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RECEPTORS AND SIGNAL TRANSDUCTION

-Adrenergic receptor-stimulated apoptosis in adult cardiac myocytes involves MMP-2-mediated disruption of ₁ integrin signaling and mitochondrial pathway

¹Department of Physiology, James H. Quillen College of Medicine, James H. Quillen Veterans Affairs Medical Center, East Tennessee State University, Johnson City, Tennessee; and ²Department of Medicine, University of California San Diego School of Medicine, and Veterans Administration San Diego Healthcare System, San Diego, California

Submitted 13 May 2005 ; accepted in final form 1 September 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Stimulation of -adrenergic receptors (-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits -AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with ₁ integrins. Herein we tested the hypothesis that MMP-2 impairs ₁ integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited -AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated -AR-stimulated decreases in mitochondrial membrane potential. Overexpression of ₁ integrins using adenoviruses expressing the human _1A-integrin decreased -AR-stimulated cytochrome c release and apoptosis. Overexpression of ₁ integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the ₁ integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis.

matrix metalloproteinases; focal adhesion kinase; c-Jun NH₂-terminal kinase; cytochrome c

Matrix metalloproteinases (MMPs), a large family of endopeptidases, are capable of degrading a wide variety of the extracellular matrix proteins (26). MMPs are present in the heart, and changes in the levels of MMPs have been described in a variety of animal models of heart failure (26). Inhibition of activity of MMPs through pharmacological interventions is demonstrated to attenuate the process of ventricular remodeling after infarction in mouse and rat (21, 30), and in the rapid cardiac pacing model of heart failure (27). Adult cardiac myocytes express MMP-2, and neurohormonal stimulation increases its expression and activity (6, 16, 18).

Integrins play a crucial role in the organization of the extracellular matrix, which undergoes extensive reorganization during disease. Integrins serve as adhesion receptors for extracellular matrix proteins, transduce biochemical signals into the cell, and regulate a variety of cellular functions, including spreading, migration, proliferation, and apoptosis (4). Cardiac myocytes predominantly express ₁ integrins (23). Upon integrin occupancy and clustering, focal adhesion kinase (FAK), an important mediator of the ₁ integrin signaling pathway, undergoes autophosphorylation and associates with other intracellular signaling molecules (1). Inhibition of FAK phosphorylation by adenovirally mediated overexpression of FAK-related nonkinase has been shown to induce apoptosis in neonatal cardiac myocytes (11).

We have previously shown that stimulation of ₁ integrins plays an antiapoptotic role in -AR-stimulated apoptosis of ARVMs (9). Recently, we have shown that MMP-2 plays a proapoptotic role in -AR-stimulated apoptosis, possibly via its interaction with ₁ integrins (18). Herein we demonstrate that MMP-2 interferes with the ₁ integrin-mediated survival signals involving FAK and activates JNK-dependent mitochondrial death pathway.

	MATERIALS AND METHODS

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Cell isolation and culture. Calcium-tolerant ARVMs were isolated from the hearts of adult male Sprague-Dawley rats (240–280 g) as described previously (7). Briefly, hearts were perfused retrogradely with Ca²⁺-free Krebs-Henseleit bicarbonate buffer for 5 min. Hearts were then perfused with Krebs-Henseleit bicarbonate buffer containing 0.04% collagenase type II for 20 min. After the atria and great vessels were removed, the hearts were minced and dissociated in the same buffer containing trypsin (0.02 mg/ml) and DNase (0.02 mg/ml). The cell mixture was filtered and sedimented through a 6% bovine serum albumin cushion to remove nonmyocyte cells. The cell pellet was resuspended in Dulbecco’s modified Eagle’s medium (Mediatech) supplemented with 25 mM HEPES, 0.2% albumin, 5 mM creatine, 2 mM L-carnitine, 5 mM taurine, and 0.1% penicillin-streptomycin. The ARVMs were then plated in the above media at a density of 30–50 cells/mm² on 100-mm tissue culture dishes (Fisher Scientific) or coverslips precoated with laminin (1 µg/cm²). The investigation protocol conformed to the guidelines in the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996), and the animal protocol was approved by the University Committee on Animal Care.

