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Cardiovascular Research 2006 69(3):697-705; doi:10.1016/j.cardiores.2005.08.005
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Copyright © 2005, European Society of Cardiology

Degraded collagen induces calpain-mediated apoptosis and destruction of the X-chromosome-linked inhibitor of apoptosis (xIAP) in human vascular smooth muscle cells

Karin von Wnuck Lipinskia, Petra Keula, Susann Luckea, Gerd Heuscha, Jeremias Wohlschlaegerb, Hideo A. Babab and Bodo Levkaua,*

aInstitute of Pathophysiology, Center of Internal Medicine, Hufelandstraβe 55, 45122 Essen, Germany
bInstitute of Pathology, University Hospital Essen, Hufelandstraβe 55, 45122 Essen, Germany

* Corresponding author. Tel.: +49 201 723 4414; fax: +49 201 723 4413. Email address: levkau{at}uni-essen.de

Received 7 July 2005; revised 17 August 2005; accepted 18 August 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The extracellular matrix (ECM) of the atherosclerotic lesion is a crucial determinant of its stability, while its degradation by matrix metalloproteinases (MMPs) has been implied in plaque rupture. As accumulation of both MMP-derived collagen fragments and apoptotic smooth muscle cells (SMC) is observed at sites of plaque rupture, we tested the effect of polymerized and degraded type I collagen on the susceptibility of SMC to apoptosis.

Methods: Human SMC were cultured on monomeric or polymerized collagen, and collagen gels were degraded by collagenase. Apoptosis was evaluated using antibodies to active caspases and their substrates. Calpain and caspase activity were measured using fluorogenic substrates.

Results: Culture of SMC on polymerized collagen led to increased apoptosis compared to culture on monomeric collagen. In addition, we observed a distinct proteolytic degradation of the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis (xIAP). As MMP-1 was strongly activated in SMC on polymerized collagen, we examined the effect of degraded collagen fragments on xIAP cleavage and apoptosis. Degraded collagen induced rapid proteolytic processing of xIAP identical to that on polymerized collagen. We identified calpains as the proteolytic enzymes responsible for xIAP processing as: i) they were rapidly activated by degraded collagen; ii) recombinant calpain II processed xIAP in an identical manner, and iii) inhibition of calpains by BAPTA or calpeptin abrogated xIAP degradation in intact cells. The functional consequence of xIAP processing by calpains was a loss of its caspase-inhibitory potential. Calpain activation distinctly preceded caspase activation, and inhibition of calpains suppressed apoptosis.

Conclusions: Collagen fragments proteolytically released from the ECM by MMPs may propagate apoptosis of SMC by calpain-mediated inactivation of anti-apoptotic proteins such as xIAP. This may be a novel mechanism of SMC apoptosis in biological settings of enhanced collagen degradation such as vascular remodeling, neointima formation, and atherosclerotic plaque rupture.

KEYWORDS Apoptosis; Smooth muscle; Calpain; Extracellular matrix; Matrix metalloproteinases


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
During the pathogenesis of atherosclerosis, degradation of the extracellular matrix by matrix metalloproteinases (MMPs) has been implied both in smooth muscle cell (SMC) migration from the media to the neointima and in the destabilization of the atherosclerotic lesion leading to plaque rupture [1]. Conversely, the synthesis, composition and integrity of the extracellular matrix of the atherosclerotic lesion are viewed as crucial determinants of its stability [2]. Up to 60% of the extracellular matrix of the plaque is constituted of type I collagen abundantly synthesized by the neointimal SMC [3]. SMC bind to type I collagen through several members of the integrin family such as {alpha}1β1, {alpha}2β1, and {alpha}vβ3, as well as a new family of collagen receptors, the discoidin domain receptors [4]. At the same time, SMC constantly modify and degrade the collagen matrix surrounding them through the action of several MMPs they secrete (MMP-1, -2, -3, -9, and MT1-MMP). The expression and activity of these MMPs are increased after vascular injury, in arterial remodeling and at sites of plaque rupture [5,6].

