Published online before print November 1, 2007, doi: 10.1161/CIRCRESAHA.107.150573
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© 2008 American Heart Association, Inc.
Molecular Medicine |
Novel Mode for Neutrophil Protease Cathepsin G–Mediated Signaling
Membrane Shedding of Epidermal Growth Factor Is Required for Cardiomyocyte Anoikis
From the Cardiovascular Research Center (K.R., M.H., A.S.), Department of Anatomy & Cell Biology, Temple University, Philadelphia, Pa; Department of Radiation Oncology (K.V.), Virginia Commonwealth University, Richmond; and Department of Pharmacology (S.F.S.), Columbia University, New York, NY.
Correspondence to Abdelkarim Sabri, PhD, Cardiovascular Research Center, Temple University, 3420 N Broad St, Philadelphia, PA 19140. E-mail sabri{at}temple.edu
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Abstract |
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Neutrophils are thought to orchestrate myocardial remodeling during the early progression to cardiac failure through the release of reactive oxygen species, antimicrobial peptides, and proteases. Although neutrophil activation may be beneficial at early stages of disease, excessive neutrophil infiltration can induce cardiomyocyte death and tissue damage. The neutrophil-derived serine protease cathepsin G (Cat.G) has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis. However, the involved signaling mechanisms for Cat.G are not well understood. This study identifies epidermal growth factor receptor (EGFR) transactivation as a mechanism whereby Cat.G induces signaling in cardiomyocytes. Cat.G induced a rapid and transient increase in EGFR tyrosine phosphorylation, and inhibition of EGFR kinase activity, either with AG1478 or by expression of kinase inactive EGFR mutants (EGFR-CD533), markedly attenuated EGFR downstream signaling and myocyte anoikis induced by Cat.G. Consistent with this effect of EGFR, high level expression of wild-type EGFR was sufficient to promote myocyte apoptosis. We also found that matrix metalloproteinase–dependent membrane shedding of heparin-binding EGF was involved in Cat.G signaling and that membrane type 1 matrix metalloproteinase activation may constitute a potential target that entails matrix metalloproteinase activation induced by Cat.G. The paradoxical proapoptotic effect of EGFR appeared to be dependent on protein tyrosine phosphatase SHP2 (Src homology domain 2–containing tyrosine phosphatase 2) activation and focal adhesion kinase downregulation. These results show that Cat.G-induced cardiomyocyte apoptosis involves an increase in EGFR-dependent activation of SHP2 that promotes focal adhesion kinase dephosphorylation and subsequent cardiomyocyte anoikis.
Key Words: cathepsin G • EGF receptor • focal adhesion • protein tyrosine phosphatase • cardiomyocytes • apoptosis
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Introduction |
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Inflammation is thought to be a critical mediator of myocardial remodeling in response to cardiac injury.1,2 Although beneficial at early stages after myocardial injury, inflammatory cells release free radicals and proteolytic enzymes within the myocardium that exacerbate cardiomyocyte death and increase myocardial dysfunction.1–3 Cathepsin-G (Cat.G), a major serine protease released by activated neutrophils, has been proposed to play an important role in tissue remodeling at sites of tissue injury.3–5 Cat.G has been shown to hydrolyze a host of proteins including chemoattractants, extracellular matrix (ECM) and hormonal factors.5 In addition, Cat.G might cleave and activate G protein–coupled protease-activated receptors as a mechanism to modulate coagulation and tissue remodeling at sites of injury and inflammation.6 In cultured neonatal rat cardiomyocytes (NRCM), pathophysiological concentrations of Cat.G activate signaling pathways that culminate in myocyte detachment and apoptosis. Some facets of Cat.G signaling in cardiomyocytes are independent of protease-activated receptor-1 and -4 activation.7 Recent work emphasizes the role of focal adhesion (FA) signaling downregulation and protein tyrosine phosphatase (PTPs) activation as a mechanism whereby Cat.G modulates normal cell–cell or cell–ECM interactions that are necessary for cell survival.8