Cell treatment. After the ARVMs were cultured for 24 h, they were then treated with isoproterenol (ISO, 10 µM; Sigma) in the presence of ascorbic acid (100 µM) for 15 min to study activation of FAK and JNKs, for 6 h to study cytosolic cytochrome c release, or for 24 h for TdT-mediated dUTP nick-end labeling (TUNEL) and JC-1 staining. To inhibit MMP-2, cells were pretreated with SB3CT (1 nM; Calbiochem) or TIMP-2 (50 ng/ml; Calbiochem) for 30 min before treatment with ISO. The inhibitors were maintained in the medium during the treatment period with ISO. Cells were also treated with activated MMP-2 (1 nM; Calbiochem) to study cytochrome c release, apoptosis, and FAK phosphorylation.

Adenovirus infection of ARVMs. Adenoviruses expressing the human _1A integrin (Ad_1A) (24) were propagated using human embryonic kidney-293 cells. The adenoviral titer was determined using the end-point dilution method. ARVMs plated on laminin-coated dishes were infected with the adenovirus, with a matched multiplicity of infection of 50–100/cell, in modified Dulbecco’s modified Eagle’s medium for 24–48 h before treatment with ISO or MMP-2. Cells infected with equal multiplicities of infection of green fluorescent protein (GFP) adenoviruses served as controls.

TUNEL assay. TUNEL staining was performed on ARVMs plated on Thermanox coverslips using an in situ death detection kit according to the manufacturer’s instructions (Roche Molecular Biochemicals). The percentage of TUNEL-positive cells (relative to total ARVMs) was determined by counting 200 cells in 10 randomly chosen fields per coverslip for each experiment.

Preparation of cytosolic fraction for cytochrome c release. To prepare cytosolic fraction, cells were washed with PBS and scraped into the lysis buffer (250 mM sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 17 µg/ml PMSF, 8 µg/ml aprotinin, and 2 µg/ml leupeptin) and homogenized gently using a glass homogenizer. The cell suspension was centrifuged at 3,500 g for 5 min to pellet nuclei and other cell debris. The supernatant was centrifuged at 14,000 g for 10 min to pellet mitochondrial fraction. The collected supernatant was then centrifuged at 100,000 g for 30 min at 4°C. The supernatant (cytosolic fraction) was examined using Western blot analysis with anti-cytochrome c antibody (Santa Cruz).

Immune complex kinase assay. Activation of JNKs was studied with the use of a SAPK/JNK assay kit (Cell Signaling Technology). Total cell lysates (250 µg) were incubated overnight with 2 µg of c-Jun fusion protein beads. The beads were washed twice with lysis buffer and twice with kinase buffer (25 mM Tris, pH 7.5, 5 mM glycerophosphate, 2 mM DTT, 0.1 mM Na₃VO₄, and 10 mM MgCl₂) and then suspended in 50 µl of kinase buffer containing 100 µM ATP. After incubation for 30 min at 30°C, the reaction was terminated using 2x SDS sample buffer. The immunoprecipitates were examined using Western blot analysis with anti-phospho-c-Jun (Ser63) antibody.

Western blot analysis. To analyze FAK phosphorylation, ARVMs were lysed in cell lysis buffer (10 mM Tris·HCl, pH 7.6, containing 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.4 mM PMSF, and 1% Triton X-100). The protein content in cell lysates was measured using Bradford assay (Bio-Rad). Equal amounts of proteins (50–100 µg) were resolved by SDS-PAGE (Bio-Rad). Proteins from the gel were electrophoretically transferred to a PVDF membrane (Hybond-P; Amersham Biosciences). The membranes were stained with Ponceau S to confirm equal loading of proteins in the samples. After being destained, the membranes were incubated overnight in the blocking buffer, which was composed of TBST (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk. The membranes were then incubated with primary [anti-FAK-pY397 and anti-FAK-pY576 (Biosource)] antibodies diluted as suggested by the vendor in blocking buffer. After being washed with TBST, the membranes were incubated with a diluted peroxidase-conjugated secondary antibody. The immune complexes were detected using chemiluminescence reagents (Pierce Biotechnology). The membranes were stripped and probed with antibodies against actin or FAK to optimize loading.

JC-1 staining. To study mitochondrial membrane potential, ARVMs were incubated with JC-1 dye (Cell Technology) for 15 min at 37°C in the dark. The cells were then washed in PBS twice to remove excess dye, and the images were immediately captured using a fluorescence microscope (Nikon). The fluorescent images were analyzed using Bioquant Image analysis software, and the ratio of red (aggregate) to green (monomer) fluorescence (F₅₈₅/F₅₁₀) was calculated.