However, the collagen matrix of the vessel wall is by no means an inert scaffold subject merely to synthesis and degradation: it actively participates in the biological processes regulating SMC behavior [7]. Proteolytic digestion of collagen by MMPs leads to generation of degradation products with distinct properties very different in nature from that of the native, intact molecules. The responses of the SMC to such denatured or degraded collagen fragments are mediated by composite signals resulting from an interplay between the "regular" integrin-binding sites and novel, cryptic sites exposed only after collagen degradation such as the RGD sequence {alpha}vβ3 binds to [4,8]. In addition, the spatial and temporal distribution of the integrins across the cell, together with their propensity to bind either a single large molecule or its fragments via identical binding sites allow a high degree of cell shape malleability and changes in tensegrity [9,10].

Culture of human SMC on polymerized type I collagen has been shown to profoundly suppress their proliferation in the G1 phase of the cell cycle due to increased levels of the p21 and p27 cyclin-dependent kinase inhibitors [11]. Blocking the {alpha}2-integrin on monomeric collagen in this study simulated a quiescent phenotype similar to that on polymerized collagen by inhibiting the assembly of focal adhesions [11]. Downregulation of focal adhesion proteins by culture on polymerized collagen type I has also been observed in epithelial and fibroblastic cells, where reduced synthesis and enhanced degradation by calpains have been implied [12]. Our previous studies have shown that extended culture of SMC on polymerized type I collagen profoundly altered their focal adhesions due to calpain-mediated proteolysis of the focal adhesion kinase (pp125FAK), talin and paxillin, and that this process was mediated by collagen fragments generated by MMPs [13]. Little is known about the effect of polymerized type I collagen and its proteolytic fragments on the susceptibility of SMC to apoptosis. Several studies in fibroblasts cultured either inside or on top of type I collagen gels have shown enhanced apoptosis that differed from anoikis and was dependent on integrin-mediated gel contraction and changes in cell shape [14,15]. Vascular rat SMC cultured on floating type I collagen gels have been reported to undergo apoptosis due to lack of MMP-generated collagen fragments that, readily produced by SMC on anchored collagen gels, conferred protection against apoptosis through induction of the synthesis and secretion of the survival factor tenascin-C via the {alpha}vβ3 integrin [16].

In our study, we tested the susceptibility of SMC to apoptosis when cultured on a polymerized type I collagen gel compared to denatured monomeric collagen, and the role collagen degradation plays in this process. We have observed that both polymerized type I collagen and proteolytic fragments derived from collagen gels induce SMC apoptosis by activating intracellular calpains that degrade anti-apoptotic molecules such as xIAP prior to and necessary for caspase activation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Reagents and antibodies
The antibodies used for Western blot analysis were: xIAP and PARP (Transduction Laboratories), active caspase-3, caspase-9 and cleaved lamin A/C (Cell Signaling), MMP-1 (Chemicon), pp125FAK and cyclin A (Santa Cruz Biotechnology), and β-actin (Sigma). Anti-mouse and anti-rabbit peroxidase-conjugated secondary antibodies were purchased from Vector Laboratories Inc. Rat recombinant calpain II (m-calpain), BAPTA and Calpeptin were purchased from Calbiochem. Calpain inhibitor I (N-acetyl-leucinyl-leucinyl-norleucinal), the calpain substrate Succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin (Suc-LLVY-AMC), and the caspase-3 substrate Ac-Asp-Glu-Val-Asp-aminomethylcoumarin (Ac-DEVD-AMC) were obtained from Alexis.

2.2 SMC culture, preparation of monomeric collagen, type I collagen gels, and degraded collagen fragments
Human newborn arterial SMC were isolated from the thoracic aorta as previously described [17], and were a kind gift of Elaine W. Raines, University of Washington, Seattle. SMC were cultured in 10% foetal bovine serum (FBS)/Dulbecco's modified eagle's medium (DMEM). Type I collagen gels (1.0 mg/ml final collagen concentration) were prepared by neutralizing the bovine skin collagen solution (Vitrogen 100, Cohesion) with one-sixth volume of a seven-fold concentrated DMEM to a final one-fold DMEM solution, and incubating at 37 °C for 12 h. Monomeric collagen-coated dishes were prepared by incubating 0.1 mg/ml collagen solution in 0.1 M acetic acid at 37 °C for 12 h, and were washed twice with DMEM before cell seeding. Degraded type I collagen was prepared by incubating polymerized collagen gels (prepared as above) with 2 mg/ml collagenase type 3 (Worthington Biochemical Corp.) at 37 °C for 30 min. After digestion, collagenase activity was inhibited by the addition of an equal volume of one-fold DMEM containing 10% FBS. For calpain inhibition studies in the presence of degraded collagen and BAPTA, SMC were cultured in Ca2+-free Hank's balanced Salt Solution. The investigation conforms with the principles outlined in the Declaration of Helsinki.