Several studies have shown the role of epidermal growth factor receptor (EGFR) transactivation in mediating serine protease–induced signaling and growth.9,10 Transactivation of EGFR is mediated, at least in some cases, by the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF), which is cleaved from its membrane-anchored form (proHB-EGF) in a process termed "ectodomain shedding".11 HB-EGF is a member of the EGF family with a strong affinity for immobilized heparin.12 Like other members of the EGF family of ligands (EGF, neuregulin, and heregulin), HB-EGF interacts with the EGFR family, which comprises, in addition to erbB1/EGFR, 2 direct receptors (erbB3 and erbB4), and 1 coreceptor (erbB2).13,14 HB-EGF has been shown to bind and stimulate erbB4 as well as erbB111,13,14 and can also form a complex with integrins at sites of cell–cell contact, reiterating the importance of HB-EGF in cell-to-cell communication.15 Secreted HB-EGF is a potent mitogen for many cell types,12,16 and membrane shedding of HB-EGF and/or stimulation of EGFR, erbB2, or erbB4 has been shown to promote cardiomyocyte hypertrophy.17–19
Moreover, proHB-EGF is also biologically active with variable functions, ranging from stimulating cell growth and suppressing cell death to inhibiting cell growth and inducing apoptosis, depending on the type of target cells.12,16 Whereas the proapoptotic effect of proHB-EGF is paradoxically unusual, several other reports have shown that increased levels of EGFR expression leads to ligand-dependent apoptosis.16,20,21 Although this apparent discrepancy remains unresolved, in many of these reports, cell death may be the result of FA signaling downregulation and cell anoikis associated with hyperstimulation of erbB signaling.21,22
Based on the findings that proHB-EGF expression is enhanced in various tissues after injury and inflammation and that ectodomain shedding occurs in response to cellular stress and inflammation,23,24 we tested the hypothesis that neutrophil-derived protease Cat.G is responsible for membrane shedding of proHB-EGF, leading to cardiomyocyte apoptosis.
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Materials and Methods |
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Myocyte Preparation
Cardiac myocytes were isolated from the ventricles of neonatal Sprague–Dawley rats (Charles River Laboratories, Inc, Wilmington, Mass) by collagenase digestion as previously described.7,8 After 30 minutes of preplating (to eliminate adherent fibroblasts), cells were plated at a density of 160 000/cm2 in 10% FBS DMEM supplemented with 10 mg/mL cytosine arabinoside, 1 mmol/L L-glutamine, and antibiotic/antimycotic solution. Under these high-density conditions, the myocytes form cell–cell contacts and display spontaneous contractile activity within 24 hours of plating. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Temple University.
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
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Results |
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EGFR Activation Mediates Cat.G-Induced Extracellular Signal-Regulated Kinase 1/2, p38 Mitogen-Activated Protein Kinase, and AKT Activation
Transactivation of EGFR can be initiated by a variety of ligands, including serine proteases.9,10 To ascertain whether early signaling pathways induced by Cat.G are mediated through EGFR activation, we examined the effect of tyrphostin AG1478, a potent inhibitor of EGFR tyrosine kinase activity, on key signaling molecules emanating from EGFR (ie, extracellular signal-regulated kinase [ERK]1/2, p38 mitogen-activated protein kinase [MAPK], and AKT). As we have shown previously,7 myocytes treated with Cat.G for 5 and 30 minutes showed an increase in ERK1/2, p38 MAPK, and AKT phosphorylation but with different kinetics. ERK1/2 phosphorylation was rapid and transient, whereas p38 MAPK and AKT phosphorylation was relatively slow and sustained over time. Treatment with AG1478 effectively abrogated Cat.G-induced ERK1/2, p38 MAPK, and AKT phosphorylation (Figure 1).
