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【摘要】
Objective- The extracellular N terminus of the endothelin B (ET B ) receptor is cleaved by a metalloprotease in an agonist-dependent manner, but the physiological role of this N-terminal proteolysis is not known. In this study, we aimed to determine the functional role of the ET B receptor and of its N-terminal cleavage in vascular smooth muscle cells (VSMCs).
Methods and Results- VSMCs expressing either the full-length ET B receptor or an N-terminally truncated ET B receptor (corresponding to the N-terminally cleaved receptor) were analyzed for ligand-induced mitogen-activated protein kinase activation and expression of contractile proteins. In VSMCs expressing the full-length ET B receptor, IRL1620 (an ET B -selective agonist) induced a biphasic extracellular signal-regulated kinase 1/2 (ERK1/2) activation and increased expression of contractile proteins (smooth muscle myosin-1 [SM-1]/SM-2, SM22, and -actin). Interestingly, the second phase of ERK1/2 activation required metalloprotease activity, epidermal growth factor (EGF) receptor transactivation, and predominantly activation of G i proteins. In contrast, in VSMCs expressing N-terminally truncated ET B receptors, IRL1620 did not elicit EGF transactivation and failed to increase contractile protein expression.
Conclusions- This study is the first to show that stimulation of full-length ET B receptors promotes expression of contractile proteins and may thus participate in the differentiation of VSMCs.
The ET B receptor undergoes agonist-induced N-terminal proteolysis. Postreceptor signaling comprised biphasic ERK1/2 activation, EGF receptor transactivation, and an increased expression of contractile proteins in vascular smooth muscle cells (VSMCs). Thus, the ET B receptor may participate in the differentiation of VSMCs.
【关键词】 transactivation EGF receptor ERK/ differentiation
Introduction
Endothelin-1 (ET-1), one of the most potent vasoactive peptides, acts on 2 G protein-coupled receptors (GPCRs): the ET A receptor mainly expressed in vascular smooth muscle cells (VSMCs) and the endothelin B (ET B ) receptor predominantly expressed in endothelial cells. 1,2 Whereas VSMCs normally express only low levels of ET B receptors, a dramatic upregulation occurs during atherosclerosis, hypercholesterolemia, and in models of focal ischemia. 3-6 In atherosclerotic plaques, VSMCs close to foam cells show a predominant expression of ET B receptors and an enhanced immunoreactivity of ET-1. It was therefore suggested that ET-1 and ET B receptors may play a role in atherogenesis. 4 A protective role for the ET B receptor has been reported for the monocrotalin-induced pulmonary hypertension in mice. 7 Similarly, inhibition of the ET B receptor deteriorated remodeling after vascular injury in mice, 8 pointing to a protective role of the ET B receptor in atherosclerosis. However, the precise role of ET B receptors in VSMCs remains elusive.
VSMCs in native vessels are characterized by a slow proliferation rate and an abundant expression of contractile proteins, such as smooth muscle (SM) myosin and SM -actin. On vascular injury, VSMCs show a loss of contractile protein expression and increased proliferative and migratory responses to mitogens or cytokines. 9,10 However, within 3 to 6 months after vascular injury, VSMCs may regain a contractile phenotype through yet unknown mechanisms. 11 It is notable that thrombin via the protease-activated receptor 1 (PAR1) 12,13 and rapamycin, a blocker of the mammalian target of rapamycin, induce differentiation of VSMCs by inhibiting growth and increasing the expression of SM myosin heavy chain (SM-MHC), SM -actin, and calponin. 14
Several features of the ET B receptor are unique and not shared by the ET A receptor. For example, the ET B receptor (but not the ET A receptor) activates G Leibriz-Institut proteins of the G i family. Thus, the ET B receptor may induce differentiation in VSMCs similar to the PAR1 receptor. 12,13 A further unique feature of the ET B receptor is agonist-induced proteolysis of its N terminus by a metalloprotease between residues 64 and 65 (SLAR/SLA). 15 However, the physiological significance of N-terminal proteolysis is not known. Interestingly, incubation of purified ET B receptors with trypsin or thermolysin also resulted in N-terminal proteolysis, 16 suggesting that other proteases, too, can induce N-terminal proteolysis.