2.3 Protein analysis
Cells were washed twice with ice-cold PBS and lysed in 100 µl of buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 5 mM EDTA, 5 mM EGTA, 10 mM NaF, 10% glycerol, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaVO4, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) for 15 min on ice. Cell lysates were cleared by centrifugation at 15,000 x g for 5 min, and protein concentrations were determined using the BCA protein assay (Pierce). Lysates were separated by SDS-PAGE under reducing conditions, transferred to an Immobilon polyvinylidene difluoride membrane (Millipore) and subsequently immunoblotted with specific antibodies prior to visualization by enhanced chemiluminescence (ECL, Amersham Biosciences).

2.4 Calpain activity assays, and treatment of cell extracts and recombinant xIAP with calpains
For measurement of calpain activity, SMC treated with or without degraded collagen fragments were washed with ice-cold PBS and resuspended in calpain extraction buffer (10 mM HEPES pH 7.5, 5 mM DTT, and 1% NP-40). After 15 min incubation on ice, cell lysates were centrifuged (15,000 x g for 5 min) and the supernatants were used for the assay: 40 µg of total protein were diluted to a total volume of 200 µl with calpain reaction buffer (50 mM Tris–HCl pH 7.5, 10 mM CaCl2, 30 mM NaCl, 5 mM DTT) containing 50 µM Suc-LLVY-AMC. Production of fluorescent AMC was monitored continuously (excitation 360 nm and emission 460 nm) by use of a fluorescent plate reader (KC4; Biotek). For calpain cleavage studies, cell lysates of untreated SMC were isolated in calpain extraction buffer and 40 µg protein were incubated with 0.250 and 3.2 µg recombinant calpain II, respectively, in calpain reaction buffer for the indicated times. The reaction was stopped by addition of SDS-PAGE sample buffer. For recombinant xIAP cleavage studies, 50 µg of GST-xIAP prepared as previously described [18] were incubated with 3.2 µg recombinant calpain II in calpain reaction buffer at 37 °C for 20 min and stopped by addition of 50 mM EDTA.

2.5 Caspase-3 activity assays
Cell lysates were prepared from SMC treated with or without degraded collagen fragments as described above and 80 µg of cell lysate was incubated in 200 µl caspase reaction buffer (25 mM HEPES pH 7.5, 0.1% CHAPS, 50 mM KCl, 5 mM β-mercaptoethanol) with 10 µM Ac-DEVD-AMC at 37 °C. Production of fluorescent AMC was monitored continuously (excitation 360 nm and emission 460 nm) by use of a fluorescent plate reader (KC4; Biotek). Purification of recombinant active human caspase-3 was performed as previously described [18]. For caspase inhibition studies using GST-xIAP, 0.63 pM recombinant caspase-3 was incubated with 50 µg native GST-xIAP or calpain-digested GST-xIAP in 200 µl caspase reaction buffer and 10 µM Ac-DEVD-AMC at 37 °C.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Culture of smooth muscle cells on polymerized type I collagen induces apoptosis
Human SMC cultured on polymerized type I collagen have been shown to arrest in the G1 phase of the cell cycle as compared to monomeric collagen [11]. In our study, we have used the identical system for the comparison of SMC apoptosis on polymerized and monomeric collagen as exemplified by the well-known dramatic downregulation of cyclin A on type I collagen gels (Fig. 1). We observed an activation of caspase-3 and distinct cleavage of the caspase substrates lamin A/C in SMC on polymerized type I collagen after 12 h that increased over the following 36 h (Fig. 1). This cleavage was not present in cells cultured on monomeric collagen (Fig. 1). Interestingly, when probing for the potent endogenous caspase inhibitor X-chromosome linked inhibitor of apoptosis (xIAP), we observed several crossreactive bands of smaller molecular weight suggesting processing and/or degradation of the protein (Fig. 1). Other members of the inhibitor of apoptosis protein (IAP) family such as c-IAP1, c-IAP2, and survivin were not affected (data not shown). In addition, a distinct proteolytic cleavage of the focal adhesion kinase (pp125FAK) occurred on polymerized type I collagen as we have previously observed [13]. Over time, SMC cultured on polymerized collagen degraded the gels through the action of endogenously produced and activated MMPs as shown for MMP-2 in this system [13]. We observed a robust activation of MMP-1 that coincided with the gradual degradation of the gel (Fig. 1).