Statistical analyses. All data are expressed as means ± SE. Statistical analysis was performed using the Student’s t-test or a one-way ANOVA and a post hoc Tukey’s test. P values <0.05 were considered to be significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Role of MMP-2 in FAK phosphorylation. Previously, we (18) have shown that -AR stimulation increases interaction of MMP-2 with ₁ integrins. Inhibition of MMP-2 inhibits interaction of MMP-2 with ₁ integrins and plays antiapoptotic role in -AR-stimulated apoptosis. To study the role of MMP-2 in FAK phosphorylation, cell lysates were analyzed by Western blotting using phospho-specific antibodies. This analysis demonstrated basal phosphorylation of FAK (Tyr397 and Tyr576). -AR stimulation (ISO, 10 µM, 15 min) increased FAK phosphorylation. Inhibition of MMP-2 alone or in combination with ISO increased FAK phosphorylation at both Tyr397 (relative change vs. control; ISO, 3.0 ± 0.3; SB, 2.6 ± 0.4 SB + ISO, 4.2 ± 0.1; P < 0.05 vs. control; 4.2 ± 0.1; P < 0.05 vs. ISO; n = 3) and Tyr576 (relative change vs. control; ISO, 3.2 ± 0.3; SB, 3.6 ± 0.3; SB + ISO, 4.5 ± 0.4; P < 0.05 vs. control; 4.5 ± 0.4; P < 0.05 vs. ISO; n = 3; Fig. 1A). SB3CT is a specific inhibitor of gelatinases with inhibition constant values of 13 nM for MMP-2 and 600 nM for MMP-9. We have shown that SB3CT at 1 nM concentration inhibits -AR-stimulated increases in MMP-2 activity as well as interaction of MMP-2 with ₁ integrins after -AR stimulation (18). Pretreatment with purified TIMP-2, tissue inhibitor of MMP-2, resulted in significant increases in -AR-stimulated FAK phosphorylation at Tyr397 (relative change vs. control; ISO, 2.1 ± 0.06; TIMP-2, 1.9 ± 0.1; TIMP-2 + ISO, 3.0 ± 0.2; P < 0.05 vs. control; 3.0 ± 0.2; P < 0.05 vs. ISO; n = 3) and Tyr576 (relative change vs. control; ISO, 2.2 ± 0.14; TIMP-2, 2.3 ± 0.15; TIMP-2 + ISO, 3.4 ± 0.14; P < 0.05 vs. control; 3.4 ± 0.14; P < 0.05 vs. ISO; n = 3; Fig. 1B). On the other hand, treatment of ARVMs with purified active MMP-2 significantly decreased FAK phosphorylation at Tyr397 (0.25 ± 0.02-fold vs. control; P < 0.05, n = 3) and Tyr576 (0.55 ± 0.01-fold vs. control; P < 0.05, n = 3; Fig. 2).

View larger version (30K): [in this window] [in a new window]   Fig. 1. Inhibition of matrix metalloproteinase-2 (MMP-2) increases -adrenergic receptor (-AR)-stimulated focal adhesion kinase (FAK) phosphorylation. Adult rat ventricular myocytes (ARVMs) were pretreated with SB3CT (1 nM; A) or tissue inhibitor of MMP-2 (TIMP-2; 50 ng/ml; B) for 30 min, followed by treatment with isoproterenol (ISO; 10 µM) for 15 min. Cell lysates were analyzed by Western blot using phosphospecific focal adhesion kinase (FAK) antibodies (FAK pY397 and FAK pY576). Bottom, intensity of p-FAK as relative increase vs. control (CTL). Equal protein loading in each lane was verified using anti-FAK antibodies. *P < 0.05 vs. control; n = 3; #P < 0.05 vs. ISO; n = 3.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Treatment with purified active MMP-2 inhibits FAK phosphorylation. ARVMs were treated with active MMP-2 enzyme (1 nM) for 15 min. Cell lysates were analyzed by Western blot using phosphospecific FAK antibodies (FAK pY397 and FAK pY576). Bottom, intensity of p-FAK as relative change vs. control. Equal protein loading in each lane was verified using anti-FAK antibodies. *P < 0.05 vs. control; n = 3.

Inhibition of MMP-2 inhibits -AR-stimulated increases in the activation of JNK.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Activation of JNKs. ARVMs were pretreated with SB3CT (1 nM; A) or TIMP-2 protein (50 ng/ml; B) for 30 min, followed by treatment with isoproterenol (ISO, 10 µM) for 15 min. Activation of JNKs was analyzed using immune-complex kinase assay. Bottom, p-c-Jun levels expressed as relative change vs. control. *P < 0.05 vs. control; n = 6; #P < 0.05 vs. ISO; n = 3.