To extend our understanding of the role of the ET B receptor and of its N-terminal proteolysis in VSMCs, we mimicked the upregulation of ET B receptors (as observed in diseased vessels) by transfecting rat neonatal VSMCs with plasmids encoding the full-length ET B receptor or a mutant 2-64 ET B receptor (lacking the N-terminal portion removed by metalloproteases). We demonstrate that stimulation of the full-length ET B receptor induces a biphasic extracellular signal-regulated kinase 1/2 (ERK1/2) activation involving epidermal growth factor (EGF) receptor transactivation, whereas the 2-64 ET B receptor fails to transactivate the EGF receptor and elicits only monophasic ERK1/2 activation. Importantly, only the full-length ET B receptor, but not the 2-64 ET B receptor promotes the expression of differentiation markers, such as SM myosin, SM22, and -actin.
Methods
Materials
125 I-ET-1 (2000 Ci/mmol), and thrombin were from Amersham Biosciences. [ 3 H]thymidine and [ 3 H]chloramphenicol were from Perkin-Elmer. AG1478 and G418 were from Merck Biosciences. GM6001 was from Biomol. All other reagents were from Sigma. Ro28-2653 and Ro32-7315 were provided by Roche Diagnostics (Pharma Research Penzberg). Antibodies against SM myosin (clone SMMS-1) and SM22 were from DakoCytomation and Abcam, respectively. The -actin antibody (clone 1A4) was from Sigma. Affinity-purified polyclonal phospho ERK1/2 (pERK1/2), ERK1/2 and pElk-1 antibodies were from Cell Signaling. The monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank, maintained by the University of Iowa, Department of Biological Sciences. Matrix metalloproteinase-2 (MMP-2) small interfering RNA (siRNA; 1388356) and nonsilencing control (1022076) were from Qiagen. D-Phe-Pro-Arg chloromethyl ketone dihydrochloride-inactivated thrombin and plasmin were kindly donated by Dr Klaus T. Preissner and Tobias Schmidt-Wöll (Giessen, Germany).
Cell Culture and Transient Transfections
Human embryonic kidney 293 (HEK293) cells stably expressing the wild-type ET B ·green fluorescent protein (GFP) or the 2-64 ET B ·GFP receptor were maintained as described. 17,18 Primary cultures of VSMCs from newborn rats were established and maintained as described. 11 Growth arrest was induced by culturing cells for 48 hours in a serum-free quiescent medium containing 1% (wt/vol) BSA and 4 mg/mL transferrin. Nucleofection of VSMCs was performed using the Nucleofector technology (Amaxa) according to manufacturer suggestions. In brief, 2 x 10 6 cells were nucleofected with a mixture of solution 4837 (Amaxa) and plasmid DNA (10 µg) and siRNA (3 µg, if indicated) using program T-28.
For further experimental protocols, see the online-only data supplement, available at http://atvb.ahajournals.org.
Results
Agonist-Dependent and Agonist-Independent Cleavage of the ET B ·GFP Receptor in VSMCs
VSMCs normally express no or only low levels of ET B receptors. A dramatic upregulation is observed under pathological conditions. 3-6 The neonatal VSMCs used in this study express only ET A receptors because the nonselective agonist ET-1 but not the highly ET B receptor-selective agonist IRL1620 induced ERK1/2 activation (supplemental Figure I, available at http://atvb.ahajournals.org). To mimic the upregulation of ET B receptors in vitro, we transfected VSMCs with a plasmid encoding a fusion protein consisting of the ET B receptor and GFP. In 125 I-ET-1 saturation binding analysis with nontransfected and transfected VSMCs, maximal binding of 770±448 and 5130±1715 fmol/mg protein was found, respectively. The ET B /ET A receptor ratio was 85:15 in transfected VSMCs. 3,6
125 I-ET-1 and the ET B ·GFP receptor form tight, sodium dodecyl sulfate-stable complexes, which can be detected by autoradiography without cross-linking. 19 Therefore, VSMCs expressing ET B ·GFP receptor were incubated with 100 pmol/L 125 I-ET-1 at 4°C (to prevent receptor internalization) for 30 minutes and then either lysed or incubated for another 3 hours at 37°C. Lysates were then separated by low-temperature polyacrylamide gel electrophoresis at 4°C. With lysates from cells incubated at 4°C only, a broad band at 70 to 80 kDa was observed ( Figure 1 A, bracket), whereas in lysates from cells incubated at 37°C, 3 bands at 55, 35, and 30 kDa were detected ( Figure 1 A, arrow and arrowheads). As shown previously, the broad 70- to 80-kDa band corresponds to the full-length ET B ·GFP receptor, and the 55-kDa band corresponds to the N-terminally cleaved ET B ·GFP receptor; the 35- and 30-kDa bands result from lysosomal degradation. 