Figure 1
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  		Fig. 1 Polymerized type I collagen induces apoptosis in SMC. SMC were cultured on monomeric collagen (M) and polymerized type I collagen gels (P) in 1% FCS/DMEM for 48 h and subsequently stimulated with 10% FCS/DMEM for the indicated times. Western blot analysis was performed with total cell lysates using antibodies against cyclin A, pp125FAK, xIAP and MMP-1. Apoptosis was examined by antibodies against active caspase-3 and cleaved lamin A/C, respectively. Expression of β-actin was used as loading control.

 
3.2 Degraded collagen induces apoptosis and causes degradation of xIAP in SMC
As MMP-1 was strongly activated by culture of SMC on polymerized collagen in our system, we examined the effect of degraded type I collagen fragments generated as described in our previous studies [13] on SMC apoptosis. Degraded collagen (250 µg/ml) added to SMC cultured on monomeric collagen induced a marked activation of caspases-3 and -9 as well as cleavage of their substrates lamins A/C beginning after 120 min and increasing over time (Fig. 2). Interestingly, the same degradation of xIAP occurred as that on polymerized collagen, with rapid generation of at least one prominent fragment beginning after 5 min and lasting over the next 360 min (Fig. 2). At this time, there was no caspase activation and no apoptosis present (Fig. 2). A classical caspase substrate, poly ADP-ribose polymerase (PARP), was also distinctly processed to a major fragment of approximately 40 kDa but this fragment was different from the well-known 25 kDa caspase-generated PARP fragment associated with apoptosis (Fig. 2). In addition, robust proteolytic cleavage of pp125FAK by collagen fragment treatment occurred as we have previously described in this experimental system [13].


Figure 2
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  		Fig. 2 Degraded collagen induces apoptosis and causes degradation of xIAP. SMC cultured on monomeric collagen were incubated with 250 µg/ml degraded type I collagen for the indicated times, and cell lysates were immunoblotted against active caspase-3, caspase-9, cleaved lamin A/C, xIAP, PARP and pp125FAK. In the case of xIAP and PARP, only the degradation products {Delta}xIAP and {Delta}PARP, respectively, are shown. To confirm equal protein loading, blots were reprobed with an antibody against β-actin.

 
3.3 Calpains degrade xIAP in vitro and in intact cells after exposure to degraded collagen
Calpains are potent Ca2+-dependent cystein proteases that we have previously shown to promote focal adhesion disassembly by cleaving and degrading pp125FAK, paxillin and talin after treatment of SMC with degraded collagen fragments [13]. As the degradation of xIAP and PARP followed the same time kinetics as pp125FAK (Fig. 2), we hypothesized that calpains may be responsible for the degradation of xIAP as well. Direct measurement of calpain activity using a fluorogenic calpain substrate revealed rapid calpain activation in SMC treated with degraded collagen compared to control cells (Fig. 3A) that was inhibitable by the calpain inhibitor calpeptin (Fig. 3B). To test directly if calpains degrade xIAP, we incubated recombinant calpain II with cell lysates from control cells as a source of endogenous xIAP for 15 and 60 min (Fig. 4A). This treatment resulted in xIAP degradation and generation of fragments similar in size to those generated endogenously in SMC after treatment with degraded collagen (Fig. 4A). The degradation of xIAP was inhibited by the Ca2+-chelator EDTA and calpain inhibitor I, respectively (Fig. 4B). The functional consequence of xIAP processing by calpains was the apparent loss of its caspase-inhibitory function: calpain treatment of recombinant GST-xIAP resulted in reduced inhibition of recombinant active caspase-3 in a fluorogenic substrate assay (Fig. 4C, D). To test if calpains are responsible for the degradation of xIAP in intact cells, SMC were treated with degraded collagen in the presence or absence of the cell-permeable Ca2+-chelator BAPTA for 5, 15, and 30 min (Fig. 5A). This treatment completely abrogated the degradation of xIAP and PARP (Fig. 5A). Furthermore, preincubation of SMC with the specific calpain inhibitor calpeptin also inhibited the degradation of both xIAP and PARP induced by treatment with degraded collagen (Fig. 5B).


