment with PTX (22Sabri A. Pak E. Alcott S.A. Wilson B.A. Steinberg S.F. Circ. Res. 2000; 86: 1047-1053Google Scholar). In contrast, IP2/IP3 accumulation in response to cathepsin G is PTX-insensitive (IP2 + IP3 accumulation (CPM over basal): control 238 ± 14; PTX 253 ± 22, n = 3; not significant). Collectively, these experiments identify rather distinct IP responses to cathepsin G versus conventional PAR agonists in cardiomyocytes, suggesting that PARs are unlikely mediators of the cardiac actions of cathepsin G.Table IDistinct patterns of phosphoinositide hydrolysis in response to conventional PAR agonists (thrombin and AYPGKF) versus cathepsin GIP1IP2 + IP3Basal475 ± 4895 ± 6Thrombin1632 ± 65ap < 0.05 versus basal.144 ± 18ap < 0.05 versus basal.AYPGKF648 ± 50ap < 0.05 versus basal.126 ± 12ap < 0.05 versus basal.Cathepsin G542 ± 74327 ± 22ap < 0.05 versus basal.a p < 0.05 versus basal. Open table in a new tab The studies next examined whether cathepsin G activates ERK, p38 MAPK, and AKT, effectors previously identified as targets of PARs in cardiomyocytes. Fig. 2 shows that cathepsin G promotes a rapid and transient increase in ERK1/2 phosphorylation; the response is maximal at 5 min and wanes thereafter. The magnitude of cathepsin G-dependent activation of ERK is relatively modest, compared with the robust response induced by thrombin. In contrast, cathepsin G markedly activates p38 MAPK and AKT (to levels equivalent to or slightly exceeding the response to thrombin) but with relatively slow kinetics. Activation of p38 MAPK and AKT is detectable at 5 min and increases progressively during the first 30 min of stimulation. These results suggest that cathepsin G and PARs activate certain common target effectors. However, differences in the kinetics and the magnitude of these responses suggest distinct activation mechanisms. Fig. 3 shows that cathepsin G also depresses the amplitude of cell shortening (43.9 ± 9.6%, n = 11, p < 0.01). The PAR-1 agonist SFLLRN induces a similar decrease in contractile function (61.4 ± 10.5%, n = 13, p < 0.01). However, the effects of cathepsin G and SFLLRN on intracellular calcium regulation are quite different. SFLLRN increases diastolic and peak systolic calcium ion concentration (30.5 ± 4.7 and 28.7 ± 8.8%, respectively, n = 13, p < 0.01), whereas cathepsin G does not significantly alter intracellular calcium. The effect of cathepsin G to depress contractile function, without altering intracellular calcium, is most consistent with a mechanism that reduces myofibrillar calcium sensitivity. Stimulatory Actions of Cathepsin G Do Not Require PAR-1 or PAR-4 —Certain aspects of the cellular response to cathepsin G (in particular the unusually slow/progressive accumulation of IP2/IP3, without an associated rise in IP1) are sufficiently distinct from the response to traditional PAR agonists to suggest that cathepsin G might act via a distinct signaling mechanism. Therefore, the studies next took advantage of cardiomyocytes cultured from ventricles of wild-type and PAR-1–/– mice to investigate the role of PAR-1 in the stimulatory actions of cathepsin G. Fig. 4 shows that SFLLRN promotes IP accumulation and activates p38 MAPK in cardiomyocytes cultured from wild-type, but not PAR-1–/–, mice. Control experiments establish that the defect in SFLLRN activation cannot be attributed to a lesion in components of the signaling pathway distal to PAR-1, because IP accumulation in response to purinergic receptor activation with ATP is robust in both wild-type and PAR-1–/– cultures (Fig. 4A). In contrast to SFLLRN responses, which require expression of a PAR-1 gene product, cathepsin G promotes IP accumulation and stimulates p38 MAPK in cardiomyocytes from both wild-type and PAR-1–/– mice. These results indicate that the stimulatory actions of cathepsin G in cardiomyocytes do not require PAR-1 expression. Fibroblast cell lines derived from PAR-1–/– mice that stably express human PAR-1 or PAR-4 also were used to explore a possible PAR requirement for cathepsin G signaling. Fig. 5A shows that thrombin and PAR agonist peptides activate phosphoinositide hydrolysis, with the predicted differences in peptide agonist specificity and potency for thrombin. IP accumulation is maximally elicited by SFLLRN or 1 unit/ml thrombin in cells that overexpress PAR-1 and by AYPGKF or 10 units/ml thrombin in cells that overexpress PAR-4 (a receptor optimally cleaved by higher thrombin concentrations). In each case, receptor stimulation (proteolytically with thrombin or with peptide agonist) results in the activation of ERK, p38 MAPK, and AKT pathways (Fig. 5, B and C). In contrast, cathepsin G promotes only a very minor increase in the levels of IP1 or IP2/IP3 in cells that overexpress PAR-4; cathepsin G does not detectably elevate IP levels above basal in cells that express PAR-1 (Fig. 5A). Although cathepsin G weakly activates ERK (but not p38 MAPK or AKT) in PAR-1- and PAR-4-expressing cell lines (Fig. 5, B and C), this response displays atypically slow kinetics (peak at 10 min, relative to ERK activation by PARs which is maximal at 5 min). Cathepsin G also induces a similar weak and delayed activation of ERK in PAR–/– cells that do not respond to thrombin or peptide agonists (Fig. 5D). This indicates that the low level of ERK activation by cathepsin G cannot be ascribed to activation of either PAR-1 or PAR-4. Cells that overexpress PAR-1 maintain a spread morphology and remain adherent to substrate during incubation with cathepsin G. In contrast, cells that overexpress PAR-4 round and detach from the underlying matrix during incubation with cathepsin G. These morphological changes do not result from PAR-4 activation, because intense PAR-4 stimulation with either thrombin or AYPGKF does not induce comparable morphologic changes. More likely, the very minor signaling responses induced by cathepsin G in cells that overexpress PAR-4 (in the context of cell detachment from matrix) represent the consequences of cathepsin G-induced alterations in cell adhesion and not proteolytic activation of PAR-4 by cathepsin G. Collectively, these results raise serious doubts that the agonist actions of cathepsin G can be attributed to the activation of either PAR-1 or PAR-4. Cathepsin G Disables PAR-1 but Not PAR-2 or PAR-4 — Cathepsin G cleavage could disable, rather than activate, PARs. Fig. 6A shows that preincubation with cathepsin G for 30 min completely abrogates subsequent IP1 accumulation induced by thrombin in cells that overexpress PAR-1; responses to SFLLRN remain intact, indicating that the receptor remains otherwise structurally intact. Whereas PAR-1 is highly susceptible to cathepsin G-dependent cleavage, preincubation with cathepsin G does not impair subsequent phosphoinositide hydrolysis induced by either thrombin or AYPGKF in cells that overexpress human PAR-4 (Fig. 6B). These results provide further evidence that PAR-4 is not a direct target for cathepsin G actions. Fig. 6C also shows that cathepsin G disables PAR-1 in rat cardiomyocytes; treatment with cathepsin G renders cardiomyocyte cultures refractory to subsequent stimulation by thrombin but not by SFLLRN. These results are noteworthy, because the disabling pro
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