15 The data demonstrate that agonist-induced N-terminal proteolysis of the ET B ·GFP receptor occurs in VSMCs. In further experiments, we tested whether serine proteases can also cleave the N terminus of the receptor in an agonist-independent manner. VSMCs expressing the ET B ·GFP receptor were incubated with thrombin or plasmin for 30 minutes at 37°C and then incubated with 125 I-ET-1 for an additional 30 minutes at 4°C before lysis. Thrombin (10 and 1 U/mL) and plasmin (5 U/mL) cleaved the N terminus quantitatively, resulting in a receptor migrating as a 55-kDa band similar to that seen for agonist-induced proteolysis ( Figure 1 B; compare with Figure 1 A). D-Phe-Pro-Arg chloromethyl ketone dihydrochloride-inactivated thrombin and plasmin ( Figure 1 C) and thrombin receptor-activating peptide (data not shown) did not induce N-terminal proteolysis of the ET B receptor. GM6001, a general inhibitor of metalloproteases, inhibited ET-1-induced cleavage (incubated with 450 mmol/L sucrose to prevent ET-1-induced internalization; Figure 1 C). However, it did not prevent plasmin-induced cleavage, indicating that plasmin-induced cleavage was not subsequent to the activation of a metalloprotease. Thus, the N terminus of the ET B receptor is a substrate for serine proteases.
Figure 1. Agonist-dependent and -independent N-terminal cleavage of the ET B receptor. VSMCs expressing the full-length ET B ·GFP receptor were treated for 30 minutes at 37°C without (A) or with active (B and C) or inactive proteases and the metalloprotease inhibitor GM6001 (100 µmol/L; C) as indicated. Sucrose (450 mmol/L) was added as indicated to prevent receptor internalization. Cells were then incubated with 125 I-ET-1 for 30 minutes at 4°C or for 3 hours at 37°C and lysed. Lysates were separated by low-temperature polyacrylamide gel electrophoresis and then subjected to autoradiography. The data shown are representative of 3 independent experiments. Bracket indicates full-length ET B receptor; arrow, N-terminally truncated receptor; arrowheads, degradation products.
Full-Length and N-Terminally Truncated ET B ·GFP Receptors Differ in Their Ability to Induce ERK1/2 Activation
The functional consequences of N-terminal proteolysis of the ET B receptor are not known. In a previous analysis, we have shown that N-terminal proteolysis does not alter the affinity of the receptor to ET-1. 15 Therefore, we investigated in VSMCs whether the full-length ET B ·GFP receptor and the 2-64 ET B ·GFP receptor differ in their ability to mediate signaling events such as ERK1/2 activation. The ET B -selective agonist IRL1620 was used to avoid costimulation of endogenously expressed ET A receptors. IRL1620 treatment of VSMCs expressing the full-length ET B ·GFP receptor elicited a rapid increase in pERK1/2 formation, with a peak within the first 10 minutes after stimulation and a second peak after 80 minutes ( Figure 2 A). However, for VSMCs expressing the 2-64 ET B ·GFP receptor, IRL1620 elicited only the early phase of ERK1/2 activation. Almost identical results were obtained with HEK293 cells expressing the full-length ET B receptor or the 2-64 ET B receptor ( Figure 2 B), with the exception that ET-1 was used for stimulation (no endogenous ET A receptor expression). We further analyzed whether protease-induced removal of the N terminus of the ET B receptor also results in monophasic ERK1/2 activation similar to that found for the 2-64 ET B ·GFP receptor. Whereas pretreatment of VSMCs with thrombin already elicited ERK1/2 activation via PAR1 receptors, this was not the case for plasmin. Therefore, VSMCs expressing the full-length ET B receptor were preincubated with plasmin (5 U/mL for 30 minutes) to remove the N terminus quantitatively ( Figure 1 B). Subsequent stimulation with IRL1620 elicited only monophasic ERK1/2 activation ( Figure 2 C). Thus, the expression of an N-terminally truncated ET B receptor or plasmin-induced removal of the extracellular N terminus of the wild-type ET B receptor resulted in altered ERK1/2 activation.