Figure 3
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  		Fig. 3 Degraded collagen activates calpains. (A) SMC cultured on monomeric type I collagen were treated with 250 µg/ml degraded collagen for the indicated times. Calpain activity was determined in cell lysates using 50 µM of the fluorogenic calpain substrate Suc-LLVY-AMC. The free AMC fluorescence was continuously measured and expressed as random fluorescent units (RFU). (B) SMC were treated with or without degraded type I collagen for 30 min and calpain activity was measured in cell lysates in the presence or absence of 500 µM calpeptin. Original recordings of representative experiments (n=3) are shown.

 

Figure 4
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  		Fig. 4 Calpains degrade xIAP in vitro and in intact cells after exposure to degraded collagen. (A) Control SMC lysates (40 µg) were incubated with 250 ng recombinant calpain II for the indicated times and xIAP was detected by Western blot analysis. (B) SMC lysates (40 µg) were incubated with 3.2 µg recombinant calpain II for 120 min in the presence or absence of 20 mM EDTA or 2.5 mM calpain inhibitor I, and xIAP was detected by Western blot analysis. Cell lysates of SMC treated with type I collagen fragments for 30 min (left in [A] and right in [B]) were ran side-by-side with the calpain-treated samples to compare the sizes of ex vivo and in vitro generated xIAP fragments. Equal protein loading was confirmed with an anti-β-actin antibody. (C) Caspase substrate assays were performed using 0.63 pM recombinant active human caspase-3 and 0.25 µg/µl native and calpain-treated GST-xIAP, respectively. (D) The same probes as in (C) were subsequently analyzed by immunoblotting to verify xIAP degradation and presence of active caspase-3 (p12). Original recordings of representative experiments (n=3) are shown.

 

Figure 5
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  		Fig. 5 Calpain inhibition prevents xIAP degradation by degraded collagen. (A) SMC were treated with degraded type I collagen fragments in the presence or absence of 50 µM BAPTA under Ca2+-free conditions for 5, 15 and 30 min, and cell lysates were analyzed for xIAP and PARP degradation by immunoblotting. (B) SMC were treated with type I collagen fragments in the presence or absence of the calpain inhibitor calpeptin (400 µM) for 30 min and Western blot analysis was performed for xIAP and PARP. The same blots were reprobed with an anti-β-actin antibody to confirm equal loading of cellular proteins.

 
3.4 Calpain activation precedes caspase activation, and calpain inhibition abrogates apoptosis
As cleavage of calpain substrates preceded that of caspase substrates, we measured the kinetics of calpain and caspase activation in the same cell lysates using fluorogenic substrates. Calpain activation was dramatically enhanced after 15 min and increased further over the next 100 min (Fig. 6A). In contrast, caspase activation began later, with the first measurable increase in activity beginning after 120 min (Fig. 6A). Thus the onset and peak of calpain activity preceded that of caspase activity. To test if calpains causally promote apoptosis, we treated cells with degraded collagen in the presence and absence of BAPTA (calpain inhibitors such as calpeptin were much less efficient). While BAPTA alone had negligible effects on apoptosis, it completely inhibited apoptosis induced by degraded collagen suggesting a causal role of calpains in promoting the apoptotic process (Fig. 6B).