View larger version (20K): [in this window] [in a new window]   Fig. 4. MMP-2 affects cytosolic cytochrome c release. A: ARVMs were pretreated with SB3CT for 30 min, followed by treatment with isoproterenol (10 µM) for 6 h. B: ARVMs were treated with purified active MMP-2 (1 nM) for 6 h. Cytosolic fractions were analyzed by Western blotting, as described in MATERIALS AND METHODS. Equal protein loading in each lane was verified using anti-actin antibodies. *P < 0.05 vs. control; n = 4; #P < 0.05 vs. ISO; n = 3.

Inhibition of MMP-2 attenuates -AR-stimulated mitochondrial depolarization.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Inhibition of MMP-2 attenuates -AR-stimulated mitochondrial depolarization. ARVMs were pretreated with SB3CT (1 nM) or TIMP-2 protein (50 ng/ml) for 30 min, followed by treatment with isoproterenol (10 µM) for 24 h. Cells were incubated with JC-1 dye (1 µM) for 15 min at 37°C. The cells were visualized using epifluorescence microscopy. The fluorescent images were analyzed using Bioquant Image analysis software and the ratio of red-to-green fluorescence (F585/F510) was calculated. *P < 0.05 vs. control, n = 6; #P < 0.05 vs. ISO, n = 3.

Overexpression of ₁ integrins decreases cytochrome c release and apoptosis.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Overexpression of 1 integrins in ARVM and its role in -AR-stimulated cytochrome c release and apoptosis. ARVMs were infected with adenoviruses expressing human 1A integrin (Ad1A) or GFP for a total of 48 h. A: ARVMs were stimulated with ISO (10 µM) for 15 min. Overexpression of 1 integrins, expression of FAK, and phosphorylation of FAK (FAK pY397 and FAK pY576) were confirmed by Western blot analyses. B: cytosolic cytochrome c was analyzed with the use of Western blot analysis after 6 h of ISO treatment. C: apoptosis was measured using TdT-mediated dUTP nick end labeling (TUNEL) staining after 24 h of ISO treatment. Equal protein loading in each lane (A and B) was verified using anti-actin antibodies. *P < 0.05 vs. control, n = 3; #P < 0.05 vs. ISO, n = 3.

View larger version (11K): [in this window] [in a new window]   Fig. 7. Overexpression of 1 integrins inhibits MMP-2-induced apoptosis in ARVMs. ARVMs were infected with Ad1A for 24 h, followed by treatment with purified active MMP-2 for 24 h. The number of apoptotic cells was measured using TUNEL staining assay. *P < 0.05 vs. control, n = 3; #P < 0.05 vs. MMP-2; n = 3.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

Cardiac myocytes express and secrete MMPs, specifically MMP-2 (5, 18). MMP-2 and MMP-9 are increased in the heart under various pathological conditions (22). Inhibition of MMPs attenuates left ventricular remodeling events associated with chronic pressure overload and postmyocardial infarction (12, 19, 21). Recently, we (18) have shown that stimulation of -AR increases MMP-2 expression and activity and that inhibition of MMP-2 inhibits -AR-stimulated apoptosis. Using coimmunoprecipitation assays, we provided evidence that interaction of MMP-2 with ₁ integrins may be a mechanism by which MMP-2 plays a proapoptotic role. Several structural components, such as paxillin, talin, -actinin, and filamin, link integrin cytoplasmic tails to the actin cytoskeleton at specific sites called focal adhesions. Focal adhesions act as signaling complexes, and tyrosine phosphorylation of FAK is one of the signaling events occurring at focal adhesions (31). Integrin engagement with ligands initiates autophosphorylation of FAK at Tyr397. This autophosphorylation site provides a binding site for Src, which phosphorylates FAK at Tyr576 and Tyr577 to further activate FAK (31). Activation of FAK plays an antiapoptotic role in different cell types, including neonatal cardiac myocytes (11). Apoptosis has been shown to be associated with FAK dephosphorylation in other cell types, such as fibroblasts, endothelial cells, and neuroblastoma cells (14, 32, 33). The data presented herein demonstrate that stimulation of -AR or inhibition of MMP-2 increases FAK phosphorylation at Tyr397 as well as Tyr576. Phosphorylation of FAK was greater when cells were treated with ISO in the presence of MMP-2 inhibitors. Furthermore, FAK phosphorylation was significantly reduced when the cells were treated with purified MMP-2 protein. These data support our thesis that interaction of MMP-2 with ₁ integrins may affect intracellular signaling pathways involving FAK. It is interesting to note that inhibition of MMP-2 alone increases FAK phosphorylation, suggesting that basal MMP-2 levels have an inhibitory effect on FAK phosphorylation. Treatment with ISO alone also increases FAK phosphorylation. However, ISO still induces apoptosis in ARVMs. This could be because ISO stimulates both ₁- and ₂-ARs in ARVMs. ₁-ARs account for 80% of total -ARs. Stimulation of ₁-ARs increases apoptosis via cAMP-dependent mechanisms, whereas stimulation of ₂-ARs inhibits -AR-stimulated apoptosis via G_i-coupled pathway (8). It is possible that ₂-AR-G_i signaling activates FAK; however, this activation of FAK is not enough to inhibit apoptosis because of the predominant effects of ₁-AR stimulation on apoptosis.