Figure 2. The extracellular N terminus of the ET B receptor is required for the biphasic ERK1/2 activation. VSMCs (A and C) or HEK293 cells (B) expressing either the full-length ET B ·GFP or the 2-64 ET B ·GFP receptor were analyzed for agonist-induced activation of ERK1/2. In C, VSMCs expressing the full-length ET B ·GFP receptor were preincubated with plasmin for 30 minutes at 37°C, followed by stimulation with IRL1620. Lysates were then subjected to immunoblotting using pERK1/2 and ERK1/2 antibodies. Graphs summarize the results of 5 to 10 different experiments. QM indicates quiescent medium; P <0.05.
In immunocytochemistry, we further analyzed the temporal and spatial pattern of pERK1/2 formation in VSMCs expressing the full-length ET B receptor. Treatment with IRL1620 (10 minutes) resulted in a strong increase in cytoplasmic and nuclear pERK1/2 staining (supplemental Figure II), whereas only faint pERK1/2 signals were found in the cytoplasm after 60 minutes. After 120 minutes of IRL1620 stimulation, pERK1/2 signals were observed in the cytoplasm close to the nucleus and within the nucleus. Notably, the pERK1/2 signals during the second phase of ERK1/2 activation appeared stronger in the perinuclear region than in the nucleus. Using nuclear preparations of VSMCs, we could further demonstrate that biphasic ERK1/2 activation also results in long-lasting activation of the nuclear substrate Elk-1 (supplemental Figure II).
The Late Phase of ERK1/2 Activation Requires Transactivation of the EGF Receptor and Depends on G i Proteins
We have shown previously that the metalloprotease inhibitor Ro32-7315 blocks the agonist-induced N-terminal proteolysis of the full-length ET B receptor. 15 To test whether this inhibitor also alters biphasic ERK1/2 activation, we treated VSMCs and HEK293 cells expressing the ET B ·GFP receptor with Ro32-7315 for 30 minutes before agonist application. The selective MMP-2/MMP-9 inhibitor Ro28-2653 was included as a control (does not inhibit agonist-induced N-terminal proteolysis of the ET B receptor). 15 As shown in Figure 3 A, Ro32-7315 abolished the late phase of ERK1/2 activation. Surprisingly, the MMP-2/MMP-9-selective inhibitor Ro28-2653 also abolished the late phase, pointing to a role of MMP-2/MMP-9 in ERK1/2 activation. Analysis of gelatinolytic activity in supernatants of VSMCs expressing the full-length ET B receptor revealed that IRL1620 (4 hours; 100 nmol/L) induced the secretion of pro-MMP-2 and active MMP-2 (supplemental Figure III), which was also confirmed in ELISA activity assays (IRL1620-induced increase in MMP-2 activity from 122±64 to 400±100 pg/mL; n=3). To analyze the role of MMP-2 in biphasic ERK1/2 activation in more detail, we cotransfected VSMCs with a plasmid encoding the full-length ET B receptor and MMP-2 siRNA. IRL1620 stimulation of VSMCs transfected with MMP-2 siRNA failed to stimulate MMP-2 secretion and induced only monophasic ERK1/2 activation (supplemental Figure IIIB and IIIC). In contrast, VSMCs transfected with nonsilencing siRNA maintained IRL1620-induced MMP-2 secretion and biphasic ERK1/2 activation. The data suggest that MMP-2 contributes to the ET B receptor-mediated second phase of ERK1/2 activation.
Figure 3. The late phase of the ERK1/2 activation is mediated by a G i - and metalloprotease-dependent transactivation of the EGF receptor. VSMCs or HEK293 cells expressing ET B ·GFP receptors were either pretreated with the metalloprotease inhibitors Ro32-7315 or Ro28-2653 (10 µmol/L, 30 minutes; A) or with PTX (200 ng/mL; 18 hours), AG1478 (250 nmol/L, 30 minutes), anti-HB-EGF (5 µg/mL, 30 minutes), anti-EGF receptor (8 µg/mL; 30 minutes), or a nontoxic mutant of diphtheria toxin (CRM197, 10 µg/mL, 30 minutes; B) before stimulation as indicated. Immunoblots were probed with pERK1/2 and ERK1/2 antibodies. C, VSMCs expressing the full-length ET B receptor or the 2-64 ET B ·GFP were treated with IRL1620 as indicated. After immunoprecipitation, phosphorylated EGF receptors were detected with a phosphotyrosine antibody (C, top panel). Equal loading was verified using the EGF receptor antibody (C, bottom panel). Immunoblots are representative of 3 independent experiments. IP indicates immunoprecipitation; IB, immunoblot; QM, quiescent medium.