Figure 6
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  		Fig. 6 Calpain activation precedes caspase activation, and calpain inhibition abrogates apoptosis. (A) SMC cultured on monomeric type I collagen were treated with 250 µg/ml degraded collagen fragments for the indicated times, and caspase-3 activity (left) and calpain activity (right) were determined in aliquots of the same cell lysates. (B) SMC were exposed to degraded type I collagen fragments in the presence and absence of 50 µM BAPTA under Ca2+-free conditions for the indicated times. SMC treated with BAPTA alone were used as a control. Apoptosis was detected using antibodies to active caspase-3 and apoptotically cleaved PARP and lamin A/C, respectively.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we have observed a rapid activation of calpains after treatment of SMC with degraded collagen or following culture on polymerized collagen gels, where collagen fragments are endogenously generated by the SMC through MMPs. This is in line with our previous observations of calpain-mediated degradation of focal adhesion complex components such as pp125FAK, paxillin and talin after degraded collagen fragment treatment [13]. In our present work, we have identified two proteins closely associated with the protection of the cell against apoptosis–the potent endogenous caspase inhibitor xIAP and the DNA-repair enzyme PARP–as novel calpain substrates in SMC after treatment with degraded collagen. PARP has previously been shown to be a substrate for calpains during necrotic cell death [19], while loss of xIAP in neutrophils of patients with chronic neutrophilic leukemia has been recently attributed to calpains [20].

Interestingly, a number of other proteins associated with apoptosis (both pro- and anti-apoptotic) have been shown to be either activated or inactivated by calpains during apoptosis. This is very much dependent on the cell type and/or apoptotic stimulus applied. While calpains cleave and inactivate caspase-3, -7, and -9 in biochemical ex vivo apoptosis assays and during Ca2+ ionophore-induced apoptosis in vitro [21], they can apparently activate caspases in other experimental systems: caspase-3 during cerebral hypoxia-ischemia [22], caspase-7 in B cell apoptosis [23], and caspase-12 in amyloid β peptide-induced neural cytotoxicity [24]. Another level of complexity is added by the modification of members of the Bcl-2 family by calpains: cleavage of Bax, Bid, and Bcl-xL either generates novel pro-apoptotic fragments and/or inactivates the anti-apoptotic function of these proteins [24–26]. The role of calpains in apoptosis has still remained enigmatic similar to their apparently differing preference for individual substrates among experimental systems, and appears to be highly dependent on the cell type and apoptotic stimulus [27]. Judging mainly from the subset of substrates calpains process after a particular apoptotic stimulus, it seems that in some instances, they may inhibit apoptosis by inactivating initiator caspases, whereas in other, they may promote apoptosis by activating effector caspases or enforcing mitochondrial apoptosis [27]. The complexity of the interplay between caspases and calpains is exemplified by the activation of calpains by caspase-mediated cleavage and inactivation of their endogenous inhibitor calpastatin [28].

In our study, we have added to this complexity by identifying vice versa the potent caspase inhibitor xIAP as a calpain substrate and finding its caspase-inhibitory function abrogated after cleavage. Thus we have extended the list of apoptosis-associated calpain substrates to another key component of the programmed cell death cascade: the IAP family of caspase inhibitors. X-chromosome linked inhibitor of apoptosis inhibits highly efficiently both effector and initiator caspases, and its suppression promotes apoptosis in many experimental systems [29]. In our system, degradation of xIAP by calpains results in increased caspase activity conceivably by releasing caspases from their inhibitory complex with xIAP. This reminds of the mechanism other pro-apoptotic proteins such as Smac/DIABLO and Omi/Htr2 apply to "extract" xIAP from its complex with caspases and thus "free" the caspase from its inhibitor [30]. In the case of Omi/Htr2, this protease even degrades subsequently xIAP [31]. Loss of xIAP due to calpain action may provide an attractive explanation for the seemingly contradictive findings of both activation and inactivation of the same caspases (caspase-3 and -7) by calpains in different systems: the experiments showing activation of these two caspases in biochemical ex vivo assays have all been performed in the presence of cell lysate (e.g. S-100) [22,23]. This cell lysate may be the source of endogenous xIAP that is more sensitive to degradation than caspases thus allowing auto-processing of the caspase in the absence of its inhibitor [32]. Therefore, degradation of xIAP by calpains in our system may decrease the activation threshold of caspases normally held in check by the IAPs and/or lead to auto-activation of the caspases. Indeed, calpain activation preceded caspase activation in our system, and inhibition of the calpains inhibited both caspase activation and apoptosis, thus suggesting a causal role of calpains in the execution of the cell death program.