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Next, we examined whether Cat.G-initiated signaling is mediated by tyrosine phosphorylation of EGFR. After Cat.G exposure, EGFR was rapidly phosphorylated, as determined by blotting with anti-phosphotyrosine antibodies. This occurred within 2.5 minutes, reaching a maximum at 5 minutes and declining 10 minutes after Cat.G addition (Figure 2A). The level of EGFR phosphorylation in Cat.G-challenged cells was similar to that observed in thrombin-treated cells (another serine protease) and was significantly less pronounced than that measured in EGF-treated cells (Figure 2B). Consistent with EGFR activation, a reorganization of EGFR labeling was observed 5 minutes after Cat.G treatment (Figure 2C). In control NRCMs, EGFR staining was distributed throughout the cell with some dense staining concentrated at the perinuclear region (Figure 2Ca). Sarcomeric 
-actinin revealed the typical sarcomeric pattern of repetitive striations, with the labeled structure representing the Z-lines (Figure 2Cc). After Cat.G exposure, EGFR immunostaining was more concentrated at the level of Z-lines with some scattered labeling surrounding the nucleus (Figure 2Cb). Together, these data indicate that Cat.G triggers EGFR activation in cardiomyocytes, which, in turn, results in ERK1/2, p38 MAPK, and AKT activation.
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Membrane Shedding of HB-EGF Mediates Cat.G-Induced Signaling
EGFR transactivation has been reported to be either ligand independent25,26 or dependent on ligands such as HB-EGF.11 Therefore, we investigated whether Cat.G-induced signaling involved EGFR transactivation by membrane shedding of HB-EGF. Western blot analysis with HB-EGF antibodies revealed that Cat.G treatment initiated time-dependent accumulation of soluble 
26-kDa proteins in the conditioned media. HB-EGF–like protein was detected at 5 to 10 minutes after Cat.G treatment, and this accumulation remained elevated 2 hours after Cat.G treatment (Figure 3A). In contrast, stimulation with thrombin showed transient accumulation of HB-EGF in the conditioned media with a maximum response detected 5 to 10 minutes following treatment with thrombin.
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We next examined whether the Cat.G released form of HB-EGF possessed any biological activity. Addition of anti–HB-EGF, but not anti–transforming growth factor (TGF), neutralizing antibodies markedly reduced ERK1/2 phosphorylation induced by Cat.G. The anti–HB-EGF or anti-TGF neutralizing antibodies were highly selective, as they blocked ERK1/2 activation induced by HB-EGF or TGF, respectively (Figure 3B and Figure I in the online data supplement). Pretreatment with heparin, which blocks HB-EGF–induced EGFR transactivation by competing with cell surface heparin sulfate proteoglycans for HB-EGF binding, markedly attenuated Cat.G-induced, but not EGF-induced, ERK1/2 phosphorylation (Figure 3C). It is noteworthy that the concentration of heparin used in these experiments did not affect the proteolytic activity of Cat.G (supplemental Figure II).
It has been documented that cell surface matrix metalloproteinases (MMPs) trigger EGFR transactivation via proteolytic processing of the transmembrane form of the HB-EGF precursor into the mature soluble form of the ligand.11 We next assessed whether MMP(s) are activated in response to Cat.G and whether they are involved in Cat.G-initiated ERK1/2 activation. Figure 3D shows MMP-2 activation induced by Cat.G, as determined by in-gel zymography. This activation occurred early and was sustained for >2 hours after Cat.G treatment (Figure 3D and data not shown). Furthermore, treatment of cardiomyocytes with the potent broad-spectrum MMP inhibitor BB94 inhibited Cat.G-induced ERK1/2 phosphorylation (Figure 3C), suggesting that MMPs mediate Cat.G signaling. It is noteworthy that MMP-2 activation induced by Cat.G was markedly reduced in myocytes treated with BB94 but not affected when cells were treated with AG1478 or heparin (supplemental Figure III). However, MMP-2 is hardly activated by Cat.G,27 suggesting the involvement of an upstream activator of MMP-2. To this end, we tested whether Cat.G-induced MMP-2 activation involved activation of membrane type (MT)1-MMP, a membrane type MMP that has been shown to be activated by Cat.G and involved in MMP-2 activation.28 Figure 3E shows that myocytes treated with Cat.G increased MT1-MMP release in the conditioned medium (Figure 3E), indicating its activation. Concomitant with MT1-MMP activation by Cat.G, pretreatment of myocytes with tissue inhibitor of MMP (TIMP)2 (with high affinity toward MT1-MMP) or anti–MT1-MMP neutralizing antibodies markedly reduced MMP-2 and ERK1/2 activation induced by Cat.G treatment for 5 minutes (Figure 3F). These data show that Cat.G leads to MT1-MMP activation that entails MMP-2 cleavage and EGFR-dependent ERK1/2 phosphorylation.