Activation of metalloproteases can elicit the transactivation of the EGF receptor and subsequent ERK1/2 activation via the release of membrane-bound EGF ligands. 20 Therefore, we studied the effect of the EGF receptor tyrosine kinase inhibitor AG1478 (250 nmol/L) on ET B receptor-mediated ERK1/2 activation in VSMCs and HEK cells. For HEK293 cells, we also used CRM197, a nontoxic mutant of diphtheria toxin that binds to human (but not rat) heparin-binding EGF (HB-EGF) and stimulates its internalization. 21 AG1478 and CRM197 did not alter the early phase of ERK1/2 activation but abolished the late phase of ERK1/2 activation ( Figure 3 B). To provide further evidence that HB-EGF and the EGF ?receptor play a pivotal role in the second phase of ERK1/2 activation, we treated VSMCs with antibodies directed against HB-EGF or the EGF receptor before stimulation with IRL1620. In the presence of either antibody, the second phase of ERK1/2 activation was abolished ( Figure 3 B, left panel), suggesting that the release of HB-EGF results in an activation of the EGF receptor.
The early phase of the ET B receptor-mediated ERK1/2 activation, not affected by metalloprotease inhibitors, depends on G proteins of the G q/11 family. 22 However, it remains elusive which G proteins are involved in the regulation of the late phase of ERK1/2 activation. When we incubated VSMCs or HEK293 cells expressing the full-length ET B ·GFP receptor with pertussis-toxin (PTX; 200 ng/mL for 18 hours), the early phase of ERK1/2 activation was not affected, whereas the late phase was abolished ( Figure 3 B). These data suggest that the full-length ET B receptor stimulates the early and the late phase of ERK1/2 activation via 2 different signaling cascades (eg, G proteins of the G q/11 and the G i family, respectively).
To directly demonstrate transactivation of the EGF receptor, we immunoprecipitated endogenously expressed EGF receptors of VSMCs expressing the full-length ET B receptor after IRL1620 treatment. In immunoblot analysis, phosphorylated EGF receptors were only found in precipitates from cells stimulated for 120 minutes with IRL1620 but not in those stimulated for 5 or 60 minutes ( Figure 3 C, left panel). VSMCs expressing the 2-64 ET B ·GFP receptor did not show IRL1620-induced phosphorylation of the EGF receptor. Further analysis revealed that pretreatment of VSMCs with PTX, AG1478, or Ro28-2653 inhibited the IRL1620-mediated transactivation of the EGF receptor via the full-length ET B receptor ( Figure 3 C, right panel). In control experiments, in which the EGF receptor was stimulated by EGF, only AG1478 but not PTX or Ro28-2653 abolished EGF receptor phosphorylation. These results suggest that the late phase of ET B receptor-induced ERK1/2 activation requires EGF receptor transactivation via a G i -dependent, metalloprotease-mediated release of HB-EGF.
Full-Length ET B Receptor and Transactivation of the EGF Receptor Are Required for IRL1620-Induced Expression of Contractile Proteins in VSMCs
To determine the role of monophasic versus biphasic ERK1/2 activation for cell proliferation, we studied IRL1620-induced effects on [ 3 H]thymidine incorporation in VSMCs expressing the ET B ·GFP or the 2-64 ET B ·GFP receptor, respectively. IRL1620 induced in VSMCs expressing either the ET B ·GFP or the 2-64 ET B ·GFP receptor a similar 2.5-fold increase in DNA synthesis, whereas platelet-derived growth factor-BB elicited a 4.5-fold stimulation ( Figure 4A and 4 B). To dissect the contribution of the early (PTX-insensitive) and late (PTX-sensitive) phase of ERK1/2 activation on DNA synthesis, VSMCs were treated with PTX (200 ng/mL; 18 hours). PTX treatment had no effect on IRL1620-induced DNA synthesis ( Figure 4A and 4 B), indicating that the IRL1620-stimulated increase in DNA synthesis was dependent on the first but not on the late phase of ERK1/2 activation.