Previously, activation of JNKs was suggested to activate the mitochondrial death pathway in ARVMs (20). We have shown that inhibition of MMP-2 inhibits poly-ADP-ribose polymerase cleavage, providing evidence for the involvement of mitochondria in the signaling pathway affected by MMP-2 (18). Using two different techniques (cytochrome c release and mitochondrial membrane potential), we have confirmed our previous findings suggesting the involvement of the mitochondrial death pathway in -AR-stimulated apoptosis. Inhibition of MMP-2 inhibited -AR-stimulated increases in cytochrome c release and restored -AR-stimulated decreases in mitochondrial membrane potential. Integrin-mediated signals are suggested to engage mitochondrial function in the regulation of gene expression (35). The current study is the first to suggest that inhibition of MMP-2 may affect -AR-stimulated activation of JNKs and the mitochondrial death pathway. Activation of JNKs is suggested to induce the mitochondrial death pathway of apoptosis in cells of cardiac and noncardiac origin (17, 29, 34). However, the possible mechanisms by which activation of JNKs activates the mitochondrial death pathway are not well understood. In response to genotoxic or oxidative stress-induced apoptosis, JNKs are suggested to translocate to the mitochondria (3, 13). JNKs are demonstrated to associate with the mitochondria and activate the mitochondrial death pathway in cardiac myocytes in response to H₂O₂ (3, 20). Recent studies (10, 28) have suggested that activated JNKs also induce the permeabilization of outer mitochondrial membrane without depolarization of the inner membrane in methyl glyoxal-induced apoptosis of jurkat cells, leading to cytochrome c release.

The data presented herein suggest that impairment of integrin signaling, possibly as a result of the increased interaction of MMP-2 with ₁ integrins, may activate JNKs, thereby stimulating the mitochondrial death pathway. To support our hypothesis that interaction of MMP-2 impairs ₁ integrin signaling and leads to apoptosis, we stimulated integrin signaling by overexpression of the _1A integrin subunit. Western blot analysis with anti-₁-integrin antibodies confirmed overexpression of ₁ integrins in ARVMs. Increased downstream signaling was confirmed by the increased phosphorylation of FAK (Fig. 6A). Overexpression of ₁ integrins inhibited -AR-stimulated increases in cytosolic cytochrome c release and apoptosis. These data are consistent with our previous finding that stimulation of integrin signaling inhibits -AR-stimulated apoptosis (9). Overexpression of ₁ integrins also inhibited MMP-2-induced apoptosis in ARVMs.

In conclusion, the data presented herein suggest that interaction of MMP-2 with ₁ integrins plays an important role in the regulation of -AR-stimulated apoptosis in ARVMs. Interaction of MMP-2 with ₁ integrins disrupts the antiapoptotic signals initiated by ₁ integrin engagement and results in the activation of a JNK-dependent mitochondrial death pathway. Therefore, inhibition of MMP-2 may play a protective role against -AR-stimulated apoptosis, at least in part, by affecting focal adhesion kinase and the mitochondrial death pathway.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-071519 (to K. Singh) and HL-57872 (to R. S. Ross), a Department of Veterans Affairs Merit Review Grant (to K. Singh), and American Heart Association (Southeast Affiliate) Grant 0325298B (to B. Menon).

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. Singh, Dept. of Physiology, James H. Quillen College of Medicine, East Tennessee State Univ., PO Box 70576, Johnson City, TN 37614 (e-mail: singhk{at}etsu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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