Membrane Shedding of HB-EGF Is Involved in Cat.G-Induced Myocyte Apoptosis
NRCMs undergo retractile morphological changes and detachment from the ECM after Cat.G treatment, leading to myocyte anoikis.7,8 To determine whether EGFR transactivation is involved in Cat.G-induced myocyte apoptosis, cells were pretreated with AG1478, heparin, or BB94 before Cat.G treatment. As shown in Figure 4A, pretreatment with AG1478 significantly delayed, but did not completely block, cardiomyocyte retractile morphological changes and cell detachment induced by Cat.G (1 and 4 hours). Similar data on Cat.G-induced morphological changes were observed when cells were pretreated with heparin or BB94 (data not shown). In addition to cell detachment, myocytes undergo apoptosis when treated with Cat.G for 8 hours, as evidenced by an increase in caspase-3 activity and DNA fragmentation (Figure 4B and 4C). Interestingly, treatment with AG1478, heparin, or BB94 prevented caspase-3 activation and DNA fragmentation induced by Cat.G. These data support the hypothesis that MMP activation and subsequent transactivation of EGFR is involved in Cat.G-induced myocyte detachment and apoptosis.
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EGFR Catalytic Activity Is Required for Cat.G-Induced Myocyte Apoptosis
To gain insight into the role of EGFR in Cat.G signaling, we conducted gain- or loss-of-function experiments in which we transduced NRCMs with adenoviruses carrying wild-type EGFR (WT-EGFR) or a mutated construct expressing catalytically inactive EGFR (EGFR-CD533) (EGFR lacking the COOH-terminal 533 aa and acts as a potent inhibitor of EGFR). Myocytes infected with EGFR-CD533 at 25, 50, and 100 plaque-forming units (pfu)/cell showed an increased level of truncated mutant EGFR (110 kDa) by 4-, 7-, and 9-fold, respectively, over LacZ control, whereas the expression of the endogenous EGFR remained intact over control LacZ (Figure 5A). EGFR-CD533 overexpression significantly reduced ERK1/2 activation induced by Cat.G or EGF, with no detectable effect on ERK1/2 expression levels (Figure 5C). Myocytes infected with WT-EGFR viruses at 5, 10, 20, and 80 pfu/cell showed an increased level of EGFR expression by 4-, 6-, 7-, and 9-fold, respectively, over LacZ-infected controls (Figure 5B). Overexpression of WT-EGFR increased basal ERK1/2 phosphorylation compared with LacZ controls, and treatment with Cat.G further enhanced ERK1/2 phosphorylation (Figure 5C).
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To assess whether EGFR activation promotes myocyte anoikis induced by Cat.G, myocytes infected with recombinant adenovirus expressing WT-EGFR, dominant negative EGFR-CD533 mutants, or LacZ were evaluated for apoptotic signaling. There was an increase in both caspase-3 activity and DNA fragmentation in cells treated with Cat.G for 8 hours, indicating an increase in apoptosis (Figure 6A and 6B). WT-EGFR overexpression at low levels reduced basal myocyte apoptosis compared with control LacZ-infected cells and attenuated Cat.G-induced myocyte apoptosis. However, high-level expression of WT-EGFR promoted basal cardiomyocyte apoptosis compared with LacZ-infected controls and additional treatment with Cat.G did not further enhance myocyte apoptosis. Participation of caspases in WT-EGFR–induced apoptosis was confirmed by preemptive pharmacological inhibition with Z-VAD-FMK (a cell-permeable general caspase inhibitor), which prevented caspase-3 activity and DNA cleavage (Figure 6C and 6D). In contrast, EGFR-CD533 mutant overexpression markedly reduced caspase-3 activation and DNA fragmentation induced by Cat.G (Figure 6E and 6F). These data show that although EGFR basal activity is necessary to maintain myocyte survival, both its overexpression and activation by Cat.G are sufficient to promote myocyte apoptosis.