Figure 4. [ 3 H]thymidine incorporation and SM-MHC promoter activity in IRL1620-treated VSMCs expressing either the full-length ET B ·GFP or the 2-64 ET B ·GFP receptor. VSMCs expressing the full-length ET B ·GFP receptor (A) or the mutant 2-64 ET B ·GFP receptor (B) were incubated without or with PTX (200 ng/mL; 18 hours) before application of vehicle, IRL1620 (100 nmol/L), or platelet-derived growth factor-BB (100 ng/mL) for 24 hours. Then cells were lysed, and incorporation of [ 3 H]thymidine was determined. The values represent mean values±SEM of triplicates from 3 independent experiments. QM indicates quiescent medium. C, VSMCs were nucleofected with a promoterless plasmid pCATbasic, the plasmid pCAT-MHC, or a combination of the plasmid pCAT-MHC and pET B ·GFP or 2-64 ET B ·GFP plasmids. Cells were treated for 48 hours with PTX (200 ng/mL), Ro28-2653 (10 µmol/L), AG1478 (250 nmol/L), serum, or IRL1620, as indicated. The values represent mean values±SEM of triplicates from 3 independent experiments.
In further experiments, we aimed to investigate the role of the ET B receptor on the expression of contractile proteins in VSMCs. First, we analyzed the expression of chloramphenicol acetyl transferase (CAT) under the control of the promoter of the SM-MHC gene (pCAT-MHC). 11 In VSMCs transfected with the plasmid pCAT-MHC, serum increased the CAT activity 6- to 7-fold but not in VSMCs transfected with a promoterless pCAT plasmid (pCAT basic; Figure 4 C). In VSMCs cotransfected with the plasmids pET B ·GFP and pCAT-MHC, IRL1620 induced an 5-fold increase in CAT activity, which was abolished by pretreatment with PTX, Ro28-2653, or AG1478 ( Figure 4 C). No IRL1620-mediated increase in CAT activity was found for VSMCs expressing the 2-64 ET B ·GFP.
Because the full-length ET B receptor induced a transcriptional activation of the SM-MHC promoter, we also studied the expression of SM-MHC isoforms smooth muscle myosin-1 (SM-1) and SM-2 in immunoblot experiments. In serum-starved VSMCs expressing the full-length ET B ·GFP or the 2-64 ET B ·GFP receptor, only expression of the SM-1 isoform was observed ( Figure 5 ). On IRL1620 treatment (100 nmol/L) of VSMCs expressing the full-length ET B receptor, a prominent upregulation of SM-1, SM-2, -actin, and SM22 was seen ( Figure 5 ). Notably, this upregulation was abolished by PTX, AG1478, or Ro28-2653 treatment. As expected from the promoter studies, VSMCs expressing the 2-64 ET B receptor did not show an IRL1620-induced upregulation of SM-1/SM-2, SM22, and -actin ( Figure 5 ). The results suggest that ET B receptor-mediated signaling involving EGF receptor transactivation promotes the expression of specific markers of VSMCs and thus may contribute to differentiation ( Figure 6 ).
Figure 5. Stimulation of the full-length ET B receptor in VSMCs increases the expression of SM myosin, SM22, and -actin. VSMCs transiently expressing the ET B ·GFP receptor were either treated for 48 hours or 72 hours with vehicle (quiescent medium) or IRL1620 (100 nmol/L) and with PTX (200 ng/mL), Ro28-2653 (10 µmol/L), or AG1478 (250 nmol/L) as indicated. Cells were then processed for immunoblotting. Immunoblots were probed with a SM myosin, SM22, or -actin antibodies. Graphs summarize the results of 3 different experiments. * P <0.05.
Figure 6. Diagram of ET B receptor-mediated biphasic ERK1/2 activation. The ET B receptor activates G proteins of the G q/11 and G i families. The first phase of ERK1/2 activation is mediated via G q/11 proteins and involves a PLCß/Ca 2+ /c-Src-dependent but PKC-independent pathway. 21 The second phase of ERK1/2 activation is mediated via G i proteins and involves MMP-2, HB-EGF, and the EGF receptor. In VSMCs, the ET B receptor-mediated biphasic ERK1/2 activation leads to an increased expression of contractile proteins. Nuc indicates nucleus; PM, plasma membrane.