It appears that there is at least one more mechanism of xIAP "disposal" by calpains in addition to direct xIAP degradation: by cleaving pp125FAK, calpains may ensure inactivation of xIAP also at the transcriptional level, as pp125FAK has been shown to upregulate xIAP levels via a phosphatidylinositide 3'-OH-kinase-Akt/NF-{kappa}B-dependent survival pathway [33]. Remarkably, xIAP is the only member of the family that is destroyed as we have seen no alterations of c-IAP1, c-IAP2 and survivin. This exclusivity, again, has its precedence in several of the studies cited above, where specific subsets of calpain substrates are modified or degraded exclusively among different experimental systems. The reasons for this particular selectivity of calpains for certain substrates among the plethora of possible ones has still remained unexplored.

Activation of calpains by integrins such as {alpha}IIbβ3 on platelets has been described [34–36] as well as increases in intracellular Ca2+ levels after integrin ligation [37]. However, the downstream signaling events leading to calpain activation remain to be elucidated. In SMC, the ligation of {alpha}vβ3 by MMP-generated fragments of type I collagen promotes migration, proliferation and survival [16,38,39]. In our system, calpains are activated by collagen fragments mainly via association with {alpha}2-containing integrins as we have previously shown [13]. Thus different integrins can be engaged by the collagen fragments generated by MMPs from the collagen fibrils of the extracellular matrix. The decision of the cell between life and death would then be governed by the integration of signals from all engaged integrins with those coming from the focal adhesion complex, the assembly/disassembly of which is, remarkably, dynamically regulated by the calpains [27].

The expression and activity of MMPs are increased in lesions of atherosclerosis and their accumulation at sites of plaque rupture has been implied in the plaque destabilization process via degradation of the structural ECM [5,6]. However, little is known about the particular ECM fragments generated during plaque progression and rupture in vivo and their spatio–temporal pattern. Interstitial collagenases such as MMP-1, -8 and -13 are abundantly present in the atherosclerotic lesion, and are known to mediate the initial step of collagen degradation by cleaving the native triple-helical fibrils of type I, II, and III collagen at a specific single site (Gly775-Leu/Ile776) resulting in 3/4 and 1/4 fragments [40–42]. This cleavage-generated neoepitope is present in atherosclerotic lesions as detected by a site-specific antibody and is colocalized with the three MMPs [40,41]. The initial cleavage by collagenases makes the fragments accessible to other proteinases such as gelatinases and stromelysins which together with collagenases further catabolize the collagen fibril [43,44]. Another class of neointimal SMC proteases, the cathepsins, can also cleave collagen but at different sites than the MMPs: cathepsins L and B cleave only in the telopeptides and not in the native triple helix, while cathepsin K cleaves both in the telopeptides and at multiple sites within the triple helix similar to the collagenase type 3 we have used [45–47]. Due to the lack of epitope-specific antibodies the existence of these fragments in atherosclerotic lesions in vivo has not been demonstrated although ample biochemical evidence is available from in vitro studies [45]. However, it is more than plausible that several of these fragments are present simultaneously and/or consecutively at sites of MMP activity in the plaque in vivo.

In summary, inactivation of anti-apoptotic proteins such as xIAP by calpains may contribute to apoptosis of SMC induced by collagen fragments proteolytically released from the extracellular matrix by MMPs. This may be a novel regulatory mechanism of SMC apoptosis in biological settings of enhanced collagen degradation such as cell migration, vascular remodeling, neointima formation and at sites of atherosclerotic plaque rupture, and may contribute to the inherently increased susceptibility of neointimal SMC to apoptosis as well as its increased occurrence at sites of plaque rupture [5,48].


   Acknowledgements
 
This study was supported in part by the Deutsche Forschungsgemeinschaft (LE 940-3), and the H.-H. Deichmann Foundation for Atherosclerosis Research. We thankfully acknowledge the technical assistance of Sina Mersmann, Kerstin Abouhamed, and Vanessa Brinkmann.


   Notes
 
Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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