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Cat.G-Induced EGFR Transactivation Mediates FAK Downregulation
Previous studies have shown the role of FAK signaling downregulation in Cat.G- or EGF-induced cell retractile morphological changes and detachment from ECM.8,29 We subsequently tested whether Cat.G-induced myocyte detachment and apoptosis involved EGFR-dependent FAK downregulation. Cat.G treatment induced rapid FAK tyrosine dephosphorylation. This occurred within 10 to 15 minutes, and FAK remained hypophosphorylated for >60 minutes (Figure 7A and supplemental Figure IV). Pretreatment of cells with AG1478 or heparin significantly reduced Cat.G-induced FAK tyrosine dephosphorylation (Figure 7B and 7C). Surprisingly, EGF treatment also induced a decrease in FAK tyrosine phosphorylation. However this decrease was transient, and FAK tyrosine phosphorylation returned to baseline value 60 minutes after EGF treatment (Figure 7A and supplemental Figure IV). This FAK downregulation induced by EGF did not result from a general downregulation of EGFR signaling because ERK1/2 phosphorylation was markedly induced by EGF (supplemental Figure IV). Consistent with the involvement of EGFR kinase activity in FAK tyrosine dephosphorylation, an increase in FAK association with EGFR was observed after Cat.G treatment, as determined by immunoprecipitation with FAK and blotting with EGFR (Figure 7C) or immunoprecipitation with EGFR and blotting with FAK (Figure 7D). The FAK/EGFR association was prevented when cells were pretreated with AG1478, suggesting a role of EGFR kinase activity in FAK/EGFR interaction (Figure 7C). It is noteworthy that increased phosphorylated form of FAK interacted with EGFR in response to Cat.G (Figure 7D). However, we cannot rule out an interaction between the nonphosphorylated form of FAK and EGFR. Together, these data show the role of EGFR activation in mediating Cat-G–induced FAK downregulation that leads to myocyte detachment and apoptosis.
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Cat.G-Induced EGFR Transactivation Mediates Src Homology Domain–Containing Tyrosine Phosphatase 2 Activation
Both Cat.G- and EGF-induced loss of FAK signaling and cell detachment involved activation of PTPs.8,29 In light of our recent study showing the involvement of PTP Src homology domain–containing tyrosine phosphatase 2 (SHP2) in Cat.G-induced FAK tyrosine dephosphorylation and myocyte anoikis,8 we next tested whether SHP2 activation by Cat.G is mediated through EGFR activation. Figure 8 shows that both Cat.G and EGF induced an increase in SHP2 tyrosine phosphorylation but with different kinetics. Cat.G treatment induced a slow and sustained increase in SHP2 phosphorylation, with maximum stimulation observed 1 hour after Cat.G treatment, and SHP2 phosphorylation remained high 2 hours after Cat.G treatment (Figure 8A).8 In contrast, EGF treatment caused a rapid and transient increase in SHP2 phosphorylation that was maximal at 5 minutes and returned toward basal levels 60 minutes after EGF treatment. Interestingly, the increase in SHP2 phosphorylation induced by Cat.G required EGFR kinase activity because pretreatment with AG1478 or overexpression of Ad-EGFR-CD533 abrogated SHP2 activation induced by Cat.G treatment (Figure 8B and 8C). The involvement of EGFR activation in SHP2 phosphorylation is further supported by data showing an increase in SHP2 interaction with EGFR in response to Cat.G treatment, as determined by immunoprecipitation with EGFR and blotting with SHP2 (Figure 8D). These data indicate that EGFR activation after Cat.G treatment results in SHP2 activation, which in turn leads to FAK tyrosine dephosphorylation and myocyte anoikis.