Discussion
Stimulation of VSMCs expressing the ET B receptor causes a long-lasting, biphasic ERK1/2 activation that depends on an intact N terminus of the ET B receptor. The second phase of ERK1/2 activation depends on the transactivation of the EGF receptor. This is supported by the fact that the second phase is abolished by: (1) metalloprotease-inhibitors (Ro32-7315 and Ro28-2653), (2) CRM197, which leads to a downregulation of HB-EGF, 20 (3) AG1478, an inhibitor of the EGF receptor kinase activity, and (4) antibodies against HB-EGF or the EGF receptor ( Figure 6 ). VSMCs expressing an N-terminally truncated ET B receptor (either the plasmin-cleaved or the 2-64 ET B receptor) elicit only a transient, monophasic ERK1/2 activation and do not induce EGF receptor transactivation. The ET B receptor-mediated transactivation of the EGF receptor is effective after 80 minutes, suggesting that ET B receptor-induced activation of metalloproteases, which mediate the release of HB-EGF, represents a rather slow process.
So far, several metalloproteases have been identified to be involved in the release of HB-EGF: ADAM10, ADAM17, MMP-2/MMP-9, and MMP-7. 23-25 Our studies in VSMCs point to a role of MMP-2 in the release of HB-EGF because stimulation of the ET B receptor caused an increase in MMP-2 secretion and activity. The role of MMP-2 is highlighted by the finding that downregulation of MMP-2 expression by siRNA abolished the second phase of ERK1/2 activation. MMP-2 may be involved directly in the transactivation of the EGF receptor or mediate the activation of other metalloproteases, which, in turn, cause HB-EGF release. It is also possible that upregulation of further signaling molecules is required for the transactivation of the EGF receptor. For example, in glomerular mesangial cells, ET-1 induced an upregulation of HB-EGF and fibroblast growth factor within 2 hours. 26
Whereas the precise molecular mechanisms involved in delayed EGF receptor activation remain to be shown, the presented data provide evidence that EGF receptor transactivation in VSMCs via the ET B receptor may contribute to cellular differentiation. This is supported by the findings that ET B receptor-mediated increase in the transcriptional activity of the SM-MHC promoter and the expression of SM-1/SM-2, SM22, and -actin are abolished by substances that interfere with EGF receptor transactivation (PTX; Ro28-2653 or AG1478). In agreement with our findings, activation of the thrombin receptor PAR1 in VSMCs elicited an increased expression of contractile proteins via a PTX-sensitive biphasic ERK1/2 activation involving EGF receptor transactivation. 12,13 The findings are also in line with data obtained with PC12 cells, for which the intensity and the duration of ERK1/2 activation determined the cellular response (eg, proliferation versus differentiation). 27,28 It is intriguing to speculate that differentiation of VSMCs in general may be promoted by GPCRs, which cause biphasic ERK1/2 activation.
We show here for the first time that the ET B receptor has the potential to mediate differentiation in VSMCs similar to that reported for PAR1. 12,13 These findings favor a protective role of both receptors in vascular remodeling because both are upregulated in VSMCs of atherosclerotic lesions. 4,29 It remains to be determined whether the described ET B receptor-mediated differentiation process is preserved in vivo. High levels of serine proteases in the vessel wall during thrombosis could abolish the differentiating effect of the ET B receptor in VSMCs by an agonist-independent N-terminal proteolysis. Interestingly, it has been demonstrated that tumor necrosis factor- converting enzyme, which is upregulated in inflammation, can cleave the N terminus of PAR1 proximal to the thrombin cleavage site, resulting in a nonfunctional receptor. 30 Thus, proteases not only modulate coagulation and matrix composition but also alter the functional activity of GPCRs. These results point to a new dimension for the control of protease activity in vascular disease.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (FG 341, GRK865), the Sonnenfeld-Stiftung, and the Fonds der Chemischen Industrie. We thank Monika Bigalke and Claudia Plum for technical assistance, Dr Klaus T. Preissner and Tobias Schmidt-Wöll for inactive thrombin and plasmin preparations, and Drs Timothy Plant and Günter Schultz for critical reading of this manuscript.
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作者单位：Institut für Pharmakologie (E.G., S.G., W.R., A.O.), Charité Campus Benjamin Franklin, Berlin, Germany; Leibniz-Institut für Molekulare Pharmakologie (E.G., J.E., M.B., W.R., A.O.), Campus Berlin-Buch, Berlin, Germany; Abteilung für Klinische Pharmakologie (H.P.R.), Ruhr-Univer