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Discussion |
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We recently showed that prolonged treatment with neutrophil-derived protease Cat.G promotes myocyte anoikis.7 In the present study, we expand on these findings and show that Cat.G transactivates EGFR through a pathway that includes MMP-dependent cleavage and the release of HB-EGF, which in turn activates the EGFR pathway. The activation of EGFR was associated with its interaction with FAK and SHP2, 2 signaling molecules involved in Cat.G-induced myocyte anoikis.8 Consistent with potential involvement of EGFR in Cat.G-signaling pathways, inhibition of membrane shedding of HB-EGF or inhibition of EGFR kinase activity, either by AG1478 or by overexpression of dominant negative mutant EGFR-CD533, were both sufficient to inhibit EGFR signaling and subsequent myocyte apoptosis induced by Cat.G.
In the present study, EGFR tyrosine kinase inhibition and the blockade of the extracellular ligand-binding domain of the EGFR both abolished Cat.G-induced myocyte anoikis. This suggests that Cat.G stimulation causes binding of an extracellular ligand to EGFR. Cat.G treatment increased HB-EGF secretion and treatment with heparin or anti–HB-EGF neutralizing antibodies reduced Cat.G-induced EGFR downstream signaling molecules and blocked Cat.G-induced myocyte anoikis. These data suggest that HB-EGF is the EGFR ligand mediating Cat.G effects in cardiomyocyte anoikis. However, ectodomain shedding of HB-EGF requires an MMP-dependent cleavage of membrane-spanning proforms of EGFR ligands. Although this MMP-induced transactivation of EGFR has been well documented in response to several stimuli, including G protein–coupled receptors and stress,9,11 its implication in neutrophil-derived serine protease Cat.G-induced signaling is novel. We found that Cat.G induced early MMP-2 activation and that treatment with an MMP inhibitor significantly reduced ERK1/2 phosphorylation and attenuated myocyte apoptosis induced by Cat.G. Moreover, we found that Cat.G induced MT1-MMP activation and that inhibition of MT1-MMP, either by TIMP2 or anti–MT1-MMP neutralizing antibodies, blocked MMP-2 activation and subsequent ERK1/2 phosphorylation induced by Cat.G. These data, together with previous findings,28 strongly implicate MT1-MMP in Cat.G-induced MMP-2 cleavage and subsequent EGFR transactivation.
One paradoxical finding in this study is that EGFR activation is required for Cat.G-induced myocyte anoikis. Cat.G induced a rapid activation of EGFR that correlated with ERK1/2 phosphorylation. In addition, inhibition of EGFR kinase activity using AG1478 or overexpression of EGFR-CD533 mutant that lacks the kinase activity domain of EGFR prevented EGFR-induced downstream signaling molecules and significantly reduced myocyte apoptosis induced by Cat.G. It is plausible that the binding of HB-EGF to EGFR causes constitutive activation and/or hyperactivation of EGFR and its downstream signaling molecules. However, neither constitutive phosphorylation nor hyperphosphorylation of EGFR was observed in cardiomyocytes in response to Cat.G. In addition, significant hyperactivation of ERK1/2 was not observed, suggesting that Cat.G-induced myocyte apoptosis cannot be explained by hyperactivation of EGFR. Another possibility is that EGF-induced apoptosis is the result of a decline in cell adhesion and detachment of cells because anoikis is thought to be responsible for the paradoxical antiproliferative effects of EGF.22,29 We found that Cat.G-induced EGFR activation led to FAK tyrosine downregulation and an increase in FAK-EGFR interaction. This finding is consistent with a previous report showing FAK tyrosine downregulation and its interaction with EGFR in response to EGF.29 Interestingly, we found that Cat.G induced SHP2 activation, a PTP involved in FAK tyrosine dephosphorylation.8,30 The fact that EGFR activity was required for SHP2 activation, that EGFR formed a complex with SHP2 after Cat.G treatment, and that the kinetics of SHP2 activation correlated with the kinetic of FAK dephosphorylation induced by Cat.G strongly suggests that activated EGFR recruits and activates SHP2, leading to FAK dephosphorylation and probably to FA disruption. It is noteworthy that EGF treatment also induced SHP2 activation and FAK tyrosine dephosphorylation. However, this activation occurred early and was not sustained over time. We speculate that sustained activation of SHP2 in response to Cat.G leads to persistent tyrosine dephosphorylation of FAK, thereby inducing myocyte anoikis. In agreement to these findings, overexpression of dominant negative SHP2 mutant or wild-type FAK rescued myocyte apoptosis induced by Cat.G.8 Thus, the regulation of Cat.G-induced apoptosis appears to be a complex process involving the interplay of several regulatory molecules.
With respect to the mechanism of EGFR function in myocyte anoikis, we found that EGFR overexpression, depending on its level, caused either myocyte survival or apoptosis. Low-level expression of EGFR increased myocyte cell surface area (data not shown) and protected myocytes against apoptosis induced by Cat.G treatment. However, high-level expression of EGFR increased myocyte detachment and apoptosis, and additional stimulation with Cat.G did not significantly enhance myocyte apoptosis. The significance of this dual effect of EGFR is unclear but may be related to the nature of the WT-EGFR construct used in our study, which acts as a constitutively active mutant EGFR. Therefore, low-level EGFR activation increases survival signaling pathways, such as AKT and ERK1/2, which counteract the proapoptotic effects induced by Cat.G. In contrast, high levels of EGFR expression induce PTP activation, which modulates EGFR activity and FA signaling, leading to myocyte apoptosis.
Numerous serine proteases can initiate MMP activation, leading to collagen and ECM proteolysis and tissue remodeling. It appears that multiple pathways overlap and act synergistically to regulate ECM proteolysis. Data presented in this study provide strong additional evidence supporting a role for membrane shedding of HB-EGF as a proapoptotic factor, rather than a factor that promotes myocyte hypertrophy. Several studies in vivo found that mice lacking HB-EGF or EGFR or mice with defects in EGFR (waved2) showed grossly enlarged cardiac valves (as a result of mesenchymal cell proliferation) associated with severe dilated cardiomyopathy.31,32 Moreover, adenovirally upregulated expression of HB-EGF in the myocardial infarct area showed enhanced, rather than decreased, cell apoptosis and cardiac remodeling.33 These in vivo studies, together with our in vitro findings, emphasize that HB-EGF and EGFR may acquire proapoptotic properties in cardiomyocytes. This paradoxical effect of EGFR may be related (1) to the signaling diversity associated with the EGFR family that is dependent on the repertoire of EGFR ligands, EGFR localization, and interactions with other erbB receptors; and (2) to the EGFR association with tyrosine kinases and phosphatases that dictate the potency and duration of the signal activated by erbB ligands. Although our report focuses on membrane shedding of HB-EGF as a mediator of cardiomyocyte apoptosis induced by Cat.G, we believe the results are relevant to a wide variety of physiological and pathological conditions, including myocardial infarction, in which infiltrating neutrophils and their proteases have been shown to play an important role in cardiomyocyte death and the infarct expansion.2,3 The studies showing the cardioprotective effects of serine protease inhibitors of neutrophil Cat.G and elastase, LEX32, after ischemia/reperfusion suggest that Cat.G may be an important mediator of cardiac injury.34
In conclusion, these data indicate that EGFR activation on Cat.G treatment results in activation of PTPs and FAK tyrosine dephosphorylation, which are involved in myocyte detachment and apoptosis. This would suggest a potential role of EGFR in the regulation of cell–substratum adhesion after Cat.G treatment.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH grant HL76799 and American Heart Association Grant 0430301N.
Disclosures
None.
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Footnotes |
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Original received February 13, 2007; revision received October 4, 2007; accepted October 18, 2007.
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